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Purification and characterization of alpha-amylase from Bacteroides amylophilus strain H-18 Rahman, Sheikh Saif-Ur 1970

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PURIFICATION AND CHARACTERIZATION OF a-AMYLASE FROM BACTEROIDES AMYLOPHILUS STRAIN H-18 by SHEIKH SAIF - UR - RAHMAN B.Sc. (A.H.)-, The University of the Panjab, 1960 M.S.A., The University of B r i t i s h Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Animal Science We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1970  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  further  for  agree  scholarly  by  his  of  this  written  thesis  in  at  University  the  make  that  it  freely  for  is  financial  gain  Department  Date  5se^V,U-  of  Columbia,  British for  by  the  Columbia  shall  not  the  requirements  reference copying of  I agree and  copying or  for  that  Study.  this  thesis  Head o f my D e p a r t m e n t  understood that  permission.  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  extensive  p u r p o s e s may be g r a n t e d It  fulfilment  available  permission for  representatives. thesis  partial  or  publication  be a l l o w e d w i t h o u t  my  Chairman, Professor W. D. K i t t s  ABSTRACT  The research was undertaken to study the e x t r a c e l l u l a r a-amylase produced by the anaerobic rumen bacterium, amylophilus  s t r a i n H-18.  Bacteroides  Four active isoenzymes of a-amylase  were detected by disc electrophoresis and electrofocusing techniques.  I s o e l e c t r i c points as determined by electrofocusing  were pH 3.7, 4.5,,5.9 and 8.0.  Isoenzymes were named 1, 2, 3  and 4 with respect to their increasing i s o e l e c t r i c points. a-Amylase isoenzyme 1 was p u r i f i e d by DEAE-Sephadex and G-200 techniques.  Some of i t s general physio-chemical  t i e s were studied.  proper-  I t had maximum a c t i v i t y at pH 6.7, 44°C and  was s t a b i l i z e d by calcium ion.  It was susceptible to thermal  denaturation i n the absence of calcium.  Various other metal ions  tested could not replace the calcium i n regenerating maximum activity.  I t was found by atomic absorption  spectrophotometry  that a-amylase isoenzyme 1 contained 3 gram-atoms of calcium per mole of enzyme.  The estimated molecular weight by gel f i l t r a -  tion technique was 45,000 Daltons.  Amino acid analysis i n d i c a -  ted the absence of cysteine, therefore, disulphide linkages were not involved i n maintaining the. t e r t i a r y structure.  Tryptophan  appeared to be required for enzymic a c t i v i t y , as determined by the N-bromosuccinamide oxidation technique. The mode of action of a-amylase isoenzyme 1 was studied  using amylose and soluble starch as substrates. The products of enzymatic degradation were analysed q u a l i t a t i v e l y by thin layer chromatography.  The•maltohexaose, maltoheptaose,  maltonanaose and maltodecaose  maltoctaose,  remained i n the digest mixture for  sometime after the achroic point.  The degree of multiple attack  was 2 , as calculated by determining the r a t i o of the reducing value of the oligosaccharide f r a c t i o n to that of polysaccharide f r a c t i o n . Antisera against a-amylase isoenzyme 1, produced i n rabbits by i n j e c t i o n of a-amylase inFreund's complete adjuvant was to be mono-specific.  found  The i n h i b i t i o n of a-amylase a c t i v i t y by  antibody and i n h i b i t o r y effect of starch on the amylase-antiamylase system were'demonstrated.  The e f f e c t of anti-amylase  (isoenzyme 1) globulin on amylases of diverse o r i g i n was studied by the Ouchterlony double d i f f u s i o n technique. demonstrated  These experiments  antigenic determinants which were d i s t i n c t from those  present on the a-amylase of hog pancreas, B a c i l l u s s u b t i l i s and Aspergillus oryzae.  Immunoelectrophoretic  analysis indicated the  presence of only a single antigenic component.  Quantitative pre-  c i p i t a t i o n studies gave a t y p i c a l curve with one equivalence point with an antibody to antigen r a t i o of 2.'31.  N-Bromosuccin-  imide treated a-amylase (isoenzyme 1) exhibited s i m i l a r immunochemical behaviour to the native enzyme, but completely l o s t i t s catalytic activity. s i t e s were d i s t i n c t .  It i s possible that c a t a l y t i c and antigenic Urea treated a-amylase (isoenzyme 1) did  not show any p r e c i p i t a t e with i t s s p e c i f i c antibody and peared to have l o s t i t s antigenic structure.  thus,.ap-  Dedicated to my parents, teachers and friends who helped, each i n their own  i  way.  TABLE OF CONTENTS  Chapter I. II.  Page  INTRODUCTION  .  .  .  .  .  REVIEW OF THE LITERATURE Amylase  .  .  .  .  .  .  1.  . . . . . .  2  . . . . -  Nomenclature .  2  .• . •  2  a-Amylases and their Sources  . . .  3  Non-ruminant a-Amylase Ruminant a-Amylase .  . .  .  .  .• 3  .  4  Production and Induction of a-Amylase  . . . . . .  4  Physical Properties of C r y s t a l l i n e a-Amylase  6  Primary Structure of a-Amylases  6  .  Amino Acid Analysis  6  Functional Groups  6  Non-protein Constituents and Co-factors i n a-Amylase  .  .  .  .  a-Amylase as Metalloenzyme  .  9 .  .  . . .  .  .  . 9  Secondary and Tertiary Structure of a-Amylase .  .  .  .  . 11  Quarternary Structure of a-Amylase  .  .  .  . 13  .  .15  .  . 18  The Action Pattern of a-Amylase Immunochemical Study of a-Amylase  .  .  .  .  .  .  .  . . . . •. •.  Mammalian a-Amylase Microbial a-Amylase  18 .  . . .  18  vi  Chapter  III.  Page  MATERIALS AND METHODS .  . . .  Chemicals  •.  .  .  .  .  . . . . .  .  .. .  31 31  Organisms  .  Maintenance of Bacteroides amylophilus Strain H-18 . . . .•. . .:.  .  . . .  Growth Measurements of Bacteroides amylophilus Strain H-18  .  .  .  .  .  .31  .  32  .32  Production and P u r i f i c a t i o n of a-Amylase from Bacteroides amylophilus Strain H-18 Production of a-Amylase  .  .  .  .  33  .  .  .  .• .  .  33  P u r i f i c a t i o n of a-Amylase on DEAE-Sephadex A-50 and G-200 Sephadex  •  33  Assay of a-Amylase  •.  .  Assay of Protease  34 35  Determination of Nitrogen  .•  Determination of Protein .  .  35 .  35  Determination of Total Carbohydrate  36  Disc Gel Electrophoresis  36  Isoelectrofocusing .  . . .  .  .  .  Charcoal-Celite Column Chromatography Paper Chromatography  .  .  . . .  .  .  .  . . .  .  .  37  .  .  .• . •.  .  .  37  .  .  .  .  . . .  38  .  .  . . .  39  Thin Layer Chromatography Effect of Temperature  • .  ...  . . .  Molecular Weight Determination Calcium Content Determination  .  .  . . .  .  .  .39 .39  .  .  .  .  .  .  .  39 •  vii  Chapter  Page Amino Acid Analysis  .  .•  Immunochemical Techniques  40 .  .  40 .  Production of Antibodies  40  Determination of Enzymic Inhibition  •  .  Immunodiffusion Characteristics  41 •  Protein. Determination i n AntigenAntibody Complex . . . IV.  RESULTS AND DISCUSSION  .  .  .  .  41 -  .  •. .  .  .  .  .  .  . .  .41 .  44  Characterization of a-Amylase from Bacteroides amylophilus Strain H-18  .  .' .  .• .  .  .  .  .  .  .44  Production and P u r i f i c a t i o n of a-Amylase Production of a-Amylase  .  .  44  .  44  P u r i f i c a t i o n of a-Amylase Isoenzyme 1  54  C a t a l y t i c Properties of a-Amylase Isoenzyme 1  .  .  .  . . .  .  .  .  .  Determination of Type of Amylase  .  . . .  .  .  .  .  66  .  .  .  66  E f f e c t of pH on a-Amylase A c t i v i t y  66  E f f e c t of pH on Enzymic S t a b i l i t y  66  Effect of Temperature on a-Amylase Activity . . . .• . . . .- . E f f e c t of Temperature on Enzymic Stability  .  .  .  .  .  .  . . .  Amino Acid Determination Calcium Determination .  .  .•.  .  .  .  .  .• .  .  75  .  .  .  .  .  75  .  .  .  .81  .  .81  . . .  viii  Chapter  Page .  E f f e c t of Chemical Reagents on Enzymic A c t i v i t y .  .  .  .  .  .: .  Effect of Urea on a-Amylase A c t i v i t y  .83 83  Effect of EDTA and M e t a l l i c Ions on a-Amylase A c t i v i t y  . 87  Functional Groups Determination  .  .  .  .  .  .  Determination of Molecular Weight  93  Determination of I s o e l e c t r i c Point  .  The Action Patternrof a-Amylase Isoenzyme 1  .  ...  .  . 93  .  .  .- .  . 101  Immunochemical Studies on a-Amylase Isoenzyme 1 Inhibition of Enzymic A c t i v i t y by  113  Antibodies .  113 .  Ouchterlony Double Diffusion Analysis  116  Immunoelectrophoretic Analysis  V.  .90  -.  116  Quantitative P r e c i p i t a t i o n Analysis  121  Effect of N-bromosuccinimide and Urea on Antigenicity The Neutralization of Amylase-Antiamylase System by Starch . . . . . . . . - .  121  CONCLUSIONS  .  .  . . .  .  .  .  .  .  .  .  .  .  .  . . . 126  .  .  .  .135  ix  Chapter  Page General Properties .  .• .  . . .  Action Pattern Immunochemical Properties LIST OF REFERENCES  .  .  .  .  .  .  .  . . . .  .  .  . 135  . . .  136  . . .  :  .  136 .  .  .  .  .  .  .  Chapter I and Chapter II  22  Chapter III Chapter IV:A . B• . C  . . .  . .  42 96  .  .  .  .111 133  LIST OF TABLES  Table  Page  I. ' The Sources of a-Amylase  .  5  II.  Physical Properties of a-Amylase .• . . .  7  III.  P u r i f i c a t i o n of B_. amylophilus Strain H-18 a-Amylase Isoenzyme 1  58  Summary of the Optimum pH Range of a-Amylase from Various Sources  71  IV. V.  Summary of the Optimum pH S t a b i l i t y Range f o r Various a-Amylases .  VI. VII. VIII.  .  .  .  .  .•  Optimum Temperature f o r Various a-Amylases Calcium Contents of Various a-Amylases .  X. XI. XII. XIII. XIV.  .  . . .  .  .  . 78  .  .  .  .  . 82  .  . .  .  Effect of Reducing, Oxidizing and SH-Inactivating Agents on a-Amylase Isoenzyme 1 A c t i v i t y  IX.  74  .  .  ;  . 91  Molecular Weight of Various a-Amylases  94  I s o e l e c t r i c Points of Various a-Amylases  95  Estimation of the Degree of Multiple Attack by B^. amylophilus a-Amylase Isoenzyme 1 . .• P r e c i p i t a t i o n reaction of B_. amylophilus a-Amylase Isoenzyme 1 with i t s Antibody . . . . . . . Effect of N-bromosuccinimide on Antigenicity of a-Amylase Isoenzyme 1 . . . . . . . . . . . Effect of Urea on Antigenicity of a-Amylase. Isoenzyme 1 . . . . . . . . . . .  .  .  110 .  .  . 122  .  .  . 125  .  .  . 129  LIST OF FIGURES  Figure 1. 2.  3.  4. 5. 6.  7.  8.  9.  Page  Growth curve and production of a-amylase from Bacteroides amylophilus s t r a i n H-18  .  .  Linear relationship between the production of a-amylase and growth of Bacteroides amylophilus s t r a i n H-18 . . . . . .  .  .-  Effect of maltodextrin on the growth and production of a-amylase.from Bacteroides amylophilus s t r a i n H-18 . . . . . .  .  .  Detection of 4 isoenzymes of a-amylase by disc electrophoresis . . . : . .  . .  .  :  . •.  .  .  .45  47  . .  . .  . .  . .  . .  Detection of 4 isoenzymes of a-amylase by electrof ocusing  52  .  Flow sheet of methods for i s o l a t i o n of a-amylase isoenzyme 1 from Bacteroides amylophilus s t r a i n H-18  .  Chromatography of Bacteroides amylophilus s t r a i n H-18 a-amylase isoenzyme 1 on Sephadex G-200 . . . . . . . . . .  .  .  . . .  ...  .  60  .  .  62  Disc electrophoresis of a-amylase isoenzyme 1  64  10.  Electrof ocusing of a-amylase isoenzyme 1  11.  Optimum pH for hydrolyzing starch  12.  Effect of pH on the s t a b i l i t y of a-amylase  .- .  13.  Optimum temperature f o r hydrolysing starch  . . . . . . .  . . .  55  57  Chromatography.of Bacteroides amylophilus s t r a i n H-18 a-amylase on DEAESephadex A-50 ,. .  .  49  . 67. . . . . .  .  . . . .  .69 72 76  xii  Figure  Page  14.  Thermal s t a b i l i t y of a-amylase  15.  Effect of urea on a-amylase a c t i v i t y  16.  Effect of (A) EDTA and (B) metal ions after EDTA treatment.on reactivation of a-amylase  17.  18. 19. 20.  21.  22. 23.  24.  79 .  Thin layer analysis of the digestion of amylose by a-amylase isoenzyme 1.. .  .  .  ...  .  .  .  .  88 .  .  .  .  .  .  Thin layer analysis of the digestion of starch by a-amylase isoenzyme 1  A diagramatic representation of immunodiffusion p r e c i p i t a t i o n reaction between B_. amylophilus a-amylase isoenzyme 1 . .  .  .  .  .  . 114  ...  . 117  A diagramatic representation of Immunoelectrophoresis of j$. amylophilus a-amylase • isoenzyme 1 . . . . . . . .  119 •.  P r e c i p i t a t i o n curve of N-bromosuccinimide treated and native a-amylase with i t s antibody Inhibitory e f f e c t of starch on a-amylase isoenzyme 1 and antiamylase system  . 102  104  Neutralisation curve of a-amylase isoenzyme 1 with antiserum  P r e c i p i t a t i o n curve of a-amylase isoenzyme 1 with i t s antibody . . . . . . .  . 84  . 123  127 ., .  . 131  ACKNOWLEDGEMENTS  The author wishes to express his sincere thanks to Professor W. D. K i t t s , Chairman, Department of Animal Science, for his supervision, indispensable guidance and h e l p f u l c r i t i c i s m i n the completion of this study. The author would also l i k e to express his sincere gratitude and appreciation for the valuable suggestions and other f a c i l i t i e s provided by Dr. T. H. Blackburn, Associate Professor, Department of Microbiology, and to Dr. S. Nakai, Associate Professor, Department of Food Science. The author wishes to express his thanks to graduate students, Mr. L. E. Lesk, Department of Microbiology, Mr. R. J . Hudson, and Mr. J. A. Shelford, Department of Animal. Science for their help i n enzyme preparation, immunological studies and amino acid analysis. Thanks are due to a l l those i n Canada and i n Pakistan whose help and encouragement were of immense importance during the course of this study. Appreciation i s also expressed to Miss V. Curylo and Mrs. J . A. Shelford for typing the manuscript. Acknowledged with thanks, i s the National Research Council of Canada postgraduate scholarship and the University of B r i t i s h Fellowship.  Columbia  PURIFICATION AND CHARACTERIZATION OF a-AMYLASE FROM BACTEROIDES.AMYLOPHILUS STRAIN H-18  CHAPTER I INTRODUCTION  Much of the work on rumen bacteria has been devoted to the study o f • c e l l u l o l y t i c organisms and the mechanism by which c e l l u l o s e i s degraded.  With the recent emphasis on higher grain feeding to ruminants  i t has been important to study the breakdown.of starch i n the rumen. Though several species of amylolytic bacteria have been i s o l a t e d from the rumen and their incidence studied under a variety of dietary t r e a t ments (35), the amylolytic enzymes of these organisms have not been studied i n d e t a i l .  It i s hoped that greater knowledge of the  production  and mode of action of a-amylase by rumen bacteria may•facilitate better understanding of starch hydrolysis i n the rumen.  This i s p a r t i c u l a r l y  s i g n i f i c a n t when i t has been.shown that non-amylolytic  strains of Buty-  r i v i b r i o , Selenomonas and Eubacterium, which can ferment dextrin but not starch, are present digesters  i n the rumen i n greater proportions  than starch  (35).  Bacteriodes  amylophilus s t r a i n H-18  i s a predominant starch d i -  gester constituting 10 per cent of the t o t a l rumen b a c t e r i a l f l o r a  (35)  and secreting an active a-amylase i n s p e c i f i c laboratory growth madium (6).  Since l i t t l e i s known about the a-amylase produced by B_. amylophilus,  this organism was  selected for the present i n v e s t i g a t i o n .  It may  be men-  tioned here that with the exception of members of genera Pseudombnas and V i b r i o , most organisms producing exoenzymes are Gram p o s i t i v e (78).  13.  amylophilus,,on the other hand, deviates from this general rule i n being Gram negative.  CHAPTER II REVIEW OF LITERATURE A.  Amylases  Starch i s an important source of dietary carbon and therefore i t i s not surprising to find amylases widely distributed i n a l l Phyla. Amylase causes the hydrolysis of amylose, amylopectin, glycogen and their degraded products.  In mammals the digestion of starch i s i n i t i a t e d  by the action of salivary amylase and continued i n the duodenum by the action of amylase secreted by the pancreas and.the i n t e s t i n e . Microbial amylases are extra-cellular, i n nature and several microorganisms continue to produce e x t r a - c e l l u l a r amylase even after the f e r mentation of starch i s completed.  It might be expected that the amylase  produced during the growth period of the organism hydrolyzes starch; and sugar thus produced i s u t i l i z e d for the growth of micro-organisms.  The  species of genus B a c i l l u s (82) appear, however, to deviate from this general r u l e , v i z . , IS. stearothermophilus (112) and B^. s u b t i l i s (82) which produce e x t r a - c e l l u l a r amylase even during the stationary phase. B.  Nomenclature  Amylases were c l a s s i f i e d as a and 3 types by Khun (43) and Ohlsson (70). The a and 8 amylases y i e l d products which have a and $ configuration at C^ of the reducing sugar respectively.  Freeman and.  Hopkin (26) have confirmed the configuration of the anomeric reducing carbon atom released during the enzymatic hydrolysis of starch.  3 The second important  difference between a and g amylases i s their  mode of attack on the substrate. 1—>-4  a-Amylases, being endoenzymes, cleave  bonds located i n the inner region of the substrate.  Therefore, a-  amylases are expected to l i b e r a t e products of varying chain lengths and also rapidly decrease the v i s c o s i t y and iodine staining capacity of starch during enzymatic hydrolysis. B-Amylases have been regarded  as exo-amylases because they do not  rapidly decrease v i s c o s i t y , and iodine staining of starch during starch hydrolysis.  Since 8-amylase i s an exo-enzyme the penultimate bond at a  non-reducing chain end i s the only bond available for enzymatic hydrolysis. g-Amylase attacks i n an exclusive manner and produces 8-maltose only. Although the enzyme of 13. macerans produces c y c l i c schardinger dextrins from starch, i t i s s t i l l c l a s s i f i e d as an amylase (27).  