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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 British Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r equ i r emen t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Co lumb i a , I ag ree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S tudy . I f u r t h e r ag ree tha t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rposes may be g r an t ed by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date 5se^V,U-Chairman, Professor W. D. Kitts ABSTRACT The research was undertaken to study the extracellular a-amylase produced by the anaerobic rumen bacterium, Bacteroides  amylophilus strain H-18. Four active isoenzymes of a-amylase were detected by disc electrophoresis and electrofocusing tech-niques. Isoelectric 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 isoelectric points. a-Amylase isoenzyme 1 was purified by DEAE-Sephadex and G-200 techniques. Some of i t s general physio-chemical proper-ties were studied. It had maximum activity at pH 6.7, 44°C and was stabilized by calcium ion. It was susceptible to thermal denaturation in the absence of calcium. Various other metal ions tested could not replace the calcium in regenerating maximum activity. It 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 indica-ted the absence of cysteine, therefore, disulphide linkages were not involved in maintaining the. tertiary