The enzyme from  13. macerans, l i k e other a-amylases, renders starch achroic to iodine. Robyt and French (80) reported that the enzyme of B_. polymyxa produces mainly 8-maltose, but has the a b i l i t y to by-pass the 1—>-6 of glycogen.and amylopectin,  branch linkage  thus indicating an a-amylase action pattern.  Amylases also d i f f e r i n their action pattern on iodine-staining polysaccharides.  It i s represented graphically by p l o t t i n g the change  i n blue value against the corresponding  changes i n reducing value during  starch or amylose hydrolysis, and various amylases follow their c h a r a c t e r i s t i c curves C.  1.  own  (44). a-Amylases and their Sources  Non-ruminant a-Amylase During the l a s t twenty years a-amylases have been i s o l a t e d ,  4  p u r i f i e d and c r y s t a l l i z e d from a variety of sources.  2,  (Table I)  Ruminant a-Amylase • Rumen micro-organisms capable of hydrolyzing starch include Strep-  tococcus bovis, Bacteroides amylophilus,  Bacteroides ruminicola, Siiccin-  imonas amylolytica and Selenomonas ruminantum (35). A number of rumen c e l l u l o l y t i c micro-organisms also possess amylolytic properties such as Clostridium lachheadii, some strains of Bacteroides succinogenes and most strains of B u t y r i v i b r i o f i b r i s o l v e n s (35).  The amylolytic enzymes of rumen bacteria have not been characterized except for those of Streptococcus  bovis  ( 1 1 0 ) and Clostridium  butyricum (32). 3.  Production and Induction of a-Amylase The production c h a r a c t e r i s t i c s of a-amylase of B^. s u b t i l i s and 13.  stearothermophilus seemed c o n f l i c t i n g .  have been studied by many workers which have often J3. s u b t i l i s s t r a i n N produces e x t r a - c e l l u l a r a-  amylase predominantly after maximum c e l l growth has occurred  (66).  The  a-amylase of another s t r a i n of 13. s u b t i l i s ( 1 4 ) and of B_. stearothermophilus ( 1 1 1 ) are formed during the logarithmic phase of growth p a r a l l e l ing  the increase i n c e l l mass.  Yoshida and Tobita (115.) reported that  a-amylase i s released into the medium during the stationary phase of growth i n a leucine requiring mutant of B_.' s u b t i l i s . . Pseudomonas saccharophila produces inducible e x t r a - c e l l u l a r aamylase ( 4 9 ) .  Markovitz and K l e i n ( 4 9 , 5 0 ) , Schiff et a l . ( 8 4 ) and  5  TABLE I THE SOURCES OF a-AMYLASES  Source A.  B.  C.  D.  r-  Reference  Mammalian 1.  Human.saliva  2.  Porcine pancreas.  3.  Rat pancreas  30  4.  Human pancreas  20  25 8, 57  Plant 1.  Barley malt  22, 87  2.  Sorghum malt  16  Bacterial 1.  Bacillus subtilis  96  2.  B a c i l l u s stearothermophilus  10, 11  3.  B a c i l l u s macerans  88, 79  4.  B a c i l l u s polymyxa  80, 83  5.  Pseudomonas saccharophila  51  Fungus 1.  Aspergillus oryzae  2.  Aspergillus niger  3.  Aspergillus candidus  21, 104 102 82  j  6 Eisenstadt and Klein.(18,19) have presented  evidence f o r the de novo syn-  thesis and i n d u c i b i l i t y of a-amylase i n P_. saccharophila.  The k i n e t i c s  of enzyme formation was reported to be l i n e a r and the quantity of aamylase produced was proportional to the substrate  concentration.  Welker and Campbell.(112) also studied the induction of a-amylase of 15. stearothermophilus  by maltodextrins.  They observed that addition  of maltose,' maltotriose, maltotetraose ,'~maltopentaose and .maltohexaose to a chemically defined medium resulted i n a stimulation of the d i f f e r e n t i a l rate of a-amylase production.  D.  Physical Properties of C r y s t a l l i n e a-Amylase  Some of the general physical properties of a-amylase are summarized i n Table I I . E.1.  Primary Structure of the a-Amylases  Amino Acid.Analysis Amino acid analyses have been reported for human s a l i v a r y amylase  (62), porcine pancreatic amylase (9), 13. s u b t i l i s amylase (3,42), 13. stearothermophilus  amylase (12), and A. oryzae amylase  (1,94).  The a-amylases of J3. s u b t i l i s do not contain cysteine and cystine. _B. stearothermophilus  a-amylase i s unusual due to the absence of trypto-  phan. The amino acid sequence of a-amylases has not .been reported.  2.  Functional Groups In order to study the functional groups of a-amylases at least  7 TABLE I I PHYSICAL PROPERTIES OF a-AMYLASES  Source  Properties B.subtilis (54,56, 96)  B.stearothermophilus (11,12)  P.Sacch- A.oryzae arophila (51) (21, 104)  —  14.9(24) 13.0  15.9(24) 17.0  5.255.75  4.8-5.8 (24)  4.0-5.8 (4)  6.8  6.9  4.5-8  5.5-8.5  4.9-9.1  7.0-8.5  4.8-11  40°  35°  37°  40°  51,000 (38)  59,500  45,000 (15)  _  4.2  5.7  5.2-5.6  5.2-5.6  26(24)  26(24)  13,500  13,500  Per cent nitrogen  16(24)  15  3ptimum pH  6.0  5.0  Optimum pH s t a bility range  4.8-8.5  Optimum temperature  40°  65°  Molecu^lar weight  48,700 (23)  15,000 (47)  Isoelectric point  5.4  4.8  Absorbance % A 25.3 280 mu (24) A c t i v a t i o n (0-12°) 15,000 energy 12° 11,000  _  Barley malt (61,87)  19.7 (24) 14,000  (0-15°) 14,400 10,650 (15-40°) 8,500  7,050  Porcine pancreas (8,57, 61)  Human saliva (25,56, 58,59, 61)  8 two techniques have been,used; (a) the e f f e c t o f pH on t h e ' M i c h a e l i s constant,  Km, and the maximum v e l o c i t y , Vm and (b) c h e m i c a l m o d i f i c a t i o n  of a-amylases.• E a r l i e r work u s i n g has  given  groups.  c o n f l i c t i n g reports  c h e m i c a l m o d i f i c a t i o n of an enzyme  regarding  the p a r t i c i p a t i n g of f u n c t i o n a l  A t l e a s t p a r t of the reason f o r t h i s d i s c r e p a n c y  that the c h e m i c a l reagent used had l i t t l e w i t h many s i d e c h a i n  i s the f a c t  s e l e c t i v i t y and i s r e l a t e d  groups.  Ono e t a l . (73) i n v e s t i g a t e d the e f f e c t of pH on the Km of EL s u b t i l i s a-amylase.. T h e i r r e s u l t s i n d i c a t e d that the a p p a r e n t . r a t e cons t a n t , K^, of t h i s enzyme d i m i n i s h e d of the optimum pH.  T h i s was a s c r i b e d  on both.the a l k a l i n e and a c i d s i d e to the f o r m a t i o n  a c a t i o n which were determined to PK v a l u e values  o f 4.2 and 7.5.  These PK  along w i t h the heat of i o n i z a t i o n i n d i c a t e d t h a t the a c t i v e groups  involved  i n the c l e a v a g e of the bonds were a c a r b o x y l a t e  dazolium i o n .  The apparent M i c h a e l i s  pH range of 3.6 to 9.4, s u g g e s t i n g for  of an a n i o n and  constant  (Km) was s t a b l e over the  t h a t the s i d e chain.groups  the s u b s t r a t e b i n d i n g must i o n i z e o u t s i d e  possibility  i o n and an i m i -  responsible  the pH range s t u d i e d .  The  t h a t t y r o s y l groups may be i n v o l v e d was i n d i c a t e d because  the PK v a l u e  o f the p h e n o l i c  hydroxyl  group does not f a l l  i n the range  studied. Thoma e t a l . (100) r e p o r t e d pancreatic The  a-amylase were l i k e l y  binding  to b e , c a r b o x y l a t e and i m i d a z o l i u m  t h a t the Km changed w i t h pH, i n d i c a t i n g t h a t  l e a s t two groups w i t h PK v a l u e s  substrate  ions.  s i t e groups of t h i s a-amylase were d i f f e r e n t from those of 13.  s u b t i l i s shown by the f a c t at  t h a t the c a t a l y t i c groups of p o r c i n e  binding.  o f 5.7 and 8.7 were r e s p o n s i b l e f o r  9 L i t t l e and:Caldwell (46) inactivated porcine pancreatic a-amylase by treating with ketene, phenylisocyanate, formaldehyde and nitrous acid, and suggested that free amino groups were required for c a t a l y t i c a c t i v i t y . They also reported that p-chloromercuribenzoate, iodoacetamide and mercuric chloride did not deactivate the a-amylase.  Other work showed that  sulphydryl groups were not required f o r enzymic a c t i v i t y (7). Ikenaka (36) treated A. oryzae a-amylase with dinitrobenzene s u l phonate and fluoronitrobenzene and concluded from h i s results that the phenolic group of tyrosine was necessary for enzymic a c t i v i t y .  Ikenaka  (37) also reacted A. oryzae a-amylase with p-phenylazobenzoyl chloride and suggested that e amino groups were required for enzymic action.  3.  Non-protein Constituents and Co-factors i n a-Amylases The small quantity of carbohydrates present i n A. oryzae a-amylase  are apparently not involved i n the enzymic a c t i v i t y (2). 4.  a-Amylase as a Metalloenzyme a-Amylases so f a r investigated contain at least one atom of c a l -  cium per mole (105) which i s apparently required for enzymic a c t i v i t y (24,105).  Since no other metals could be detected i n s i g n i f i c a n t amount,  except zinc i n jB. s u b t i l i s a-amylase, i t has been suggested that a l l a amylases have certain s i t e s to which calcium i s attached s p e c i f i c a l l y (105).  13. s u b t i l i s a-amylase i s quite unique because of the presence of  four atoms of calcium per mole of protein.  I t has been suggested that  the increased amount of calcium i s required to maintain s t r u c t u r a l r i g i d i t y because the S-S linkage i s absent i n 13. s u b t i l i s a-amylase.  10 Yamamoto and Fukumoto (114) reported p a r t i a l regeneration of c a l cium depleted  s u b t i l i s a-amylase by the treatment  nesium, barium and beryllium ions.  of strontium,  Hsui et a l , (34) have suggested  mag-, that  the reagent used by Yamamoto and Fukumoto (114) was not spectroscopically pure, therefore reaction might be due to the contamination of calcium i n the reagent. Calcium can be removed from a-amylases by d i a l y s i s against sodium ethylenediamine (97).  tetra-acetate, by ammonium sulphate or by e l e c t r o d i a l y s i s  The treatment  of enzyme by phosphate, oxalate and c i t r a t e f a i l e d  to lower the calcium content below 1 gram-atom per mole of enzyme (105). Under appropriate condition of temperature, pH and ionic strength, the removal of calcium i t s e l f did not cause an i r r e v e r s i b l e denaturation of the enzyme (9,93,105).  The calcium free a-amylases were highly suscep-  t i b l e to denaturation by heat, urea and acid (93) and also were attacked e a s i l y by p r o t e o l y t i c enzymes (93). Stein et a l . (97) and Fisher and Stein (24) have reported that enzymic a c t i v i t y can be regenerated by the addition of calcium to c a l cium free a-amylases. A. oryzae ct-amylase  However, enzymic a c t i v i t y could not be revived i n  (97).  This was  point of the enzyme (pH 4.2) 5.4).  thought to be due to the low i s o i o n i c  as compared to other a-amylases (pH 5.2  to  In calcium free .B. s u b t i l i s and hog pancreas a-amylases i n s t a b i l -  i t y increased as pH increased (23).  Fisher et a l . (23) reported that no  major s t r u c t u r a l changes occurred i n calcium depleted a-amylase. The exact role of calcium i n the c a t a l y t i c a c t i v i t y of a-amylases i s not known, but i t i s indicated that calcium ions function i n a number  11 of ways; (a) i t keeps the a-amylase molecule i n compact and proper  confor-  mation for b i o l o g i c a l a c t i v i t y by forming a tight intramolecular metal chelate structure, and  (b) i t protects the native enzyme against extreme  pH, heat, and p r o t e o l y t i c enzymes (23,93,105). Myrback (64) reported that chloride ions activated the pancreatic and salivary a-amylases, whereas the data of Thoma et a l . (100) indicated that chloride ion i s not e s s e n t i a l for porcine pancreatic,a-amylase. results of Muss (63) showed that 1-10  mM  The  chloride gave maximum enzymic  a c t i v i t y for s a l i v a r y a-amylase and protected i t against the detrimental effect of high temperature and heavy metals.  The optimum sodium chloride  concentration for porcine pancreatic a-amylase was  10 mM,  and higher  concentrations than 10 mM would cause i n h i b i t i o n of enzymic a c t i v i t y . Walker and Whelan (108) reported similar relationships between the a c t i v i t y of human salivary a-amylase and chloride ion. F.  Secondary and T e r t i a r y Structure of a-Amylase  The-secondary structure of a protein i s believed to be due to the folding of the polypeptide chains into a s p e c i f i c coiled structure.  The  i n t e r r e l a t i o n s h i p and arrangement of the folded polypeptide chains into s p e c i f i c layers of crystals are called t e r t i a r y structures of the protein. It i s understood that d i s u l f i d e bonds, hydrogen bonds and hydrophobic bonds maintain the secondary and t e r t i a r y structures of proteins. Since IS. s u b t i l i s a-amylase does not have disulphide linkages' i t : i s expected to have d i f f e r e n t secondary and t e r t i a r y structures and behave d i f f e r e n t l y towards denaturing agents.  Isemura and Imanishi  (40) have  12 studied c a r e f u l l y the conformational changes i n 13. s u b t i l i s a-amylase i n alkaline and urea solution.  Their finding was  that approximately 30 per  cent of a l l the phenolic hydroxyl groups ionize freely i n alkaline pH up to 11.5.  The remaining groups appear to ionize i r r e v e r s i b l y at  apH  of 11.5 and therefore are l i k e l y to be buried i n protein molecule.  Also  at a high alkaline pH, the t e r t i a r y structure appears to be disrupted i r r e v e r s i b l y due to the dissociation of hydrogen bonds between carboxylate groups and  phenolic hydroxyl groups.  However, the enzymic•  a c t i v i t y was regenerated by d i a l y s i s after the disruption of hydrogen bonds using 8 M urea. Manning et a l . (47) reported large negative o p t i c a l rotation on 15. stearothermophilus a-amylase and this was not s i g n i f i c a n t l y affected by 8.0 M urea, 4.0 Mguanidine, or temperature  as high as 75°C.  of enzymic a c t i v i t y occurred under these conditions.  It was  No loss  concluded  that 15. stearothermophilus a-amylase i s a well hydrated molecule and has a semi-random or random c o i l i n the native state (47).  It was also sug-  gested that secondary and t e r t i a r y structures might be maintained by disulphide bonds (39). Takagi and Toda (98) investigated the effect of alkaline pH on A. oryzae a-amylase.  Their observation on o p t i c a l rotation and spectro-  photometric absorption with changes i n enzymic a c t i v i t y indicated that the modification i n enzyme structure and a c t i v i t y was  due to the dissoc-  i a t i o n of hydrogen bonds which became disrupted at pH 10.5 by i r r e v e r s i b l e i o n i z a t i o n of phenolic hydroxyl groups of tyrosine.  The a c t i v i t y of A.  oryzae a-amylase,- however, was regenerated after denaturation of a-amylase by acid (99) and 8 M urea (37).  13 Isemura et a l . (41) reduced the four disulphide groups of A. oryzae a-amylase by treating i t with sodium thioglycolate i n 8.0 M urea and found that this caused the unfolding of the l i n e a r polypeptide taining nine sulphydryl groups.  con-  The denaturation was reversible when  the enzyme was air-oxidized a f t e r the removal of urea and thioglycolate. This regenerated preparation of a-amylase had 50 per cent.of the o r i g inal activity. Toda (101) studied the e f f e c t of proteolysis on A. oryzae a amylase and reported that modified derivatives of a-amylase had a lower maximum v e l o c i t y f o r the hydrolysis of amylose as compared to the native enzyme.  He suggested that the active s i t e of the enzyme remained un-  changed and that there.was an o v e r a l l change i n the molecular configuration by the formation G.  of new secondary and t e r t i a r y structure.  Quarternary Structure of a-Amylase  The possession of quarternary  structure of a protein implies that  a protein molecule can dissociate into two or more subunits each of which retains i t s independent primary, secondary and t e r t i a r y structures.  The  13. s u b t i l i s a-amylase i n i t s native form shows the phenomena of monomerdimer transformation.  Vallee e_t a l . (105) and Stein (92) have reported  that B_. s u b t i l i s can be changed from 6S to 4S i n the presence of EDTA, and both 6S and 4S forms of the enzyme were homogeneous i n the ultracent r i f u g a t i o n (95).  Stein and Fisher (97) reported that other cation-  binding agents l i k e c i t r a t e and oxalate produce heterogeneous sedimentation patterns i n I3_. s u b t i l i s a-amylase. ' The addition of zinc would  14 restore the dissociated amylase molecule into the homogeneous o r i g i n a l form.  It was  concluded that IL s u b t i l i s a-amylase existed i n dimer form,  two units of monomer being crosslinked by an atom of zinc according to Equation 1. sequestering agent (Protein-Ca )-Zn-(Protein-Ca ) x x  2(Protein-Ca )+Zn x  „  [l]  z,n The above hypothesis was with  confirmed by treating the monomer form of enzyme  to obtain a dimer.  There was  a direct c o r r e l a t i o n between the  release of zinc from ^~*Zn l a b e l l e d enzyme when EDTA was  added and a con-  comitant conversion of the 6S into the 4S form of the enzyme (96). Stein and Fisher (96) observed I |  that other cations,such as Mn  | j  _| |_  | |  , Ni  ^ |_  , j |  Co and Cu gave some dimerization, while Mg , Ca , Ba and S i had no e f f e c t . A higher degree of association than dimerization was accomplished when the concentration of Zn  ++  was  increased to 2 X 10  -9  M.  Isemura and Kakiuchi (39) studied the effect of pH on the sedimentation v e l o c i t y of B>. s u b t i l i s a-amylase and"showed that Svedberg S decreased from 6.23  to 4.45  ing the involvement  as the pH was  changed from 6.5  to 5.0  indicat-  of the imidazole group i n the dimerization process.  Isemura and Kakiuchi'(39) further explored the possible role of imidazole groups i n dimerization process by comparing the.sedimentation pattern of the native and photo-oxidized B. s u b t i l i s a-amylase i n the presence of methylene blue.  The sedimentation c o - e f f i c i e n t was  native and photo-oxidized amylase respectively.  6.2  to 4.45 for  In the case "of photo-  oxidized a-amylase, the amino acid analysis revealed that seven out of twelve moles of h i s t i d y l residues were oxidized, while the other amino  15 acids residues were not affected. hypothesis  These results further supported the  that the imidazole groups of h i s t i d y l residue are involved i n  monomer and dimer transformation  of B_. s u b t i l i s a-amylase through the  chelating of zinc ions. Stein and Fisher (95) reported that the pure c r y s t a l l i n e a-amylases from A. oryzae, human s a l i v a and hog pancreas are normally present i n the 4S forms which remain unchanged by the addition pf zinc ions or EDTA.  H.  The Action Pattern of a-Amylase  The term "action pattern" refers here to the mechanism of cleavage of 1-4 glucans by a-amylase.  Meyer and Bernfeld  (55) and Meyer and  Gonon (60) suggested that a l l a-amylases have the same action pattern and that i t cleaved a 1-4 glycoside linkage i n amylose, except those at chain ends.  Accordingly maltose.and maltotriose would be end products  of enzymatic digestion.  Walker and Roberts (108) reported that the deg-  radation of amylose into maltose and maltotriose.indicated  semi-stable  end points, because the rate of hydrolysis of maltotriose was very low. A further evidence used.by Meyer and his colleagues  (55,60) that a l l a -  amylases have the same action pattern was the measurement of saccharogenic/dextrinogenic amylases (33).  quotient, which gave s i m i l a r values f o r d i f f e r e n t  This hypothesis  was c r i t i c i s e d because the ratios were  taken at the same stage of amylolyses, and therefore differences could not be expected.  When saccharogenic and dextrinogenic  ratios were deter-  mined near the achroic point very wide differences were apparent between various amylases (81).  The p l o t of blue value against reducing  value  16 gave c h a r a c t e r i s t i c curves for d i f f e r e n t a-amylases.  The difference was  possibly due to d i f f e r e n t chain lengths produced by the enzymic hydrolys i s of amylose by a-amylases (44).  Subsequent studies based on paper  and column chromatographic  techniques have revealed that a-amylases of  d i f f e r e n t origins produced  low molecular weight products with molecular  size d i s t r i b u t i o n c h a r a c t e r i s t i c of i n d i v i d u a l enzymes  (17,76,79,110).  Robyt and French (81) reported that pancreatic and human salivary a amylase produced very s i m i l a r end products from amylose.  However, the  curves r e l a t i n g drop i n blue values to the corresponding increase i n the reducing values were d i f f e r e n t .  In the l i g h t - o f these results these  authors did not accept the explanation offered by Kung et a l , (44) r e garding differences between various amylase curves r e l a t i n g drop i n blue values to corresponding increases i n the reducing value.  Bird and  Hopkins (5) reported another aspect of action pattern i n which d i f f e r ences were observed even with a-amylase from the same source when the substrate concentration was changed.  These results indicated that  Meyer's hypothesis regarding equal rate of hydrolysis of a l l but end linkage was not v a l i d for various a-amylases. Robyt and French (79), and Bird and Hopkins (5) have reported that eventually a l l the amylose would be hydrolyzed to maltose and glucose by a-amylases through d i f f e r e n t action, patterns.  The l i n e a r portion  of glycogen and amylopectin e s s e n t i a l l y follows the same fate as amylose to produce maltose and glucose. In the case of amylopectin, which i s a branched polymer, the l i m i t dextrin produced by the action of s a l i v a r y a-amylase was found to  17 contain 1—>k  and•1—*6 bonds, ranging.from the pentasaccharide upwards,  and moreover these large molecules have two and three 1—^6  bonds (68,76).  Wheal and Roberts (113) have suggested that s a l i v a r y a-amylase cannot cleave certain 1—>4  bonds i n the v i c i n i t y of the 1—>6  branched points,  and this concept has been extended to other a-amylases as well (28). Robyt and French (81) have considered  the action pattern of a-  amylases on amylose i n terms of single chain, multichain, and multiple attack.  They suggested that porcine, pancreatic, human s a l i v a r y and  oryzae a-amylases follow multiple attack patterns during amylolysis  A. and  they also calculated the degree df multiple attack by these enzymes. Leach and Schoch (45) studied the action of various a-amylases on starch granules and found that d i f f e r e n t types of starches have varying degrees of s u s c e p t i b i l i t y to amylases.  In addition they observed no  c o r r e l a t i o n between granule size and the extent of. s o l u b i l i z a t i o n . l a r differences have been reported by Walker and Hope (109) c e p t i b i l i t y of starches of d i f f e r e n t o r i g i n to amylases.  Simi-  i n the sus-  Their results  also indicated that porcine pancreatic and human s a l i v a r y a-amylases were adsorbed on the surface of the corn starch granules, while the sweet potato B-amylase and A. oryzae a-amylase were not adsorbed.  The  a-amylase from S_. bovis and C_. butyricum can also degrade corn starch granules (109).  Nordin and Kim  (69) observed an apparent increase i n  the amylose content during the i n i t i a l period of degradation of starch granules, as measured by the potentiometric It was  t i t r a t i o n of bound iodine.  concluded that amylopectin must be degraded f i r s t , indicating that  i t constituted the external covering of starch granules.  The location  18 of amylopectin with respect to amylose i n starch granules i s i n agreement with the hypothesis of Ulmann (103). I. 1.  Immunochemical Study of a-Amylase  Mammalian a-Amylase In recent years antibodies have been produced against a number of  mammalian enzymes, for example, phosphorylase  (31), lactate dehydrogenase  (65), alkaline phosphatase (85) and ribonuclease (13).  Antisera thus  ob-  tained have been used to compare and contrast enzymes from d i f f e r e n t organs or species (31,48,86). McGeachin and Reynolds (52) were the f i r s t workers to report that mammalian a-amylase could act as an antigen to produce antibodies.  They  used amylase antiserum to study the relationship of hog pancreatic amylase to the amylase of other hog organs and to amylases of other species. McGeachin (53) has reported immunological techniques f o r determining  dif-  ferences and s i m i l a r i t i e s among amylases of various species and also of' a given species. 2. • Microbial a-Amylase Wada (107) demonstrated that when c r y s t a l l i n e Taka a-amylase was injected into rabbits the antibody was  formed against the enzyme.  He  also studied the s e r o l o g i c a l properties of the anti-sera produced and found only a single homogenous antibody,. but this antibody could not i n h i b i t enzyme a c t i v i t y completely.  He further observed  that starch and  starch hydrolysates i n h i b i t e d the amylase-antiamylase reaction.  It was  found that anti-Taka-amylase antibody s p e c i f i c a l l y inhibited the a c t i v i t y  19 of a-amylase from Aspergillus species.  On the other hand, a-amylase  a c t i v i t y of other molds, bacteria and a-amylase of a l l other sources tested were unaffected (107).  Heat denatured Taka-a-amylase did not i n -  h i b i t the reaction between Taka-a-amylase and i t s antibody. Nomura and Wada (67) obtained antibodies by i n j e c t i n g c r y s t a l l i n e —' s u b t i l i s a-amylase into rabbits.  Antiserum produced i n rabbits by  i n j e c t i o n of c r y s t a l l i n e amylase neutralized the enzymic a c t i v i t y to about 90 per cent.  A competitive i n h i b i t i o n of the action of antisera  by the substrate, starch and i t s hydrolysed products (67,108,109) was also noted. Onoue elt -al. (74) modified the 13. s u b t i l i s a-amylase by treating i t with N-bromosuccinimide to study the molecular configuration between modified and native a-amylase by immunochemical analysis.  They reported  that a n t i - b a c t e r i a l a-amylase gave almost i d e n t i c a l p r e c i p i t a t i o n curves when treated with b a c t e r i a l a-amylase and modified N-bromosuccinimideb a c t e r i a l a-amylase.  In.addition, by.using the agar-gel immunodiffusion  technique, they observed a single sharp p r e c i p i t a t i o n l i n e between Nbromosuccinimide-bacterial a-amylase and a n t i - b a c t e r i a l a-amylase, and the p r e c i p i t a t i o n l i n e fused together with the. l i n e between b a c t e r i a l a amylase and a n t i - b a c t e r i a l a-amylase.  Onoue e_t a l .  (74) reported that  N-bromosuccinimide treatment did not change the molecular configuration of the enzyme and the loss of enzymic a c t i v i t y was due to oxidation of one tryptophan residue.  These results indicated that the c a t a l y t i c s i t e  of b a c t e r i a l a-amylase might be d i f f e r e n t from that of the antigenic site.  Onoue ej: a l .  (75) prepared p u r i f i e d antibodies against 13. s u b t i l i s  20 a-amylase and the p u r i f i e d antibodies neutralized the a-amylase a c t i v i t y completely.  The antibody i n the antigen-antibody complex could not be  displaced by substrate.  These results are not consistent with the ear-  l i e r work of Nomura and Wada (67) i n which they reported that 10 per cent enzymic a c t i v i t y remained after antibody treatment. also demonstrated body was  Onoue et a l . (75)  that the neutralizing a b i l i t y of papain treated a n t i -  less than that of the intact antibody, though the papain digested  antibody had the capacity to combine with the antigen. ' It was  suggested  from these results that the antibody affected the i n t e r a c t i o n of amylase and starch by s t e r i c hindrance and therefore would be expected to decrease when the molecular size of antibody i s reduced.  Okada,et a l . (71) re-  ported that photo-oxidized a-amylase of .13. s u b t i l i s did not form a prec i p i t a t e with I3_. s u b t i l i s a-amylase antibody.  It was further demonstra-  ted that i h the presence of calcium, photo-oxidized 13. s u b t i l i s a amylase was not susceptible to proteinase indicating that photo-oxidation did not grossly change the molecular configuration (29). Okada et a l . (72) reported that a-amylase a c t i v i t y of both Takaamylase A and p-phenylazobenzyl-Taka-amylase  A was inhibited up to the  same degree by anti-TakaTamylase A and by anti-p-phenylazobenzyl-Takaamylase A.  It was suggested that antibody to the altered protein moiety  of p-phenylazobenzoyl-Taka-amylase  A was produced  (72).  Okada et a l .  (72) further observed that the maltosidase a c t i v i t y of Taka-amylase A was p a r t i a l l y inhibited by anti-Taka-amylase A and anti-p-phenylazobenzyl-Taka-amylase  A was i n e f f e c t i v e to i n h i b i t the enzyme a c t i v i t y .  Moreover, the maltosidase a c t i v i t y of p-phenylazobenzyl-Taka-amylase  A  21 was not neutralized by anti-Taka-amylase A or anti-p-phenylazobenzyl-Takaamylase A.  Since the neutralizing a b i l i t y of the antibody depends on the  molecular size of the substrate (starch, phenyl maltoside) i t was suggested that the antibody inhibited enzymic a c t i v i t y by s t e r i c hindrance (72). S i r i s i n h a and A l l e n (90) used immunochemical methods to study the structure of Aspergillus a-amylase.  Urea treated native enzyme under  various conditions resulted i n a preparation which gave a reaction partly i d e n t i c a l with the non-treated enzyme during immunodiffusion  analysis.  Quantitative p r e c i p i t a t i o n curves with urea treated enzyme preparation indicated that only a p a r t i a l loss of immunochemical r e a c t i v i t y occurred even with prolonged treatment.  The appearance of several bands of pre-  c i p i t a t i o n with urea treated enzyme preparation suggested that various intermediate states exist between the f u l l y unfolded structure of protein and the native protein (90).  Immunochemical changes were also observed  with enzyme preparation treated with EDTA alone or i n combination with .1 M mercaptoethanol. S i r i s i n h a and A l l e n (91) reported marked differences regarding immunochemical behaviour between urea treated and oxidized a-amylase from A. oryzae.  Although oxidized a-amylase would p r e c i p i t a t e the same amount  of antibody, the e f f i c i e n c y of oxidized enzyme decreased per unit weight. On the other hand, urea treated a-amylase would p r e c i p i t a t e only a certain portion of antibody from a s p e c i f i c antiserum.  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Effect of denaturing agents and.proteolytic enzymes on the immunochemical r e a c t i v i t y of a-amylase from Aspergillus oryzae. Arch. Biochem. Biophys. 112:137.  91.  . 1965. Immunochemical studies on a-amylase. II. Examination of immunochemical and enzymic a c t i v i t i e s of native and modified a-amylase from Aspergillus oryzae. Arch. Biochem. Biophys. 112:149.  29 92.  S t e i n , E. A.  1957.  t i o n Proc.  S t r u c t u r e of  s u b t i l i s a-amylase.  Federa-  16:254.  93.  and.E. H. F i s c h e r . 1958. The r e s i s t a n c e of a-amylase t o wards p r o t e o l y t i c a t t a c k . J . B i o l . Chem. 232:867.  94.  , J . M. Junge and E. H. F i s h e r . 1960. The amino a c i d comp o s i t i o n of a-amylase from A s p e r g i l l u s o r y z a e . J . B i o l . Chem.  235:371. 95.  and E..H. F i s c h e r . 1960. B a c i l l u s s u b t i l i s a-amylase^ a z i n - p r o t e i n complex. Biochem. Biophys. A c t a , 39:287.  96. '  . 1961. a-Amylase from B a c i l l u s Biochemical Preparation. 8:34.  97.  , J . H s u i and E. H. F i s h e r . 1964. A l p h a amylase as c a l cium-metalloenzymes. I . P r e p a r a t i o n of c a l c i u m - f r e e apoamyl a s e s by c h e l a t i o n and e l e c t r o d i a l y s i s . Biochemistry. 3:56.  98.  T a k a g i , T. and H. Toda. 1960. S t u d i e s on the amphoteric p r o p e r t i e s of taka-amylase A. I . I o n i z a t i o n of p h e n o l y i c h y d r o x y l groups. J . Biochem. Tokyo.8:781.  99.  . 1962. S t u d i e s on the d e n a t u r a t i o n of t a k a amylase A and on i t s r e v e r s i b i l i t y . J . Biochem. Tokyo.  subtilis.  52:16. 100.  Thoma, J . A., J . Wakim and L. Stewart. 1963. Comparison of the a c t i v e s i t e s of a l p h a and b e t a amylase. Biochem. Biophys.  Res. Comm.  12:350.  101.  Toda, H. 1963. amylase A.  Enzymatic m o d i f i c a t i o n of p h e n y l a z o b e n z o y l - t a k a J . Biochem. Tokyo. 53:425.  102.  T s u c h i y a , H. M. , J-. Corman and H. J . K o e p s e l l . 1950. Production of mold amylases i n submerged c u l t u r e . I I . Factors a f f e c t i n g the p r o d u c t i o n of alpha-amylase and maltase by c e r t a i n Aspergilli. C e r e a l Chem. 27:322.  103.  Ulmann, M. 1957. Bestimmung der chemischen n a t u r der h i l l l e c i n e s gerguollenen starkekornes. K o l l o i d . Z. 150:128.  104.  U n d e r k o f l e r , L. A. and.D. K. Roy.. 1951. C r y s t a l l i z a t i o n of f u n g a l alpha-amylase and l i m i t d e x t r i n a s e . C e r e a l Chem.  28:18. 105.  V a l l e e , B. L., E. A. S t e i n , W. N. Summerwell and.E. H. F i s c h e r . 1959. M e t a l content of a-amylases of v a r i o u s o r i g i n s . J.  B i o l . Chem.  234:2901.  30 106. 107. 108.  109. 110. 111.  112.  Wada, T. and M. Nomura. 1958. An immunochemical study of microb i a l amylase (1). J . Biochem. 45:639. .  . 1959. J. Biochem.  An immunochemical study of microbial amylase (11) 46:239.  Walker, G. J . and W. J . Whelan. 1960. The mechanism of carbohydrase action. VII. Stages i n the salivary a-amylosis of amylose, amylopectin and glycogen. Biochem. J . . 76:257. and P. M. Hope. 1963. The action of some a-amylases on starch granules. Biochem. J . 86:452. -. - •;• 1965. The c e l l bound a-amylase of Streptococcus bovis. Biochem. J . 94:289. Welker, N. E. and L. L. Campbell. 1963. Effect of carbon source on formation of a-amylase by Bacillus stearothermophilus. J . Bact; 86:681. . 1963. Induction of a-amylase of B a c i l l u s stearothermophilus. J . Bact. 86:687.  113.  Whelan, W. J . and P. J . P. Roberts. 1952. Action of salivary a amylase on amylopectin and glycogen. Nature. 170:748.  114.  Yamamoto, T. and J . Fukumoto. 1960. Enzymatic properties of b a c t e r i a l a-amylase reactivated with various alkaline earth metals. B u l l . Agr. Chem. Soc.- Japan. 24:16.  115.  Yoshida, A. and T. Tobita. 1960. Studies on the mechanism of protein,synthesis. Non-uniform incorporation of [c!4] leucine into a-amylase and the presence of a-amylase precursor. Biochem. Biophys. Acta. 37:513.  CHAPTER III MATERIALS AND METHODS  A.  Chemicals  The sources from which the substrates and chemicals were obtained are as follows: starch ( B r i t i s h Drug House, Poole, England), amylose  (Stein-Hall and Co., New York, U.S.A.), maltose, technical and  reagent grade (Fisher S c i e n t i f i c Co., New Jersey, U.S.A.), bovine serum albumin (Calbiochem, Los Angeles, C a l i f o r n i a , U.S.A.), casein hydrolysate, and a-amylases from Ii. s u b t i l i s , A. oryzae and hog pancreas (Sigma Chemical Company, St..Louis, U.S.A.).  A l l chemicals used during this  investigation were of the highest purity grade and were obtained through A l l i e d Chemical Company Canada, L t d . , Vancouver, B.C., and Fisher S c i e n t i f i c Co. Ltd., Vancouver,  B.C.  DEAE Sephadex A-50 and Sephadex G-200 were purchased from Pharmacia, Uppsala, Sweden. B.  Organism  The organism used i n this investigation was Bacteroides amylophilus s t r a i n H-18, kindly supplied by Dr. T. H. Blackburn, Department of Microbiology, University of B r i t i s h Columbia, Vancouver 8, Canada. sheep.  B.C.,  Blackburn and Hobson (2) isolated this s t r a i n from the rumen of  32 C.  Maintenance of Bacteroides Strain H-18  amylophilus  The complete chemically defined basal medium used during this investigation was  that developed by Hungate (8).  (g/1): K HP0 , 0.45; 2  0.09;  4  CaCl^, 0.09;  KH P0 , 0.45; 2  4  This medium contained  ( N H ^ S O ^ 0.9;  NaCl, 0.9;  HgS0 , 4  resazurin, .001; L-cysteine hydrochloride, 0.5.  The  resazurin and mineral solutions, or any additions to the medium were placed i n a screw capped b o t t l e and d i s t i l l e d water added to give a f i n a l volume of 900 ml.  The medium was  autoclaved for f i f t e e n minutes  at 120°C and on removal of the b o t t l e from the autoclave the cap immediately screwed t i g h t .  F i f t y ml. of 1 per cent (w/v)  hydrochloride and f i f t y ml. of 10 per cent (w/v)  was  L-cysteine  sodium bicarbonate  sol-  ution were steam autoclaved separately at 100°C for f i f t e e n minutes and then added to the remaining medium under a stream of CO2. of the medium was of the culture was  6.7.  The f i n a l pH  A l l the dispensing of the medium and incubation  done under oxygen-free CO^ as described by  Blackburn  (3). Stock cultures were maintained  on nutrient agar slopes which  contained i n addition to the basal medium 2 per cent agar and 0.5  per  cent each of maltose and casein.  and  The cultures were stored at 4°C  transfers were made each week to fresh slopes by stab innoculation under an atmosphere of CO^. D.  A l l cultures were grown at 38°C.  Growth Measurements of Bacteroides amylophilus Strain H-18  The growth of _. amylophilus was  measured i n a Bausch and Lomb  33 Spectronic 20 Colorimeter (Bausch and Lomb, Rochester, New York, U.S.A.) at 660 nm.  E.  Production and P u r i f i c a t i o n of a-Amylase from Bacteroides amylophilus Strain H-18  The amylase was a by-product of protease p u r i f i c a t i o n undertaken by Lesk (9) who kindly denoted the f r a c t i o n III a-amylase (Figure 6) at the point where i t was separated from the protease.  1.  Production of a-Amylase Twenty-nine l i t e r s  of growth medium were prepared i n a 32 l i t r e  stainless s t e e l milk can and inoculated with 1 l i t r e of log phase culture of .B. amylophilus.  After anaerobic incubation f o r 23 hours at 38°C the  can and i t s contents were cooled immediately with a waterhose.  The c e l l  was removed by continuous flow centrifugation (8700 x g at 4°C) using a S e r v a l l Centrifuge (Servall type SS-34, equipped with a KSB:R S e r v a l l continuous flow adopter from S e r v a l l , Norwalk, Connecticut). The supernatant had a pH of 5.5 which was the optimum for the attachment  to DEAE  Sephadex A-50 for p u r i f i c a t i o n .  2.  P u r i f i c a t i o n of a-Amylase on DEAE-Sephadex A-50 and G-200•Sephadex • DEAE Sephadex A-50 (0.2 g dry weight/100 ml. of supernatant) was  gradually added to the supernatant and CO 2 was bubbled through i t f o r twelve hours at 4°C to ensure proper mixing.  The DEAE suspension was  allowed to s e t t l e ; the supernatant decanted and the DEAE collected on a sintered glass f i l t e r .  The DEAE was mixed thoroughly i n 500 ml. of 1 M  34 NaCl and centrifuged. The supernatant was This procedure was  repeated s i x times.  then decanted and stored.  The f i r s t f i v e fractions  (total  volume 2540 ml.) were pooled and dialyzed against 0.05 M phosphate buffer (pH 7.0) overnight. The dialysed preparation was  further fractionated by chromatog-  raphy on a 100 x 5 cm. column of DEAE Sephadex equilibrated with 0.05  M  phosphate buffer (pH 7.0).' a-Amylase was  eluted with l i n e a r gradient of 0.2 M to 1.0 M NaCl  i n phosphate buffer (pH 7.0).  The fractions were tested for a-amylase  a c t i v i t y and three fractions having enzymic a c t i v i t y were collected. Volumes of 538 ml, ,500 ml and 840 ml were collected for f r a c t i o n I, I I , and III respectively.  Each f r a c t i o n was  phate buffer (pH 7.0) overnight at 4°C.  dialysed against 0.05 M phosFraction I I I was  concentrated  to 40 ml with a D i a f l o U l t r a - f i l t r a t i o n C e l l (Diaflo Model 50 U l t r a f i l t r a t i o n C e l l from Amicon Co., Lexington, Mass., U.S.A.) equipped with a Pm - 10 f i l t e r (exclusion limit;10,000 MW) The concentrated a-amylase f r a c t i o n III was  under a pressure of 40 p . s . i . further p u r i f i e d on a 2.5 x  50 cm column of Sephadex G-200 which had been equilibrated and eluted with 0.05 M phosphate buffer (pH 7.0).  A l l of the a-amylase a c t i v i t y  obtained i n a single peak and the enzyme solution was pressure d i a l y s i s to 45.0  F.  was  concentrated by  ml.  Assay of a-Amylase •  The a-amylase a c t i v i t y i n the sample was the amount of reducing sugars produced  assayed by determining  from starch or amylose using the  35 3.5 d i n i t r o s a l i c y l i c acid method of Fisher and Stein (5).  The assay  medium consisted of 1.0 ml of properly diluted enzyme i n an equal volume of 2 per cent (w/v) soluble starch or amylose buffered to pH 6.7 with 0.2 M T r i s and 0.1 M maleate.  Unless otherwise indicated the time of  incubation was f i f t e e n minutes at 44°C. When i t was desired to calculate the degree of multiple attack, the a-amylase a c t i v i t y was determined by the Nelson Copper method (16). A unit of a-amylase a c t i v i t y was defined as the amount of enzyme that would produce the equivalent of 1.0 mg. maltose i n one minute under standard.conditions. The method of Robyt and Whelan (21) was used to determine the blue values. G.  Assay of Protease  The determination of protease a c t i v i t y was done according to the method of Blackburn (3). H.  Determination of Nitrogen  The indophenol colorimeter method of Nakai and Tsuchiya (14) was used for nitrogen determination.  Bovine serum albumin was used as the  standard. I.  Determination of Protein  The method of Lowrey et_ a l . (11) , was used to measure the protein concentration i n the extracts.  Bovine serum was used as the standard.  36 J.  Determination of Total  Carbohydrates  Total carbohydrates were determined by phenol and sulphuric acid procedure as described by M i l l e r (13). K.  Disc Gel Electrophoresis  Disc g e l electrophoresis apparatus was constructed by Mr. R. J . Hudson and Mr. James A. Shelford, i n the Department of Animal Science Laboratory, University of B r i t i s h Columbia, following the procedure of Davis (4). Disc electrophoresis chemical r e f i l l pack containing standard 7 per cent acrylamide g e l and premixed stock reagents, acrylamide, b i sacrylamide (N, N'-methylenebisacrylamide)  and (N, N, N', N'-tetramethyl-  ethylene diamine) TEMED was purchased from Canalco, Rockville, Maryland, U.S.A. Disc gel electrophoresis was carried out i n a standard g e l (7 per cent) according to the method described by Davis (4). The gels were stacked at pH 8.9 and run at pH 9.5 during routine work. samples were run i n duplicate.  The enzyme  After g e l electrophoresis f o r two hours  (5 mA per column) one gel was stained immediately  f o r protein with Amido  black 10B. The second gel.was used to detect a-amylase a c t i v i t y by l a y ering the gel.on starch coated glass s l i d e s .  The glass slides were  coated f i r s t with 1.0 per cent agar and then with 1.0 per cent starch plus 1.0 per cent agar made i n 2 M TRIS-Maleate buffer at pH 6.7.  After  being superimposed with the g e l , the s l i d e s were placed i n p e t r i dishes and.incubated  at 40°C f o r 15 minutes.  After the incubation the gels were  removed and the s l i d e s dipped momentarily i n Lugol's iodine solution to  37 s t a i n the unhydrolysed starch.  The clear band (S) indicating hydrolysis  of starch was v i s i b l e against the blue s t a i n produced by the starch.  L. Isoelectrofocusing The LKB f r a c t i o n c o l l e c t o r with Uvicord (0.3 on l i g h t path), and Ampholine Electrofocusing equipment (LKB Produkter AB, Stockholm, Brouma, Sweden) were used. The Ampholine column and sample were prepared according to the instructions given i n LKB 8100 Ampholine Instruction Manual.  The gradient  mixer equipped with s t i r r e r motor was used to f i l l the Ampholine column, No. 8102, pH 3.0  capacity 440 ml.  to 10.0  Low molecular weight ampholines i n the range  (4 per.cent).were M.  used.  Charcoal-Celite Column Chromatography  The technique was  e s s e n t i a l l y the same as described by Whistler  and Duro (22) using a charcoal-celite column to i s o l a t e and detect o l i g o saccharides found i n technical grade Maltose. the sugar was  A 50 per cent solution of  autoclaved for 20 minutes at 40 l b s . (p.s.i.) and f i l t e r e d  to remove the p r e c i p i t a t e . A chromatographic  column (4.5 x 50 cm.) was  40 cm. with charcoal-celite mixture.  The column was washed with  l i t e r s of 0.1 N HCl to remove basic ash; acid was haustively with d i s t i l l e d water.  f i l l e d to a height of 1.5  removed by washing ex-  The sugar and oligosaccharides were  eluted by passing two l i t e r s of each of water, 5 per cent, 15 per cent, 30 per cent, and 95.per cent ethanol through the column.  The effluent  38 was collected In 100 ml. fractions and sugars were detected by the 3.5 d i n i t r o s a l i c y l i c acid method. and oligosaccharides was  Qualitative detection of various sugars  done by paper chromatography.  The water f r a c -  tion contained glucose and varying amounts of maltose and trisaccharides. The 5 percent, 15 per cent, 30 per cent and 95 per cent ethanol fractions contained maltose, trisaccharides, a mixture of t r i - , t e t r a - and pentasaccharides and a mixture of hexasaccharides and high molecular weight oligosaccharides, respectively.  N.  Paper Chromatography  Whatman No. 1 f i l t e r paper was used during this investigation. The containers used were wide mouth cabinet with screw l i d s Specialties Co., New  (Research  J e r s e y , U.S.A.).  This technique was e s s e n t i a l l y used to purify malto-oligosaccharide. Resolution of the sugar and oligosaccharide was  achieved by multiple  ascent technique (18) using solvent (10) n- butanol-pyridine-water (6:4:3 v/v).  It was  found that an ascent of 30 cm. was  s u f f i c i e n t to  separate the f i r s t seven members of homologous series of malto-oligosaccharide. The spots on the paper chromatograms were detected by spraying with a n i l i n e phosphate reagent (6) and.heating at 115°C f o r 20 minutes. These spots were used as markers for sectioning the remaining portions of the chromatograms.  The i n d i v i d u a l sugars were extracted from the  paper and concentrated i n vacuo.  They were further dried with acetone '  and washed with a small amount of n- butanol.  The syrup was  dissolved  39 i n 50 ml. of water and freeze-dried.  The freeze-dried fractions were  stored i n a vacuum desiccator. *  0.  Thin Layer Chromatography  Desaga thin layer chromatography apparatus (Canadian Laboratory Supplies Limited, Vancouver, B.C.) was used.  The coating material was  Kieselgel G ( S i l i c a gel) or Kieselgur G (Merck and Co.). A suitable technique f o r the separation of oligosaccharides by TLC was developed (20) using the solvent systems, isopropanol-t^O-ethylacetate (2:1:2 v/v) or 1-propanol-nitromethane-water (5:2:3) as reported by Huber _ _ _ 1 . (7). P.  Effect of Temperature  The e f f e c t of temperature on a-amylase a c t i v i t y under various treatment was studied i n a thermal gradient apparatus.  The temperature  range could be adjusted from 20° to 80°C. Q.  Molecular Weight Determination  Molecular weight estimation was done by gel chromatography according to the method of Andrews (1), on a Sephadex column (2.5 cm x 50 cm). R.  Determination of Calcium Content  SP 90 Atomic Absorption Spectrophotometer (Unicam Instrument, Ltd., York St., Cambridge, England) was used to determine the amount of calcium in a-amylase.  A known amount of a-amylase was dialysed against  40 demineralized water for 48 hours.  The amount of Calcium was  determined  according to the instructions given i n the SP 90 spectrophotometer manual.  S.  Amino Acid Analysis  The amino acid analysis of the p u r i f i e d a-amylase was Phoenix Amino Acid Micro-Analyser was  (Model M-7800).  done on a  The enzyme preparation  f i r s t dialysed against deionized water for 48 hours at 5°C.  ysis was  Hydrol-  performed i n hydrolysis tubes containing 1.0 mg.of protein i n  1.0 ml. of deionized water and 1.0 ml. of concentrated ysis tubes were put i n an oven set at 110°± Hydrochloric acid was  removed by repeated  sure by a rotary evaporator. sodium c i t r a t e buffer pH 2.2  column for amino acid analysis. system was  The hydrol-  1°C for a period of 24 hours.  evaporation under reduced pres-  The residue was and 0.95  HCl.  dissolved i n 1.0 ml. of  ml. was  f i n a l l y applied to the  A Piez-Morris  (19) accelerated buffer  used for e l u t i o n of the amino acids.  *T.  Immunochemical Techniques  The various immunochemical techniques  as detailed below were used  to study a-amylase immunochemistry and homogeneity.  1.  Production of Antibodies Antibodies against the a-amylase of _. s u b t i l i s , A. oryzae,  hog  pancreas and B. amylophilus . (isoenzyme 1) were prepared i n rabbits.  Each  of the four rabbits received, by subcutaneous i n j e c t i o n over a course of three weeks, a t o t a l of 4.0 mg.  of protein i n complete Freund's adjuvant.  41 The animals were bled two weeks after the l a s t i n j e c t i o n to provide immune serum.  The-antisera was inactivated by heating and i t was  stored  i n frozen state (15).  2.  Determination of Enzymic I n h i b i t i o n The determination of the percentage of the inhibited a c t i v i t y of  a-amylase was done according to the method of McGeachin and Reynolds (12).  Homologous normal rabbit serum was included as a control i n a l l  experiments.  Control with homologous normal rabbit serum showed no i n -  h i b i t i o n of a-amylase a c t i v i t y .  The 3 to 5 d i n i t r o s a l i c y l i c acid  method was used to measure the a-amylase a c t i v i t y .  3.  Immunodiffusion  Characteristics  Antigenic relationships were studied by Immunoelectrophoresis  and  double d i f f u s i o n i n agar gel (17). 4.  Protein Determination i n Antigen-Antibody Complex Protein of antigen-antibody p r e c i p i t a t e was determined according  to the method of Lowry et a l . , using bovine albumin as standard (11).  REFERENCES I I I  1.  Andrews, P. 1965. The gel f i l t r a t i o n behaviour of proteins r e lated to their molecular weights over a wide range. Biochem. J. 96:595.  2.  Blackburn, T. H. and P. N. Hobson. 1962. Further studies on the i s o l a t i o n of p r o t e o l y t i c bacteria from the sheep rumen. J . Gen. Microbiol. 29,69.  3.  .  . 1968. Protease production by Bacteriodes amylophilus Strain H 18. J. .Gen. Microbiol. 53:27.  4.  Davis, B. J . 1964. Disc electrophoresis I I . Method and application to human serum proteins. Ann. N.Y. Acad. S c i . 121:404.  5.  Fisher^ E.- and E. A. Stein. 1961. a-Amylase from human s a l i v a . Biochem. Preparation. 8:27.  6.  Frahn, J . L. and J . A. M i l l s . 1959. Paper ionophoresis of carbonydrates. I. Procedures, and results for four e l e c t r o l y t e s . Aust. J . Chem. 12:65.  7.  Huber, C. N., H. D. Scobell and E. E. Fisher. 1968. Thin layer chromatography of the malto-oligosaccharides and megalosaccharides with mixed support and multiple i r r i g a t i o n . Anal. Chem. 40:207.  8.  Hungate, R. E. 1950. The anaerobic mesophilic c e l l u l o l y t i c bacteria. Bact. Rev. 14:1.  9.  Lesk, E. M. • 1969. P u r i f i c a t i o n and characterization of Proteolytic enzymes from _. amylophilus Strain H-18. M.Sc- Thesis. Univers i t y of B r i t i s h Columbia, Vancouver, B.C.  10.  Jeanes, A., C. W. Wise and R. J . Dimlee. 1951. Improved techniques i n paper chromatography of carbohydrates. Anal. Chem. 23:415.  11.  Lowry,. 0. H. ,• N. J . Rosebrough, A. L. Farr and R. J . Randall. 1951. Protein measurement with the f o l i n protein reagent. J . B i o l . Chem. 193:265.  (  43 12.  McGeachin, R. J . and J. M. Reynolds. 1959. Differences i n mammalian amylases demonstrated by enzyme i n h i b i t i o n with s p e c i f i c antisera. J . B i o l . Chem. 234:1456.  13.  M i l l e r , G. L. 1960. Micro-column chromatographic method for analysis of oligosaccharides. Anal. Biochem. 2:133.  14.  Nakai, S. and F. Tsuchiya. 1961. Improved method of nitrogen determination by indophenol reaction. Jap. Analy. 10:387.  15.  Nomura, M. and T. Wada. 1958. Studies on amylase formation by B a c i l l u s - s u b t i l i s . V. Immunochemical studies of amylase produced by B a c i l l u s - s u b t i l i s . J . Biochem. 45:629.  16.  Nelson, N, 1944. A photometric adaptation of the Somogyi method for determination of glucose. J . B i o l . Chem. 153:375.  17.  Ouchterlony, 0. 1968. two dimensions, and Immunodiffusion and Science Publishers,  18.  Pazur, J . and D. French. 1952. The action of transglucosidase of Aspergillus oryzae onmaltose. J . B i o l . Chem. 196:265.  19.  Piez, K. A. and L. Morris. 1960. A modified procedure for the automatic analysis of amino acids. Anal. Bio. Chem. 1:187.  20.  Rahman, Sh. Saif-Ur-, C. R. Krishnamurti and W. D. K i t t s . 1968. Separation of Cello-saccharides by thin layer chromatography. J. Chromat. 38:400.  21.  Robyt, J . F. and W. J . Whelan. 1968. "The a-amylase." In the Starch and i t s Derivatives. Ed. by J . A. Radley, 4th Ed., Chapman and H a l l Ltd. , London, EC4.  22.  whistler, R. L. and D. F. Durso. 1950; Chromatographic separation of sugars on charcoal. J . Amer. Chem. Soc. 72:677.  "The techniques of double d i f f u s i o n i n Immunoelectrophoresis." In Handbook of Immunoelectrophoresis. Ann. Arbor Michigan, 48106.  \  CHAPTER IV RESULTS AND DISCUSSION A.  1.  Characterization of a-Amylase from Bacteroides amylophilus Strain H-18  Production a'nd P u r i f i c a t i o n of a-Amylase  a.  Production of a-Amylase  Figure 1 shows that a-amylase was produced e x t r a c e l l u l a r l y during the logarithmic, and stationary phase of growth by 13. amylophilus. results are i n agreement with the findings of Lesk (20).  These  In this regard  ]3. amylophilus i s s i m i l a r to .B. stearothermophilus which starts producing a-amylase during the logarithmic.period of growth (51) , b u t . i t i s d i f f e r ent from B. s u b t i l i s which produces a-amylase during the stationary phase of growth (36). The growth curve (Figure 1) of 13. amylophilus i s c h a r a c t e r i s t i c of the other rumen bacteria (3). Seventy-eight and twenty-two per. cent of a-amylase was released into the medium during the logarithmic and stationary phase of growth respectively (Figure 1). During the midlogarithmic growth phase the' amount of a-amylase liberated was l i n e a r (Figure 2). Since a-amylase did not contain cysteine (see IV:A:2:f) and i t s production began during the logarithmic phase.of growth, i t has the c h a r a c t e r i s t i c features of other e x t r a c e l l u l a r enzymes (41). The effect of maltodextrins on a-amylase formation i s shown i n Figure 3.  When maltodextrins were added to the growing culture which  45  Figure 1  Growth curve and production of a-Amylase from Bacteroides amylophilus Strain H-18. Growth curve, o—o  a-Amylase a c t i v i t y released into the culture Supernatant.  O--O  Percentage of maximum a-Amylase a c t i v i t y i n culture Supernatant.  a-Amylase a c t i v i t y i n Supernatant (units/ml) to o  46 to  LO O  LO Ul  Ul  Ul  o c  to O  t-i to Ui  LO O  Lo Ul  to O  O  ON  o  00 o  Percentage of Maximum A c t i v i t y (32.5 units/ml) J I J 00 O.D,660  o o  —J 4>  47  Figure 2  Linear relationship between the production of a-Amylase and growth of Bacteroides amylophilus Strain H-18.  a -Amylase (units/ml) tO 1  Ul  O  1—  1  Ul  NJ O  NJ Ul  49  Figure 3  Effect of Maltodextrin on the growth and production of a-Amylase from Bacteroides amylophilus  Strain  Maltose o—o  Maltotriose  A-—A  Maltotetraose  O—O  Maltopentaose  H-18.  a-Amylase (units/ml)  50  had just entered the logarithmic phas.e, there was production of amylase.  an increase i n the  However when maltodextrins were replaced by glu-  cose, sucrose, and cellobiose, there was no change i n the amount of amylase produced.  These results, are e s s e n t i a l l y i n agreement with the  findings of Blackburn (2) and Hungate (19) i n which the a 1-4  linked  glucose polymers were the only carbohydrate substrates metabolized by B_. amylophilus. Under the experimental conditions used I5_. amylophilus appeared  cells  to be permeable to maltose and maltotriose. After incubation  for 6 hours with maltodextrins, maltose was  the only sugar detected extra-  c e l l u l a r ly by thin layer chromatography, and glucose was never found.  It  was not investigated to find i f I3_. amylophilus takes up other maltooligosaccharides d i r e c t l y l i k e Micrococcus Sp 40 (53) or hydrolyses them to maltose or maltotriose. These results indicate that 1$. amylophilus c e l l s are permeable to maltose and maltotriose and therefore the maltose and maltotriose up-take systems i n this micro-organism  are constitutive  l i k e Micrococcus Sp 40 (53) but unlike that of E_. cold (52) which i s adaptive. The results i n Figure 1 indicated fluctuations of the production of a-amylase into the. medium during the stationary phase of growth.  This  suggested that there may be more than one a-amylase produced.by IS. amylophilus.  In order.to assess this hypothesis,.a-amylase  was  analysed  by disc electrophoresis and electrofocusing techniques.. Four active i s o enzymes of a-amylase (Figure 4) accompanied by 12 other bands of protein were detected by disc electrophoresis.  Four peaks of a c t i v i t y were also  52  Figure 4  Detection of 4 Isoenzyme of a-Amylase by disc electrophoresis. I l l u s t r a t i o n A. The- separation of 4 i s o enzyme of a-Amylase by disc electrophoresis on acrylamide gel. The d i r e c t i o n of migration was from the top of the figure. I l l u s t r a t i o n B. The starch s l i d e a f t e r i n cubation at 38°C for 15 minutes with acrylamide g e l . After incubation the unhydrolysed starch was stained with Lugol's iodine. The clear bands i n d i c a t i n g hydrolysis of starch was v i s i b l e against the blue s t a i n produced by the starch and iodine complex.  Stained acrylamide gel  Stained s l i d e with 1% starch  A  B  Amylase A Amylase B Amylase C Amylase D  54 detected by iso-electrofocusing (Figure 5).  I s o e l e c t r i c points as de-  termined by electrofocusing were pH 3.7, 4.5, 5.9 and 8.0.  The i s o -  enzymes were named 1, 2, 3 and 4 with respect to their increasing i s o e l e c t r i c points (Figure 5). The reasons why  this organism produces four a-amylases i s not .  clear at the present time, but p o s s i b i l i t i e s may be suggested.  Since  they have d i f f e r e n t i s o e l e c t r i c points they should have d i f f e r e n t amino acid compositions.  It has been reported recently that d i f f e r e n t a-,  amylases have d i f f e r e n t a f f i n i t i e s towards various starches (5,28). Evidently .B. amylophilus i s a very v e r s a t i l e organism and may  control  secretion of d i f f e r e n t isoenzymes depending upon the nature of starch i n the diet of the animal. b.  P u r i f i c a t i o n of a-Amylase Isoenzyme 1  The methods used f o r p u r i f i c a t i o n of a-amylase isoenzyme 1 are summarized i n Figure 6 and the results are presented i n Table I I I .  The  stepwise p u r i f i c a t i o n process was conducted as follows:  Step 1 - A good quantity of a-amylase was culture for 23 hours.  obtained by growing the  In order to measure the protein  content correctly, a sample of supernatant was  dialysed  against 0.05 M phosphate buffer pH 7.0 to remove cysteine and tryptose peptides (20). Step 2 - The enzyme solution was operation.  concentrated by DEAE-Sephadex batch  This step reduced the volume of the enzyme s o l -  ution from 29000 ml to 2540 ml, and gave a 5 f o l d p u r i f i c a tion.  55  Figure 5  Detection of 4 isoenzymes of a-Amylase  (x—x)  by electrofocusing with superimposed pH curve (.—.).  The p i values of separated components  are obtained by taking the pH of the corresponding f r a c t i o n  at the maximum a c t i v i t y .  p i value of the components were 3.7, 4.5, and 8.2.  The 5.9  The isoenzymes were named 1, 2, 3  and 4 with respect to their increasing i s o e l e c t r i c points.  The figures above the enzyme  a c t i v i t y peaks give the p i values of a-Amylase isoenzymes.  56  Relative a c t i v i t y (%)  < o M C  B  fD  pH Gradient  57 24 hours culture supernatant 29000.0 ml  DEAE-Sephadex batch operation. The enzyme was eluted from DEAE-Sephadex s l u r r y with 1.0 M NaCl on a Buchner funnel 2540.0 ml ;  D i a l y s i s overnight•against  0.05 M phosphate buffer, pH 2600.0 ml  7.0  DEAE-Sephadex column chromatography. Enzyme was eluted with a l i n e a r gradient of 0.2 M to 1.0 M NaCl i n 0.05 M phosphate buffer, pH 7.0  Fraction II 57-84 (Fig.7)* 500.0 ml  Fraction I 27-56 (Fig. 7)"* 538.0 ml  This indicates Fraction number i n Figure VII .  Figure 6  Fraction III 85-131 (Fig.7)* a-amylase Isoenzyme 1. Total volume collected, 840.0 ml. It was reduced by pressure d i a l y s i s to 40 ml.  Sephadex G-200 column chromatography. Total volume c o l l e c t e d , 760.0 ml. It was reduced by pressure d i a l y s i s to 45 ml.  Flow sheet of methods for the i s o l a t i o n of a-amylase isoenzyme 1 from Bacteroides amylophilus Strain  H-18.  58 TABLE I I I PURIFICATION OF a-AMYLASE ISOENZYME 1 FROM B. AMYLOPHILUS STRAIN H-18  Procedure  Volume ml  24 hour superrnatant  29000.0  Concentration units/ ml  Total Units (xlO-3)  Protein mg/ml  4.0  116.00  0.30  13.3  100.00  1  DEAESephadex batch operation 2540.0  40.0  101.60  0.60  66.6  87.5  5.00  Dialysis against P0 buffer 2600.00  35.0  91.00  0.50  70.0  78.4  5.26  DEAESephadex Column Fraction No. I l l  840  75.0  65.52  0.15  550.00  56.00  Sephadex G-200 Column Fraction and Pressure Dialysis  45  45.00  0.7  Specific Y i e l d Activity % (units/ mg protein  Purification  4  1000  1428.5  38.8  41.35 .  107.4  59 Step 3 - The enzyme solution was dialysed against 0.05 M phosphate buffer pH 7.0, to remove NaCl.  The step due to some unknown  reasons decreased the enzymic a c t i v i t y .  Since the amount  of protein present decreased from 0.60 mg to 0.50 mg per ml, the decrease i n enzymic a c t i v i t y may.be due to denaturation of the a-amylase protein. Step 4 - The enzymic preparation was fractionated by DEAE-Sephadex chromatography using a l i n e a r sodium gradient (Figure 7). At this stage the volume of enzyme solution was reduced to 840 ml and p u r i f i c a t i o n obtained was 41 f o l d .  The solution  was further concentrated to 40.00 ml by pressure d i a l y s i s . Step 5 - The enzyme solution obtained i n Step 4 was subjected to g e l f i l t r a t i o n on Sephadex G-200.  An e l u t i o n p r o f i l e of a-  amylase a c t i v i t y i s shown i n Figure 8. a c t i v i t y , was obtained i n a single peak.  A l l of a-amylase At this stage the  recovery was 38.8 per cent and p u r i f i c a t i o n obtained was 107- f o l d .  This enzyme preparation was free from protease  activity.  The enzyme solution was concentrated by pressure  d i a l y s i s to 45.0 ml, freeze-dried and stored at 4°C for further use. Step 6 - The purity of this preparation was checked by disc e l e c t r o phoresis.  A single band of.protein, was obtained indicating  homogeneity of a-amylase protein (Figure 9). Step 7 - To further assess the homogeneity of this a-amylase, this enzyme was subjected to isoelectrofocusing.  A single sharp  60  Figure 7  Chromatography of Bacteroides amylophilus Strain H-18 a-amylase on DEAE-Sephadex A-50. Enzyme was eluted with a l i n e a r NaCl gradient (0.2 to 0.75 M) i n phosphate buffer (0.05 M, pH 7.0).  a-Amylase a c t i v i t y (Units/ml)  NaCl (M)  62  Figure 8  Chromatography of Bacteroides amylophilus Strain H-18 a-amylase isoenzyme 1 on Sephadex G-200.  Enzyme was eluted with phos-  phate- buffer (0.05 M, pH 7.0).  700, cn vO  600  500  400  •300  200  100  /  /  /  -z 50  100  150  200  250  300  Fraction Number (5.0 ml/Fraction)  350  400  1  450  64  Figure 9  Disc electrophoresis of a-Amylase Isoenzyme 1 . I l l u s t r a t i o n A.  Stained disc g e l .  I l l u s t r a t i o n B.  Densiometric tracing.  65  +  td  66 peak was obtained (Figure 10).  Therefore-it i s concluded  from these results that a-amylase isoenzyme 1 i s a single homogeneous protein.  2.  C a t a l y t i c Properties of a-Amylase Isoenzyme 1 a.  Determination of Type of Amylase  To determine whether the amylase i n question i s of the a or 3 type the enzymic digestion of starch and amylose was examined by thin layer chromatography.  Thin layer analysis revealed the. presence of high  molecular weight reducing dextrin and a series of malto-oligosaccharides (see IV:B).  These results indicate that the amylase isoenzyme 1 studied  i s of the a-type.  b.  Effect of pH on.a-Amylase A c t i v i t y  The pH p r o f i l e s of the a c t i v i t y of a-amylase are shown i n Figure 11.  The pH of the maximum enzymic a c t i v i t y was found to be 6.7 at 44°C.  The optimum pH values for a-amylases from other sources as reported i n the l i t e r a t u r e are i n the acid region between 4.5 and 7.0.  These r e -  sults are summarized i n Table IV. c.  Effect of pH on Enzymic S t a b i l i t y  As seen i n Figure 12, the a-amylase i s stable i n the pH range of 6.2 to 7.6.  The stable pH i s narrow on both a c i d i c and alkaline side.  The pH s t a b i l i t i e s of a-amylases from other sources are reviewed i n Table V for purposes of comparison.  67  Figure 10  Electrofocusing of a-Amylase Isoenzyme 1 ( x — x ) with superimposed pH gradient (.—.)•  The figure above the enzyme  a c t i v i t y peak gives the p i value of a-Amylase.Isoenzyme  1.  pH Gradient  69  F i g u r e 11  Optimum pH f o r h y d r o l y z i n g s t a r c h . enzymatic a c t i v i t y was  determined a t 44°C  f o r 15 minutes a f t e r i n c u b a t i o n w i t h s t a r c h at r e s p e c t i v e pH  The  values.  Relative A c t i v i t y (%)  71 TABLE IV SUMMARY OF THE OPTIMUM pH RANGE OF a-AMYLASE FROM VARIOUS SOURCES  Source of a-amylases  Optimum pH range  References  Porcine pancreas  6.8  4,31,34  Monkey small intestine  6.8 .  43  Human s a l i v a  6.9  32,34  4.8-5.8  12  Bacillus subtilis  6.0  29  Pseudomonas saccharophila  5.25-5.75  27  Bacillus  5.0  6,7  4.5-6.5  37  Streptococcus bovis  4.6-6.1  17  Clostridium  5.5-6.5  17  6.2-7.5  42  6.7  S.U.R.  Aspergillus  oryzae  stearothermophilus  butyricum  B. polymyxa  Bacteroides  amylophilus  72  Figure  12  E f f e c t of pH on the s t a b i l i t y o f a-amylase. The b u f f e r s o l u t i o n used was 0.02 M T r i s maleate (pH 5.8 to.8.6). To 1.0 ml of each of the above b u f f e r s o l u t i o n s 0.2 ml o f a 1 per cent s o l u t i o n of the enzyme ( i n d i s t i l l e d water) was added and the m i x t u r e was kept a t 37°C f o r 24 h o u r s . A f t e r a d j u s t i n g the pH to 6.7, the f i n a l volume was made up t o 4.0 ml. The enzymic a c t i v i t y was determined b e f o r e and a f t e r treatment and the percentage of the a c t i v i t y which remained was c a l c u l a t e d .  % of Residual A c t i v i t y 73  O  O  ON  O  00  o  O O  74 TABLE V SUMMARY OF THE OPTIMUM pH STABILITY RANGE FOR VARIOUS a-AMYLASES  Source of a-amylases  pH s t a b i l i t y range  References  Barley malt  4.9-9.1  34  Porcine pancreas  7.0-8.5  4,34  Human s a l i v a  4.8-11  13,32,34  Aspergillus oryzae  5.5-8.5  10,49  Bacillus stearothermophilus  6-11  37  Pseudomonas saccharophila  4.5-8  27  Bacillus s u b t i l i s  4.8-8.5  30.46  Bacteroides amylophilus  6.2-7.6  S.U.R.  75 d.  Effect of Temperature on a-Amylase A c t i v i t y  The temperature p r o f i l e s of the a c t i v i t y of a-amylase isoenzyme 1 are shown i n Figure 13.  Temperature•for the maximum a c t i v i t y i s 44°C  as compared to a-amylases f o r other sources (Table VI).  e.  Effect of Temperature on Enzymic S t a b i l i t y  Figure 14 (curve B) i l l u s t r a t e s that a-amylase isoenzyme 1 r e tained 100 per cent of i t s o r i g i n a l a c t i v i t y after heat treatment up to 42°C f o r 15 minutes.  In this regard thermophilic a-amylase from B.  stearothermophilus retained 100 per cent of i t s o r i g i n a l a c t i v i t y at 65°C f o r 15 minutes and the mesophilic a-amylase from B_. s u b t i l i s maintained 100 per cent of i t s o r i g i n a l a c t i v i t y at 43°C f o r 15 minutes (38). It was found that a-amylase isoenzyme 1 contained 3 gram atoms of calcium per mole of enzyme (see IV:A:2).  a-Amylases of various  have been shown to contain calcium which i s essential  origins  i n the c a t a l y t i c  a c t i v i t y and s t a b i l i z a t i o n of the enzyme molecule (12,18,22,50).  There-  fore the effect of calcium ions on the s t a b i l i t y of a-amylase isoenzyme 1 was studied f o r comparative purposes. CaC^,  In the presence of 0.02 M  the thermal s t a b i l i t y of a-amylase isoenzyme 1 increased from  42°C to 58°C (Figure 14, Curve A).  This result indicates that the  property of a-amylase isoenzyme 1 i s similar  to that of other a-amylases.  Pretreatment of a-amylase isoenzyme 1 with 0.02 M EDTA decreased the thermal s t a b i l i t y of the enzyme (Figure 14, Curve C).  Increased  s u s c e p t i b i l i t y of a-amylase to temperature was caused by the nona v a i l a b i l i t y of calcium which was chelated by EDTA.  Similar results have  76  Figure 13  Optimum temperature for hydrolysing starch. The reaction mixture contained 1 ml of a 2 per cent solution of starch at pH 6.7 and 0.3 mg of enzyme i n 0.02 M Tris-maleate buffer (pH 6.7).  The-reaction was carried  out at various temperatures  for 10 minutes.  Relative A c t i v i t y (%) 77  78 TABLE VI OPTIMUM TEMPERATURE FOR VARIOUS a-AMYLASES  Source of a-amylase  Optimum Temperature  References  Barley malt  35°C  34  Porcine pancreas  37°C  4,31,34  Human s a l i v a  4 0 °C  13,32,33,34  Aspergillus oryzae  40°C  10,49  Bacillus s u b t i l i s  40°C  29,30,46  43°-58°C  37  65°C .  6,7  65°-73°C  37  B a c i l l u s stearothermophilus  Streptocuccus bovis  , 48°C  17  Clostridum butyricum  48°C  17  Bacteroides amylophilus  44°C  S.U.R.  ;  79  Figure 14  Thermal s t a b i l i t y of a-amylase. Enzyme (0.03 mg/ml) was treated at various temperatures as indicated. After 15 minutes each solution was immediately cooled. The residual a c t i v i t y was determined and percentage of the a c t i v i t y which remained was calculated with respect to each treatment. The various treatments were' as follows: Curve A. 0.02 M C a C l buffer, pH 6.7. Curve B.  2  i n 0.02 M Tris-maleate  0.02 M Tris-maleate b u f f e r , pH 6.7.  Curve C. Enzyme was dialysed against 0.22 M EDTA i n Tris-maleate b u f f e r , pH 6.7, 20°C.  % of Residual A c t i v i t y  80  81 been reported by EDTA treatment f o r human s a l i v a , hog pancreas, B. subt i l i s and A. oryzae a-amylases (50), _. stearothermophilus a-amylase (38)  and a-amylase from monkey small intestine  f.  (43).  Amino Acid Determination  The amino acid analysis of a-amylase isoenzyme 1 was p r i n c i p a l l y done to determine the presence or absence of cysteine and cystine.  The  amino acid analysis showed complete absence of cysteine and cystine. Therefore disulphide linkages and sulphydryl groups are not involved i n maintaining the enzymic a c t i v i t y and t e r t i a r y structure of a-amylase molecules.  The absence of sulphydryl groups i s i n agreement with the  finding that p-chloromercuribenzoate did not inactivate the enzyme (IV:A:2).  It i s also interesting to note that protease from B. amylo-  philus H - 1 8 i s completely void of cysteine and cystine (20).  In this  regard a-amylase isoenzyme 1 i s comparable with _. s u b t i l i s a-amylase which does not contain cystine (25). B a c t e r i a l a-amylase from B_. stearothermophilus does contain cysteine, but completely lacks tryptophan (6).  Pollock (41)  has reported the absence of disulphide linkages as a  c h a r a c t e r i s t i c feature of various b a c t e r i a l exqenzymes.  g.  Determination of Calcium  a-Amylases isolated from various sources have been reported to contain a few atoms of firmly bound calcium (Table VII).  When _. sub-  t i l i s a-amylase was dialysed continuously against chelating agents, (a) calcium was not removed completely, indicating i t was bound very firmly; (b)  and there was a reversible loss of enzymic a c t i v i t y when calcium  82  TABLE VII CALCIUM CONTENTS OF VARIOUS a-AMYLASES  Source of a-amylase  Amount of Calcium Present*  References  Human s a l i v a  1-2  50  Hog pancreas  1-2  50  A. oryzae  2-3  50  B. s u b t i l i s  3.0  50  Bacteroides amylophilus  3.0  S.U.R.  The results are reported in. gram-atom per mole (50,000 g) of enzyme.  83 contents were lowered below 1 gram atom per mole of enzyme, thus, i n d i c a t ing a functional, role, of calcium (50) .  The findings of Hsiu.et al_. • (18) ,  are similar for 13. s u b t i l i s a-amylase and human salivary a-amylase. By spectrophotometric analysis i t was  found that p u r i f i e d a-  amylase isoenzyme 1 from B. amylophilus contains 3 gram-atom of calcium per mole of a-amylase.  This value i s i n the range of reported values  for the other a-amylases (Table VII).  When a-amylase isoenzyme 1 was  dialysed against 0.02 M EDTA (Figure 14), the enzyme retained 30 per cent of i t s o r i g i n a l a c t i v i t y and the calcium content at this stage was 0.8 gram atom per mole of a-amylase.  When I3_; s u b t i l i s a-amylase was  dialysed against 0.01 M EDTA for 50 hours, the enzyme retained 40 per cent of i t s o r i g i n a l a c t i v i t y and calcium content after d i a l y s i s was gram-atom per mole of enzyme (50) .  0.4  These results indicate that while  isoenzyme 1 binds calcium more strongly than 13. s u b t i l i s a-amylase, i t i s less stable at low calcium levels than I3_. s u b t i l i s a-amylase. h.  Effect of Chemical Reagents on Enzymic A c t i v i t y (i)  Effect of Urea on a-Amylase A c t i v i t y  The effect of urea on a-amylase a c t i v i t y i s shown i n Figure 15. The results (Curve A) indicate the remaining enzymic a c t i v i t y at d i f f e r ent concentrations of urea. inhibited.  In 8.0 M urea the a c t i v i t y was  completely  After removal of urea by d i a l y s i s against Tris-maleate buffer  of pH 6.7, p a r t i a l regeneration of the enzyme was obtained (Curve B, Figure 15).  After incubation i n 8.0 M urea (pH 8.5) at 30°C for 30 min-  utes, 13. s u b t i l i s a-amylase l o s t .40 per cent of i t s o r i g i n a l a c t i v i t y ,  84  Figure 15  Effect  of urea on a-amylase a c t i v i t y .  Curve A. Various concentrations of urea as indicated were added to standard assay mixture and the percentage of the a c t i v i t y r e maining was calculated. Curve B. Solutions of enzymes (0.03 mg/ml), containing various concentrations of urea were kept at 37°C f o r 16 hours at pH 7.0. After d i a l y s i s i n cold against 0.02 M T r i s maleate buffer (pH 6.7) for 24 hours, the. percentage of the remaining a c t i v i t y was calculated.  86 whereas B. stearothermophilus a-amylase l o s t 10 per cent of the o r i g i n a l a c t i v i t y (38).  Imanishi et a l . (23) recovered about:80 per cent of the  o r i g i n a l enzymic a c t i v i t y of _. s u b t i l i s a-amylase after treating with 8.0 M urea containing EDTA.  Maximum recovery of a-amylase isoenzyme 1  was 68 per cent i n 1.0 M urea.  I t appears from the results that a-amylase  isoenzyme 1 was more sensitive to urea under the experimental conditions used.  During the urea treatment the temperature was held at 37°C f o r 16  hours, which was higher than that used by other workers as noted above. _. aeruginosa protease was completely inhibited i n a solution of 8.0 M urea, after treatment-at 37°C f o r 16 hours and reactivation could not be achieved after a removal of urea (35) .  Treatment  of a-amylase from  monkey intestine with 5.0 M urea resulted i n i n a c t i v a t i o n which was apparently i r r e v e r s i b l e (43).  Fukushi e_ a l . (14) , also recovered 60 to 90  per cent of denatured B_. s u b t i l i s a-amylase a c t i v i t y at pH 8.5 i n 8.0 M urea at room temperature.  These workers further reported that changes  i n o p t i c a l rotatory dispersion and spectral s h i f t took place much more rapidly, reaching almost f i n a l values immediately after the onset of regeneration.  Therefore, denatured protein after the removal of urea  rapidly resumed a three dimensional structure closely resembling that of the native protein.  This refolding i s followed by a slower i n t r a -  molecular rearrangement  to a c h a r a c t e r i s t i c native structure responsible  for a native and b i o l o g i c a l l y active protein.  A c t i v i t y obtained was r e -  ported to be due to p a r t i a l regeneration of enzyme molecules having the same s p e c i f i c a c t i v i t y as that of native a-amylase (14).  The f a i l u r e of  regeneration may be due to the chemical binding of cyanate which i s  87 present i n urea with, amino groups t o . y i e l d carbamyl derivatives (8,44). The other possible causes of i r r e v e r s i b l e i n a c t i v a t i o n of denatured a-amylase reported by others may be due to incorrect refolding and i n t e r molecular aggregation (23) , and p a r t i a l l y due to p r o t e o l y t i c degradation of the unfolded enzyme molecule by p r o t e o l y t i c contaminants  (23,45).  Re-  generation of the enzymic a c t i v i t y also depends on the pH value and i o n i c strength of the solution and concentration of enzyme (23).  (ii)  Effect of EDTA and M e t a l l i c Ions on a-Amylase A c t i v i t y  As seen i n Figure 16, treatment with EDTA reduced the enzymic a c t i v i t y to 30 per cent of the o r i g i n a l a c t i v i t y . various metals the enzymic a c t i v i t y was  By the addition of  regenerated (Figure 16, B).  Treatment with calcium restored the enzymic a c t i v i t y completely while magnesium reactivated the enzymic a c t i v i t y up to 90 per cent.  It may  be  noted that d i a l y s i s had no effect on the a-amylase isoenzyme a c t i v i t y . The a c t i v i t y remained constant during the period of d i a l y s i s . Treatment with EDTA removed the calcium from the various a-amylases, thus decreasing their a c t i v i t y (18,50).  Addition of calcium re-  sulted i n the restoration of enzymic a c t i v i t y (18,48).  In this regard  the results presented i n this thesis are similar to the findings of other workers (18,38,46).  The evidence has been presented that the c a l -  cium atom i s necessary i n maintaining the c a t a l y t i c a l l y active conformation of the amylase molecule  (18).  The role of calcium becomes important  p a r t i c u l a r l y i n .B. s u b t i l i s a-amylase which lacks intramolecular d i s u l phide linkage and free sulfhydryl groups (1,25).  In such cases the  88  Figure  16  E f f e c t of (A) EDTA and (B) m e t a l i o n s a f t e r EDTA treatment on r e a c t i v a t i o n of a-amylase. The enzyme s o l u t i o n was d i a l y s e d a g a i n s t M EDTA i n 0.02 M t r i s - m a l e a t e b u f f e r a t 5°C f o r 100 h o u r s . further dialysis  (pH 7.0)  EDTA was removed by  f o r 24 hours a g a i n s t  m e t a l s o l u t i o n s as i n d i c a t e d i n 0.02 M maleate b u f f e r i t y was  0.02  various tris-  (pH 6.7) and the enzyme a c t i v -  determined.  % of the A c t i v i t y Remained  /—\ bd  X) C tn Mi •~J •  o  g  r-  1  M l fD (D fo H rt fD  to O  • O to  O  ON  o  89 oo o  t-  c c  S H M  I  o (S3  g  W  o  1-3  >  Reactivation to  o  o • o o n 4> ro  n • o  01 O M ho  4S g  3  O  M  g  n *~  g  •  CM  O  M  g  ISJ  o  (%) ON  o  oo o  c  "7  90 active conformation of protein may  be maintained by c e r t a i n intramolecu-  l a r non-covalent linkages rather than usual disulphide bridges (18). Actually the non-existence of disulphide linkage appears to be i s t i c of various b a c t e r i a l exoenzymes (41). maintaining  character-,  The role.of calcium i n  enzymic a c t i v i t y has also been suggested i n several other  b a c t e r i a l exoenzymes, v i z . proteinase (16) and ribonuclease  (18).  In  this regard a-amylase isoenzyme 1 i s an exo-enzyme, lacks disulphide linkage and free s u l f h y d r y l groups and requires calcium for i t s a c t i v i t y . Imanishi  (22) has reported that removal of calcium does not cause detec-  table changes i n protein conformation and suggested that calcium ions may be located on.the surface of the protein. that calcium forms a tight metal-chelate  It appears, therefore^  structure with the protein  molecule to maintain a proper configuration for b i o l o g i c a l a c t i v i t y (18,50).  Takagi and Iseumura (48) also indicated the role of calcium  ions i n refolding the reduced taka-a-amylase A.  As noted i n Figure 16  (B), the treatment with cobalt, zinc and magnesium could not regenerate the enzymic a c t i v i t y completely.  The reason may  be that c e r t a i n metals  could not form-a correct metal chelate structure with protein, thus res u l t i n g i n unstable or incomplete secondary and t e r t i a r y structures, which did not have complete b i o l o g i c a l a c t i v i t y . i.  Functional Groups Determination  The results on the e f f e c t of reducing, oxidizing and ing agents are presented  i n Table VIII.  SH-inactivat-  The a-amylase activity, remained  unaffected a f t e r the treatment with reducing agents, v i z . , cysteine, sodium cyanide, sodium thioglycolate, mercaptoethanol.  91  TABLE VIII EFFECT OF REDUCING, OXIDIZING AND SH-INACTIVATING AGENTS ON a-AMYLASE ISOENZYME 1 ACTIVITY  Reagents  Concentration* (M)  Cysteine  5 x 10"  Sodium cyanide  Residual A c t i v i t y (%)  3  100.00  5 x IO  - 3  100.00  Sodium thioglycolate  5 x IO  - 3  102.00  Mercaptoethanol  5. x I O  - 3  100.00  p-Chloromercuribenzoate  5 x IO  - 3  98.00  Monoiodoacetic acid  5 x IO  - 3  92.0  Potassium permanganate  N-Bromosuccinimide  IO"  5 x IO  3  0  - 3  0  Solutions of enzyme (6 units/ml) containing various concen-r trations of d i f f e r e n t reagents at pH 7.0 are kept f o r one hour at 37°C. The percentage of the a c t i v i t y which r e mained after the treatment was calculated.  92 Further the enzymic a c t i v i t y i s not inactivated by s p e c i f i c SHi n a c t i v a t i n g agents such as p-chloromerciiribetizoate and monoiodoacetic acid.  These results indicate the noninvolvement of sulfhydryl groups i n  enzymic reaction mechanisms, and are i n agreement with the finding that a-amylase isoenzyme 1 did not contain cystine.  The s l i g h t i n h i b i t i o n may  be due to the reaction with methionine, serine or imidazole-group of histidine.  Since the i n h i b i t i o n observed was s l i g h t these.amino acids  may be located near the active center. It i s to be noted that enzymic a c t i v i t y was completely l o s t by oxi d i z i n g agents v i z . , potassium permanganate Although potassium permanganate  and N-bromosuccinimide.  i s a non-specific oxidizing agent, N-  bromosuccinimide i s more s p e c i f i c with controlled conditions i n i t s reaction with tryptophan. (39).  Okada et^.a_. (39) used N-bromosuccinimide  to oxidize tryptophan and suggested that i t was involved i h the active center of ._. s u b t i l i s ar-amylase.  It has been reported by Sugae (47)  that B. s u b t i l i s a-amylase l o s t i t s a c t i v i t y when an azo-group.was duced into the a-amylase molecule.  intro-  He suggested that a peptide group i n  the neighbourhood of a p a r t i c u l a r tyrosine residue which was modified by the azo group was closely related with the a c t i v i t y of b a c t e r i a l ct-. amylase.  In general, the active center includes, besides the c a t a l y t i c  s i t e , the grouping conforming to the substrate s p e c i f i c for the enzyme (40); the two groups are s u f f i c i e n t l y close to each.other.  It i s possible  that the active center of b a c t e r i a l a-amylase may have two groups,.tryptophan (39) and tyrosine (47). Yamato (54) indicated that.the tryptophan and tyrosyl group a-amylase obtained from B, amyloliquefaciens  Fukumoto was e s s e n t i a l f o r i t s enzymic a c t i v i t y .  Ikenda (21) reported  that i n case of A. oryzae, the phenolic group of tryosine was essential for enzymic a c t i v i t y .  The a c t i v i t y of a-amylase isoenzyme was lost with  the treatment.of N-bromosuccinimide (Table VIII).  It i s suggested from  this result that tryptophan i s essential f o r the c a t a l y t i c a b i l i t y of a-amylase isoenzyme 1.  j . Determination of Molecular Weight The estimated molecular weight f o r a-amylase isoenzyme i s 45,000. The'molecular weight of various a-amylases are given i n Table IX. k.  Determination of I s o e l e c t r i c Point  I s o e l e c t r i c point as determined by electrofocusing technique was pH 3.7.  I s o e l e c t r i c points of other a-amylases are also i n the acidic  region as indicated i n Table X.  94  TABLE IX MOLECULAR WEIGHT OF VARIOUS a-AMYLASES  Source  Molecular Weight  References  Barley malt  59,500  34  Porcine pancreas  45,000  9  Aspergillus oryzae  51,000  24  Bacillus subtilis  48,700  11  Bacillus saccharophila  15,600  26  Bacteroides amylophilus  45,000  S.U.R. •  95  TABLE X ISOELECTRIC POINTS OF VARIOUS a-AMYLASES  Source  I s o e l e c t r i c points  References  Barley malt  5.7  34  Porcine pancreas  5.2-5.6  4,31,34  Human s a l i v a  5.2-5.6  32,34  Aspergillus oryzae  4.2  10,49  Bacillus subtilis  5.4  29,30,46  Bacillus thermophilus  4.8  6,7  Bacteroides amylophilus  3.7  S.U.R.  REFERENCES IV:A  1.  Akabori, S., Y. Okada, S. Fujiwara and K. J . Sugae. 1965. Studies on b a c t e r i a l amylase. I. Amino acid composition of c r y s t a l l i n e b a c t e r i a l amylase from B_. s u b t i l i s N. J . Biochem. 43:741.  2.  Blackburn, T. H. 1968; Protease production by Bacteroides amylophilus s t r a i n H-18. J . Gen. Microbiol. 51:27.  3.  Bryant, M. P. and I. W. Robinson. 1961. Some n u t r i t i o n a l requirements of genus Ruminococcus. Apply Microbiol. 9:91.  4.  Caldwell, M. L., M. Adams, J . F. Kung and G. C. T o r a l b a l l a . 1952. C r y s t a l l i n e pancreatic amylase. I I . Improved method for i t s preparation from hog pancreas glands and additional studies of i t s properties. J . Amer. Chem. Soc. 74:4033.  5.  Clary, J . J j G. E. M i t c h e l l , J r . and C O . L i t t l e . 1968. Action of bovine and ovine a-amylases on various starches. J . Nut. 95:469.  6.  Campbell, L. L. and G. B. Manning. 1961. Thermostable a-amylase of Bacillus stearothermophilus. I I I . Amino acid composition. J. B i o l . Chem. 236:2962.  7.  and P. D. Cleveland. 1961. Thermostable a-amylase of B a c i l l u s stearothermophilus. C r y s t a l l i z a t i o n and some general properties. J . Biol..Chem. 236:2952.  8.  Cole, R. D. 1961. On the transformation of i n s u l i n i n concentrated solution of urea. J . B i o l . Chem. 236:2670.  9.  Danielsson,. C.' E. 160:899.  10.  11.  1947. Molecular weight of a-amylase;  Nature.  Fischer, F. H. and R. DeMontmollin. 1951. P u r i f i c a t i o n et cryst a l l i s a t i o n de 1'a-amylase d.'Aspergillus oryzae. Sur les enzymes amylolytiques. Helv. Chim. Acta. 34:1987. • , W. N. Summerwell, J . M. Junge and E. A. Stein. 1958. Proceedings of Symposium VIII, IVth International Congress of Biochemistry, Vienna, Pergamon Press.  97 12.  13.  Fischer, E. H. and E. A. Stein. ' 1960. "a-Amylases." In the enzymes.Ed. P. D. Boyer, H. Lardy, and K. Myrback. Vol. 4. Academic Press. . 1964 a-Amylase from human s a l i v a . cal Preparation. 8:27.-  Biochemi-  14.  Fukushi, T., A. Imanishi and T. Isemura. 1968. Changes i n enzymat i c a c t i v i t y and conformation during regeneration of native b a c t e r i a l amylase from denatured form. J . Biochem. 63:409.  15.  Hagihara, B. 1954. C r y s t a l l i n e b a c t e r i a l amylase and proteinase. Ann. Rep. S c i . Osaka Univ. 2:35.  16.  . 1960. "Bacterial and mold.proteases." In the enzyme. Ed. by P. D. Boyer, H. Lardy, and K. Myrback. Academic Press Inc. New York. Biochem. Biophys. Acta. 52:176.  17.  Hobson, P. N. and M. Macpherson. 1952. Amylases of Clostridium butyricum and a streptococcus isolated from rumen of the sheep. J. Biochem. 52:671.  18.  Hsin, J.-, E. H. Fisher and E. A. Stein. 1964. Alpha-amylase as calcium-metalloenzyme. I I . Calcium and c a t a l y t i c a c t i v i t y . Biochem. 3:61.  19.  Hungate, R. E. - 1966. "The rumen b a c t e r i a . " In the rumen and i t s microbes. By R. E. Hungate. Academic Press. New York and London.  20.  Lesk, E. M. 1969. P u r i f i c a t i o n and characterization of proteolytic enzymes from _. amylophilus s t r a i n H-18. M.Sc. Thesis. Univers i t y of B r i t i s h Columbia, Vancouver, B.C.  21.  Ikenda, T. 1959. Chemical modification on taka-amylase A. I I . Phenylazobenzoylation of taka-amylase A. J . Biochem. 46:297.  22.  Imanishi, A. Biochem.  23.  24.  1966. Calcium binding by b a c t e r i a l a-amylase. 60:381.  J.  , K. Kakiuchi and T. Isemura. 1963. Molecular s t a b i l i t y and r e v e r s i b i l i t y of denaturation of _. s u b t i l i s a-amylase. I I . Regeneration of urea denatured enzyme by removal or d i l u t i o n pf urea. J . Biochem. 54:89. Isemura, T. andS. F u j i t a . 1957. Physicochemical studies on takaamylase A. I. - Size and shape determination by the measurement of sedimentation, d i f f u s i o n c o e f f i c i e n t and v i s c o s i t y . J . Biochem. 44:443.  98 25.  Junge, J . M. , E. A. Stein, J . Neurath and E. H. Fischer. 1959. The amino acid composition of a-amylase from Bacillus s u b t i l i s . J. Biochem. 234:556.  26.  Manning, G. B., L. L. Campbell and R. J . Foster. 1961. Thermostable a-amylase of B a c i l l u s stearothermophilus. I I . Physical properties and molecular weight. J . B i o l . Chem. 236:2958.  27.  Markovitz, A., H. P. K l e i n and E. H. Fisher. 1956. P u r i f i c a t i o n , c r y s t a l l i z a t i o n , and.properties of the a-amylase of Pseudomonas saccharophila. Biochem. Biophys. Acta. 19:267.  28.  Meites, S. and S. Rogols. 1968. Serum amylases, isoenzymes, and p a n c r e a t i t i s . I. Effect of substrate v a r i a t i o n . C l i n . Chem.  14:1176.  29.  Menzi, R., E. A. Stein and E. H. Fisher. 1957. Proprietes de deux • a-amylase de .B. s u b t i l i s . Sur les enzymes amylolytiques. Helv. Chim. Acta. 40:534.  30.  Meyer, K. H., M. Fuld and P. Bernfeld. 1947. P u r i f i c a t i o n et c r i s t a l l i s a t i o n de 1'a-amylase de bacterie. Experentia. 3:411.  31.  32.  .  , E. H. Fischer and P. Bernfeld. 1947. Sur les enzymes amylolytiques (1). L'isolement de l'a-amylase de pancreas. Helv. Chim. Acta. '30:64. , , A. Stauband P. Bernfeld. 1948. Proprietes de l'a-amylase de s a l i v e humainine c r i s t a l l i s e e . Helv. Chim. Acta.  31:2165.  33.  , , . 1948. Sur les enzymes amylolytiques. Isolement et c r i s t a l l i s a t i o n de l'a-amylase.de s a l i v e humaine. Helv. Chim. Acta. 31:2158.  34.  . 1952. The'past and present of starch chemistry. entia. 8:405.  35.  Exper-  Morihara, K. 1963. Pseudomonas aeruginosa proteinase. I. P u r i f i c a t i o n and general properties. Biochem. Biophys. Acta.  73:113.  36.  Nomura, M., B. Maruo and S.Akabori. 1956. Studies on amylase formation by Bacillus s u b t i l i s . I. Effect of high concentration of polyethylene g l y c o l on amylase formation by B a c i l l u s s u b t i l i s . J . Biochem. 43:143.  37.  Ogasaharaj K. , A. Imanishi and T. Isemura. 19.70. Studies o r i t h e r mophilic a-amylase from B a c i l l u s stearothermophilus. I. Some' general and physico-chemical properties of thermophilic aamylase. J . Biochem. 67:65.  99 38.  Ogasahara; K. , A. Imanishi and T. Isemura. 1970. Studies on thermophilic a-amylase.from Bacillus stearothermophilus. I I . Thermal s t a b i l i t y of thermophilic a-amylase. J . Biochem. 67:77.  39.  Okada, Y. , K. Onoue, S.Nakashima and Y.Yamamura. 1963. Studies on the enzyme-antienzyme system. I I . N-bromosuccinimide modified b a c t e r i a l a-amylase. J . Biochem. 54:477.  40.  Pechere, J . F. and H. Neurath. 1957. "Proteolytic enzyme." In Symposium on protein structure.. Ed. by A. Neuberger. Internat i o n a l Union of Pure and Applied Chemistry. Paris.  41.  Pollock, M. R. 1962. "Exoenzyme." In the Bacteria. Ed. by I. C. Gunsalus and R. Y. Stainer. V o l . 4. New York and London. Academic Press, Inc.  42.  Rose, D.  1948. The amylase of B a c i l l u s polymyxa.  Arch. Biochem.  16:349. 43.  Seetharam, B., N. Swaminathan and A. N. Radhakrishna. 1969. P u r i f i c a t i o n and properties of a-amylase from monkey small i n t e s t i n e . Indi. J . Biochem. 6:51.  44.  Stark, G. R., W. H. Stein and S. Moore. 1960. Reaction of the cyanate present i n aqueous urea with amino acids and protein. J. B i o l . Chem. 235:3177.  45.  Stein, E.A., and E. H. Fisher. 1958. The resistance of a-amylase towards p r o t e o l y t i c attack. J . B i o l . Chem. 232:867.  46.  . 1961. a-Amylase from B a c i l l u s s u b t i l i s . Biochemical preparation. 8:34.  47.  Sugae, K. I960; . Studies on b a c t e r i a l a-amylase. V. Chemical modification o f - b a c t e r i a l amylase and coupling of taka-amylase A with p. sulfobenzene-dizonium-chloride. J . Biochem. 48:790.  48.  Takagi, T. and T. Isemura. 1965. Necessity of calcium for the r e generation of reduced Taka-amylase A. J . Biochem. 57:89.  49.  Underkofler, L. A. and D. K. Roy. 1951. C r y s t a l l i z a t i o n of fungal alpha-amylase and l i m i t destrinase. Cereal Chem. . 28:18.  50.  Vallee, B. L., E. A. Stein, W. N. Summerwell and E. H. Fisher. 1950. Metal content of a-amylases of various o r i g i n s . J . B i o l . Chem. 234:2901.  51.  Welker, N. E. and L. L. Campbell. 1963. Effect of carbon source on formation of a-amylase by B a c i l l u s stearothermophilus. J . Bact. 86:861.  100 52.  Wiesmeyer, H. and M. Cohn. 1960. The characterization of the pathway of maltose u t i l i z a t i o n by Escherichia C o l i . I I I . A desc r i p t i o n of the concentrating mechanism. Biochem. Biophys. Acta. 39:440.  53.  Williams,. P. J . and J . J . McDonald. 1966. Permeability of a.micrococcal c e l l to maltose and some related sugars. J . Canad. Microbiol. 12:1213.  54.  Yamamoto. T. 1955. Studies on s e n s i t i v e groups o f . c r y s t a l l i n e b a c t e r i a l a-amylase. B u l l . Agr. Chem. Soc. Japan. 19:121.  55.  . 1955. Studies on the a-amylase destroying enzyme. Part I. Occurrence and some properties of the enzyme. B u l l . Agr. Chem. Soc. Japan. 19:22.  101 B.  The Action Pattern of a-Amylase Isoenzyme 1  In this section the mode of action of _. amylophilus a-amylase isoenzyme 1 was  examined with two substrates, amylose and.soluble  starch.  The products of digestion were examined q u a l i t a t i v e l y by thin layer chromatography.  The importance of the iodine-staining characteristics  i s reported i n terms of possible mechanisms for a-amylase action. differences and s i m i l a r i t i e s of a-amylase isoenzyme 1.action are  The discussed  with respect to other a-amylases. The progress of hydrolysis of amylose and starch i s shown chromatographically  i n Figures 17 and 18.  ysis beyond the achroic point and this was utes. point.  In Figure 18 hydrolysis was  Figure 17 represents  hydrol-  reached by about t h i r t y min-  also extended beyond the achroic  The reference samples i n each case were obtained by p a r t i a l acid  hydrolysis of amylose. Due :  to the following two observations,  the spots  were regarded as l i n e a r oligosaccharides of maltose s e r i e s :  I.  The experimental spots from enzymatic digests had a corresponding Rf with regard to reference sample (3).  II.  The introduction of branch point (4) retards the mobility i n such a way  that l i n e a r oligosaccharides are separated  branched oligosaccharides of equal D.P.  from  For example, a  branched dextrin of four glucose units f a l l s at a point i n t e r mediate i n distance between maltopentaose arid  maltotetraose  These experimental results suggest that s i g n i f i c a n t amounts of branched oligosaccharides were not present at the achroic point.  The  102  Figure 17  Thin layer analysis of the digestion of Amylose.by a-Amylase Isoenzyme 1. The f i r s t column contained reference compounds, obtained by the acid hydrolysis of Amylose. The remaining columns contained.the products of the progressive hydrolysis of Amylose taken from the digestion mixture at various time intervals as indicated.on the chromatograms. The chromatogram was obtained by the method of Rahman et a l . (11) with a solvent system, n-propanol ethyl acetate-water (6:1:3,v/v). C.  Control of pure sample.  DP.  Degree of polymerization.  «  5  — ,  •  10  15  •  20  •  25  Tine (Minute)  30  3 5  u  0  104  Figure 18  Thin layer analysis of the digestion of starch by an a-Amylase Isoenzyme 1. The f i r s t column contained reference compounds, obtained by the acid hydrolysis of Amylose. The remaining columns contained the products of the progressive hydrolysis of Amylose taken from the digestion mixture at various time intervals as indicated on the chromatogram. The chromatogram was obtained by the method of Rahman et a l . (11) and solvent system was 1-propanol-nitromethane-water (5:2:3) as reported by Huber ejt.al. (8). C.  Control of pure sample.  DP.  Degree of polymerization.  106 appearance of thin layer chromatogram developed with the digestion products of amylose and starch were the same, and could not be d i f f e r e r i t i a ted from one another.  It i s suggested  from these results that the pro-  duction of higher malto-dextrin i s a c h a r a c t e r i s t i c pattern of B. amylophilus a-amylase isoenzyme 1. Figures 17 and 18 show the complete maltodextrin spectrum from D.P.  2 to 14.  from D.P.  The early products i n the digest are maltodextrin mixtures  5 to 14.  There are, f i r s t of a l l , traces of maltopentaose,  maltohexaose and maltoheptaose, and predominance of higher saccharides. Maltose appeared only after twenty-five minutes of digestion and i t s amount increased steadily as.enzymatic reaction progressed. of D.P.  Maltodextrin  12 to 14 started disappearing after the achroic point, and corres-  pondingly the i n t e n s i t y of maltodextrin to D.P.  7 to 11 started decreasing.  No glucose could be detected during the course of enzymatic reaction. This can be contrasted and compared with the c h a r a c t e r i s t i c maltodextrin spectrum produced by other a-amylases.  B_. s u b t i l i s a-amylase produces  mainly maltotriose, maltotetraose and maltoheptaose during early stages of amylose, amylopectin and starch hydrolysis (12,14) and near the achroic point maltose, maltotriose and very.small quantities of maltot r i o s e , maltopentaose, maltohexaose and maltoheptaose.(2).  During a pro-  gressive course salivary amylase produced increased amounts of maltose, maltotriose-and trace amounts of maltopentaose, maltohexaose.and higher oligosaccharides (2).  Main end products are maltose, maltotriose and a  small amount of glucose.  B_. polymyxa a-amylase produces maltose mainly,  however there are trace amounts of glucose and maltotriose formed as  107 w e l l (13).  Hanrahn and Caldwell (7) reported that with high concentra-  tion of enzyme and.if a s u f f i c i e n t time of hydrolysis were allowed, Aspergillus oryzae a-amylase degraded amylose completely into glucose and maltose.  The early stage products of the action of porcine pan-  creatic a-amylase on starch and amylose were found to be maltose and maltotriose and i t has been suggested  that mode of action of this enzyme  i s s i m i l a r to that of human s a l i v a r y a-amylase (14).  The results of  enzymatic hydrolysis shown i n Figures 17 and 18 indicate that only certain series of maltodextrin could be separated by thin layer chromatography, as reducing spots were present at the point of application on the chromatogram. In the present, investigation the changes i n the polysaccharideiodine spectrum were studied during hydrolysis of starch and amylose by a-amylase to understand  the mode of attack of substrate.-  Since the blue  colour of iodine-starch complex depends upon the degree of polymerization of straight chains of the polymer (16), a decrease i n absorption during progressive enzymatic hydrolysis has been taken as a decrease i n the average degree of polymerization of the substrate.  During the enzy-  matic hydrolysis of amylose and starch, a decline i n the absorption was recorded.  In each case the extent of this decline was comparable to the  results reported by Swanson (16), for salivary a-amylase.  I t i s suggested  that i n a q u a l i t a t i v e way the dextrinizing action of JB; amylophilus a amylase isoenzyme 1 i s comparable to salivary a-amylase; It i s generally regarded that the mode,of attack of various a amylases on substrate i s random (2,10), and the enzyme-product complex  108 dissociates i t s e l f  after single c a t a l y t i c events.  It i s also assumed  that a-amylases have equal preference for a l l a-1, 4 bonds, except  those  near the branch point and adjacent to the two ends, which are known to be more resistant to enzymatic attack.  Such an observation i s supported  by a rapid decrease i n iodine colour and v i s c o s i t y (6), but with the a v a i l a b i l i t y of chromatographic  results as reported above i t was  apparent  that such a group of highly c h a r a c t e r i s t i c malto-oligosaccharides and malto-dextrins could not be produced through random attack (1). problem has been resolved (5).  This  The i n i t i a l attack of a-amylase with the  substrate was described as cleaving of one bond or a multiple of bonds i n the proximity of the f i r s t .  The s p e c i f i c nature of this multiple  attack on substrate could be determined by enzymes with the production of c h a r a c t e r i s t i c series of malto-saccharides  (1,3).  The action pattern of  _. amylophilus a-amylase isoenzyme 1 i s i n l i n e with this hypothesis, and the concept of multiple attack at the s i t e of encounter explains the data presented for this a-amylase. Kung et a l . (9) reported that v a r i a t i o n i n the curves r e l a t i n g blue value to the increase i n the reducing values were due to the d i f f e r ent a-amylases degrading amylose i n d i f f e r e n t chain lengths.  But chroma^  tographic results have shown that.different a-amylases produced d i f f e r e n t types of digestion products and these maltodextrins are c h a r a c t e r i s t i c of i n d i v i d u a l enzymes, as mentioned above.  Robyt and French  (15) reported  that porcine pancreatic a-amylase and human salivary a-amylase digest produced very i d e n t i c a l results but their blue value-reducing value curves were very d i f f e r e n t .  These observations are not supported by the  109 explanation offered by Kung et a l . (9). . Robyt and French (15) explained these differences by multiple attack mechanism and also calculated the degree of multiple attack for d i f f e r e n t a-amylases by determining  the  r a t i o of the reducing value of the oligosaccharide f r a c t i o n to that of the polysaccharide f r a c t i o n .  In the case of JB. amylophilus  a-amylase  isoenzyme 1 the,degree of multiple attack as calculated by the method of Robyt and French (15) i s 2 at optimum pH 6.7 (Table XI).  and temperature 44°C  Under the.optimal condition of pH and temperature, porcine  pancreatic a-amylase had a degree of multiple attack of 6, three times that of human salivary and Aspergillus oryzae a-amylase (15).  r  110 TABLE XI ESTIMATION OF THE DEGREE OF MULTIPLE ATTACK BY B. AMYLOPHILUS ISOENZYME 1*  %BV  3  1  5 r  90.0  38  12.0  115.75  3.1  81.0  78.2  26.0  66.70  3.0  72.0  111.0  36.0  59.50  3.08  63.0  123.0  40.0  49.50 .  3.07 3.06**  From the r a t i o of the t o t a l reducing value to the reducing value of 67% ethanol polysaccharide p r e c i p i t a t e . " * , * • • • •  Average r determination.  Degree of multiple attack was determined by the method of Robyt and French (15). The d e f i n i t i o n s used are the same as those used by these authors. 1.  BV = (At/AO) x 100 where Ao and At are the absorbancies (620 mu) the iodine complex of the digest at zerotime and at t times of hydrolysis.  2.  RV_ = Total reducing value i s expressed as mg of apparent maltose/ml digest.  3.  RVp = Reducing value of 67% ethanol p r e c i p i t a t e i n terms of mg of apparent maltose/ml of digest.  4.  Average degree of polymerization of 67% ethanol p r e c i p i t a t e determined by the quotient: [Total carbohydrate (mg/ml)/apparent maltose (mg/ml)] x 2.:  5.  Quotient of the t o t a l reducing value divided by the reducing value of the 67% ethanol p r e c i p i t a t e (RV^RVp) .  6.  Degree of multiple attack (r-1) = (3.06-1) =  2.06.  of  REFERENCES IV:B  1.  Abdullah, M. , D. French and J . F. Robyt. 1966. Multiple attack by a-amylase. " Arch. Biochem. Biophys. 114:595.  2.  Bird, R. and R. H. Hopkins. 1952. The action of some a-amylases on amylose. Biochem. J . 56:80.  3.  Dube, S. R. and P. Nordin. 1962. The action pattern of sorghum a-amylase. Arch. Biochem. Biophys. 99:105.  4.  French, D. and G. M. Wild. Correlation of carbohydrate structure with papergram mobility. J . Amer. Chem. Soc. 75:2612.  5.  . 1957. Recent developments and theoretical aspects of amylase action. Bakers Digest. 31:24.  6.  Greenwood, C. T., A. W. Macgregor and E. Milne. of starch. Staerke. 17:219.  1965.  a-Amylosis  7.  Hanrahan, V. M. and M. L. Caldwell. 1953. A study of the action of Taka-amylase. J . Amer. Chem. Soc. 75:2191.  8.  Huber, C. N., H. D. Scobell and E. E. Fisher. 1968. Thin layer chromatography of malto-oligasaccharides and megalasaccharides with mixed support and multiple i r r i g a t i o n . Anal. Chem. 40:207.  9.  Kung, J. T., V. M. Hanrahan and M. L. Caldwell. 1953. A comparison of the action of several alpha amylases upon a linear f r a c t i o n from corn starch. J . Amer. Chem. Soc.. 75:5548.  10.  Myrback, K. 1948. Products of the enzymic degradation of starch and glycogen. Advance i n Carbohydrate Chem. 3:251.  11.  Rahman, Sh. Saif-^ur-, C. R. Krishnamurti and W. D. K i t t s . 1968. Separation of Cello-saccharides by thin layer chromatography. J. Chromat. 38:400.  12.  Robyt, J . and D. French. .1963. Action pattern and s p e c i f i c i t y of an amylase from Bacillus s u b t i l i s . Arch. Biochem. Biophys. 100:451.  112 13.  Robyt, J . and D. French. 1964. P u r i f i c a t i o n and action pattern of an amylase from B a c i l l u s polymyxa. Arch. Biochem. Biophys.  104:338.  14. 15.  16.  . Ph.D. .  1962. Action pattern of some alpha-type amylases. thesis. Iowa State University.  and D. French. 1967. Multiple attack hypothesis of ctamylase action. Action of porcine, pancreatic, human salivary and Aspergillus oryzae. Arch. Biochem. Biophys. 122:8.  Swanson, M. A. 1948. Studies on the structure of polysaccharides IV. Relation of iodine colour to structure. J . B i o l . Chem.  172:825.  113 C.  1.  Immunochemical Studies on a-Amylase•Isoenzyme 1  Inhibition of Enzymic A c t i v i t y by Antibody The results of enzymic i n h i b i t i o n by antibody are e s s e n t i a l l y  s i m i l a r to those of b a c t e r i a l and mold a-amylases (5,13).  Normal control  serum did not i n h i b i t the enzymic a c t i v i t y unlike Nomura and Wada (5) who  obtained s l i g h t i n h i b i t i o n of the enzymic a c t i v i t y .  The time course  study of the i n h i b i t i o n of the a-amylase a c t i v i t y by antibody disclosed that n e u t r a l i s a t i o n was  complete i n 1 hour.  Various concentrations of antibody were added to the constant amount of a-amylase and incubated at 37°C.  After 1 hour, the enzymic  a c t i v i t y was determined and percentage of remaining a c t i v i t y was lated.  The results are presented i n Figure 19.  The curve was  calcu-  linear  u n t i l 84 per cent of the enzymatic a c t i v i t y was neutralized, that i s , the quantity of enzyme neutralized was proportional to that antibody added i n the region of antigen excess.  A small amount of residual a c t i v i t y (16  per cent) remained i n the presence of excess antibody. This might be explained either by reversible d i s s o c i a t i o n of a n t i body-enzyme complex or that the complex exhibits amylase a c t i v i t y (13). Cinader (3) has also reported.that enzyme-antibody complexes themselves have some residual a c t i v i t y . ~ " Cinader and Lafferty (2) have.established the presence of three types of antibodies to b i o l o g i c a l l y active antigen. These antibodies can a f f e c t the a c t i v i t y of enzyme i n three d i f f e r e n t ways: antibody may  combine and i n h i b i t ; antibody may combine with a n t i -  gen but not i n h i b i t i t s a c t i v i t y ; and antibody may and may  combine, not i n h i b i t ,  i n t e r f e r e i n the combination with i n h i b i t i n g antibody.  114  Figure 19  Neutralisation curve of a-amylase isoenzyme 1 with antiserum. One ml of enzyme solution (8.0 units) was mixed with 1.0 ml of antiserum containing various amounts of o r i g i n a l antiserum, a-amylase a c t i v i t y was determined after the incubation of 1 hour at 37°C.  116 2.  Ouchterlony Double-Diffusion Analysis Results of double d i f f u s i o n i n agar gel are shown i n Figure 20.  Antisera against f_. amylophilus a-amylase isoenzyme 1 were found to be monospecific, indicating that i t was composed of a single antigenic component.  There was no p r e c i p i t a t i o n l i n e observed between B_. amylo-  philus a-amylase isoenzyme 1 and antisera to amylases of various origins (Figure 20).  Similarly the reaction between an antiserum to _. amylo-  philus a-amylase isoenzyme 1 and amylases of various origins (B. s u b t i l i s , A. oryzae and hog pancreas) was negative.  This experiment  demonstrated  that _. amylophilus a-amylase isoenzyme carried antigenic determinants which were d i s t i n c t from those present on the a-amylase of hog pancreas, _B. s u b t i l i s and A. oryzae. Antibodies were also successfully obtained against hog pancreatic a-amylase.  Although the amylases formed precipitates with their respec-  tive antisera, none of these reacted against each other, indicating that these proteins are a n t i g e n i c a l l y d i s t i n c t .  These results are i n agree-  ment with the findings of.Nomura and Wada (5). 3.  Immunoelectrophoretic  Analysis  Immunoelectrophoretic analysis, with antisera to _B. amylophilus a-amylase isoenzyme 1, revealed the presence of only a single antigenic component (Figure 21).  I t was also noted that _. s u b t i l i s a-amylase  moved to the cathode side and A. oryzae to the anode side, confirming the results of Wada (13).  117  Figure 20  A diagramatic representation of immunodiffusion p r e c i p i t a t i o n reaction between _. amylophilus a-amylase isoenzyme 1 and a n t i amylase and anti-amylase antiserum of various origins. Well 1 - _. amylophilus a-amylase i s o enzyme 1. Well 2 - A n t i - _ . amylophilus a-amylase isoenzyme 1 antiserum. Well 3 - Anti-B. s u b t i l i s a-amylase a n t i serum. Well 4 - Anti-A. aspergillus a-amylase antiserum. Well 5 - Anti-hog pancreatic a-amylase antiserum.  118  119  Figure 21  A diagramatic representation of Immunoelectrophoresis of 13. amylophilus a-amylase isoenzyme 1. Medium: 2 per cent agar i n pH 1=0.033 Veronal buffer. Antigen:  8.2,  5 mg/ml added i n central w e l l .  Electrophoresis was carried out at 5 mA per s l i d e f o r 2 hours. Bromophenol blue was used as tracking dye. After electrophoresis antiserum was added i n troughs 1 and 2. P r e c i p i t a t i o n l i n e between a-amylase. isoenzyme 1 and i t s antiserum was observed.  120  4-  I  121 4.  Quantitative P r e c i p i t a t i o n Analysis The p r e c i p i t a t i o n reaction was  studied quantitatively and a t y p i -  c a l curve with one equivalence point was presented i n Table XII and Figure 22.  obtained.  These results are  The molar r a t i o range of antibody  to antigen i n precipitates at equivalence and i n the antibody excess region were found to be between 1.8 and 2.31.  Other reported values re-  garding the molar r a t i o between 13. s u b t i l i s a-amylase and i t s antibody are 2.16 (10).  (7); 1.9  (12) and for Taka-amylase A 2.58  (8), 2.6, 2.8,  4.0  At the point of equivalence tests of the supernatant solution i n d i -  cated well defined zones of antibody excess, equivalence and antigen excess (Table XII).  The quantity of precipitate decreased i n the a n t i -  gen excess zone. During Immunoelectrophoresis  at pH 8.2 and agar gel d i f f u s i o n , a  single p r e c i p i t a t e l i n e formed between I3_. amylophilus a-amylase isoenzyme 1 and i t s antibody.  It i s suggested from the above reported results  that the a-amylase isoenzyme 1 preparation contained a single antigenic component. 5.  Effect of N -Bromosuccinimide and Urea on Antigenicity Treatment of a-amylase with N-bromosuccinimide completely des-  troyed - enzymatic a c t i v i t y (Table XIII).  This result was probably due to  the oxidation of tryptophan residues at the c a t a l y t i c s i t e of the enzyme molecule (6).  Immunochemical analysis was performed  to detect molecular  configuration differences between native and NBS-treated  enzyme.  The  quantitative p r e c i p i t a t i o n curve with the NBS-modified enzyme preparation was  found to be comparable to that given by native enzyme i n the area of  122 TABLE XII PRECIPITATION REACTION OF B. AMYLOPHILUS a-AMYLASE ISOENZYME 1 WITH ITS ANTIBODY  Antigen added ug  Total precipitate ug  Presence of Ag+AB i n supernatant Ag AB  Antibody pre- Molar r a t i o AB mole cipitate AG mole ug  50  470  -  +  420  2.31  70  631  -  +  561  2.20  90  720  -  +  630  1.90  110  821  -  -  711  1.80  150  780  +  -  200  690  +  -  300  500  +  -  400  280  +  -  +  -  500  To 0.5 ml of antibody was added 3.5 ml of t r i s maleate buffer pH 6.7 containing various proportions of a-amylase as indicated i n the table. The mixture was incubated for 1 hour at 37°C and 3 days at 5°C. The precipitates were washed with cold saline and the protein of the prec i p i t a t e was determined. a-Amylase a c t i v i t y was measured i n the supernatant. Molecular r a t i o was calculated from the following molecul a r weights: rabbit antibody (AB) 165,000 and a-amylase isoenzyme 1 (AG) 45,000.  123  Figure 22  P r e c i p i t a t i o n curve of a-amylase isoenzyme 1 with i t s antibody.  The condition of the  reaction as described i n Table XII.  125  TABLE XIII EFFECT OF N-BROMOSUCCINIMIDE ON ACTIVITY OF ct-AMYLASE ISOENZYME 1  Reagent  Treatment Molar r a t i o NBS/enzyme  Length of treatment minutes  Residual a c t i v i t y %  NBS  6  15  8  NBS  7  15  0  a-Amylase (1 mg/ml) i n 0.05 M sodium acetate buffer (pH 6.0) was treated with NBS.  The reaction was termi-  nated with Na2S0^ solution and enzymic a c t i v i t y was determined.  126 antibody excess and i n the equivalence zone (Figure 23). It i s suggested that c a t a l y t i c and antigen s i t e s are d i s t i n c t .  Similar are the results  with B. s u b t i l i s a-amylase (6), A. oryzae a-amylase (11) and bovine r i b o nuclease (1). Treatment of a-amylase with urea (8 M) completely inactivated the c a t a l y t i c a b i l i t y of the enzyme (Table XIV) .  Urea treated (8 M) ct-  amylase did not form any precipitates with the antibody.  Therefore both  enzymic a c t i v i t y and antigenic.characters were destroyed completely.  The  effect of urea i s due to an unfolding of protein molecules bringing about the loss of b i o l o g i c a l a c t i v i t y (9).  Comparable immunochemical r e -  sults have been reported with urea treated phage lysozyme (4).  It i s  interesting to note that both phage lysozyme (4) and .B. amylophilus aamylase isoenzyme 1 do not have disulphide linkage,  whether this com-  plete loss of antigenic character i s related to the absence of disulphide bonds i s not known (10).  It i s interesting that Aspergillus a-amylase,  which contains disulphide linkages, has been shown to r e t a i n part of i t s immunochemical r e a c t i v i t y following urea treatment (10).  The retention  of a certain proportion of i t s antigenic r e a c t i v i t y may be related to disulphide linkages which probably s t a b i l i z e certain sections of the enzyme molecule making i t resistant to urea denaturation (10).  6.  The Neutralisation of Amylase-Antiamylase System by Starch ' a-Amylase was incubated with antibody f o r 50 minutes at 37°C.  After t h i s , 5 per cent starch solution was added i n the incubation mixture and amylase a c t i v i t y was determined by the iodine reaction.  The  127  Figure 23  P r e c i p i t a t i o n curve of N-bromosuccinimide treated and native a-amylase with i t s a n t i body.  Reaction condition as reported i n  Table XII. x — x . NBS treated enzyme. Native enzyme.  1000  100  200  300  Antigen added (ug)  400  500  TABLE XIV EFFECT OF UREA ON. ACTIVITY OF a-AMYLASE ISOENZYME 1  Reagent  Treatment M  Length of treatment H  Residual activity  Urea  6  24  1.0  Urea  8  24  0  a-Amylase (1 mg/ml) was treated with urea at 37°C. After treatment the sample was dialysed against 0.02 M t r i s buffer (pH 6.7) f o r 24 hours and the enzymic a c t i v i t y was determined.  130 percentage of starch hydrolysis was decreased due to the presence of antibody (Figure 24, curve C), and the rate was constant at least.  f o r 150 minutes  In the next experiment enzyme was added to 5 per cent starch  solution and a f t e r 10 minutes of incubation at 37°C, the same amount of antibody was added.  As shown i n Figure 24 (curve B) the percentage of  starch hydrolysis was lower than the control curve A (Figure 24). As the starch was hydrolysed,  i t s effects as a protective agent  diminished,  and the i n h i b i t o r y effect of antibody appeared (Figure 24, curve B). Therefore,  i t appears that the products of starch digestion are not as  e f f e c t i v e as starch to neutralize the amylase-antiamylase system.  Wada  and Nomura (12), and Onoue et a l . (7) have also reported that starch did i n t e r f e r e with amylase-antiamylase system.  Antibody interference may be  due to s t e r i c hindrance or due to conformational molecule (2).  change i n the enzyme  In the present experiment i t i s possible that both factors  may be involved, as previous  experiments with NBS indicated that a n t i -  genic and c a t a l y t i c s i t e s of enzyme molecules are d i f f e r e n t .  131  Figure 24  Inhibitory effect of starch on a-amylase isoenzyme 1 and anti-amylase system. Curve A. 4.0 ml of 5 per cent starch + 3.0 ml of 0.02 M t r i s buffer pH 6.7 +1.0 ml of enzyme (8.0 u n i t s ) . Curve B. 4.0 ml of 5 per cent starch + 2.0 ml of t r i s buffer + 1.0 ml of enzyme (8.0 u n i t s ) . After incubation for 10 minutes at 37°C, 1.0 ml of 20 per cent diluted immune serum was added. Curve C. a-Amylase (8.0 units/ml) was incubated at 37°C with 1.0 ml of 20 per cent immune serum. After 50 minutes, substrate solution (4.0 ml of 5 per cent starch +2.0 ml of t r i s buffer) was added to the incubation mixture. ml of reaction mixture was removed at i n tervals and 5 ml of 0.1 N HCl was added to stop the reaction. The remaining starch was determined by measuring the blue colour at 660 mu•in iodine reaction.  50  100 Time (minutes)  150  REFERENCES IV:C  1.  Brown, Ray K. 1963. Immunological studies of bovine ribonuclease derivatives. Ann. N.Y. Acad. S c i . 103:754.  2.  Cinader, B. and K. J . L a f f e r t y . 1963. Antibody as i n h i b i t o r of ribonuclease: the role of s t e r i c hindrance, aggregate formation, and s p e c i f i c i t y . Ann. N.Y. Acad. S c i . 103:653.  3.  . 1967. "Antibodies to enzymes - a discussion of the mechanism of i n h i b i t i o n and a c t i v a t i o n . " In the proceedings of the 2nd meeting of the Federation of European Biochemical Societies, Vienna, A p r i l 21-24, 1965. Ed. by B. Cinader. Pergamon Press, Toronto.  4.  Merigan, Thomas C. and William J . Dreyer.. 1963. Studies on the antigenic combining s i t e s i n Bacteriophage lysozymes. Ann. N.Y. Acad. S c i . 103:765.  5.  Nomura, M. and T. Wada. 1958. Studies on amylase formation by Bacillus s u b t i l i s . V. Immunochemical studies of amylase produced by B a c i l l u s s u b t i l i s . J . Biochem. 45:629.  6.  Onoue, K., Y. Okada and Y. Yamamura. 1968. Modification of bact e r i a l a-amylase with N-Bromosuccinimide. J . Biochem. 51:443.  7.  , , S. Nakashima, K. Shimada and Y. Yamamura. 1963; Studies on enzyme-antienzyme system. I. Immunochemical studies on Bacillus s u b t i l i s a-amylase; J . Biochem. 53:472.  8.  Okada, Y., Y. Matsuoka, T. Yagura, T. Ikenka and Y. Yamamura. 1964. Immunochemical study of taka-amylase A and.phenylazobenzoyl taka-amylase A. J . Biochem. 55:446.  9.  Schachman, H. K. 1963. Considerations on the t e r t i a r y structure of protein., Cold Spring Harbour Symp. Quant. B i o l . , 28:409.  10.  11.  Sirishinha, S. and on a-amylase. enzymes on the oryzae.- Arch.  Peter Z. A l l e n . 1965. Immunochemical studies I. Effect of denaturing agents and p r o t e o l y t i c immunochemical r e a c t i v i t y of a-amylase from A. Biochem. Biophys. 112:137. 1965.  Immunochemical studies on a-amylase.  134 II. Examination of immunochemical and enzymatic a c t i v i t i e s of native and modified a-amylase from Aspergillus oryzae.• Arch. Biochem. Biophys. 112:149. 12. 13.  Wada, T. and M. Nomura. 1958. An immunochemical study of microbial amylase (1). J . Biochem. 45:639. ,  . 1959. J. Biochem.  An immunochemical study of microbial amylase I I . 46:329.  CHAPTER V CONCLUSIONS Bacteroides  amylophilus s t r a i n H-18  produces four isoenzymes of  a-amylase, as detected by disc electrophoresis and electrofocusing. e l e c t r i c points as determined by electrofocusing were pH 3.7, and 8.0.  4.5,  Iso5.9  Isoenzymes were named 1, 2, 3 and 4 with respect to their i n -  creasing i s o e l e c t r i c points.  a-Amylase isoenzyme 1 was  purified  and  some of i t s physico-chemical properties were examined and summarized i n a subsequent section. A. 1.  The optimum pH was  6.7  General Properties and exhibited a.narrow stable range of 6.2  to  7.6. 2.  Its optimum temperature was to 42°C.  Since optimum temperature was  range, i t was  possible that substrate  from heat, denaturation 3.  44°C and thermal s t a b i l i t y range was not within the s t a b i l i t y  (starch) protects the enzyme  during assay.  The thermal s t a b i l i t y of the enzyme was  affected, by EDTA treatment,  due to n o n - a v a i l a b i l i t y of chelated calcium. protected the enzyme from heat 4.  0  Treatment by  calcium  denaturation.•  Oxidizing agents but not reducing agents and SH-reagents inactivated the enzymic a c t i v i t y .  Enzyme was  susceptible to urea treatment.  5. ' Amino acid analysis indicated the absence of cysteine, therefore, disulphide linkages are not involved i n maintaining  the t e r t i a r y  136 structure. • Tryptophan appeared to be e s s e n t i a l for c a t a l y t i c activity. 6.  The estimated molecular weight was  7;  a-Amylase isoenzyme 1 was per mole.  45,000.  found to contain 3 gram  atoms of calcium  Various other metals tested could not replace the calcium  i n regenerating the maximum a c t i v i t y . 8.  The i s o e l e c t r i c point was  B.  1.  found to be pH  3.7.  Action Pattern  The products of enzymatic hydrolysis of starch and amylose were maltohexaose, maltoheptaose, maltoctaose, maltonanaose and maltodecaose at the achroic point and sometime after i t , as revealed by thin layer chromatography.  2.  Maltose was  the smallest disaccharide detected.  Since glucose  was  never found in-these experiments, i t appeared that a-amylase isoenzyme 1 does not hydrolyse maltotriose and maltose. 3.  The degree of multiple attack under the optimum conditions of temperature and pH was  2, as calculated by the r a t i o of the  value of the oligosaccharide f r a c t i o n to that of the  reducing  polysaccharide  fraction. C. 1.  Immunochemical Properties  Antisera against a-amylase isoenzyme l.was found to be  monospecific  and a small amount of residual a c t i v i t y remained i n the presence of excess of antibodies.  137 2.  The i n h i b i t o r y e f f e c t of starch on the amylase-antiamylase system was  3.  demonstrated.  The e f f e c t of anti-amylase (isoenzyme 1) globulin on amylase of various origins was studied by Ouchterlony double-diffusion, and the results indicated that antigenic determinants of a-amylase  isoenzyme  1 were d i s t i n c t from those present on a-amylase of hog pancreas, B a c i l l u s s u b t i l i s and Aspergillus oryzae. 4.  Immunoelectrophoretic analysis revealed the presence of only a single antigenic component.  5.  The p r e c i p i t a t i o n reaction was studied quantitatively and a t y p i c a l curve with one equivalence point was obtained.  6.  The molar r a t i o ranges of antibody to antigen i n precipitates at equivalence and i n the antibody excess zone were found to be between 1.8 and  7.  2.31.  N-Bromosuccinimide  treated a-amylase.(isoenzyme  a c t i v i t y , but exhibited comparable enzyme.  1) had no enzymic  immunochemical behaviour to native  It i s possible that antigenic and c a t a l y t i c s i t e s are d i s -  tinct. 8.  Urea treatment destroyed the a b i l i t y of the enzyme to p r e c i p i t a t e with i t s s p e c i f i c antibody.  

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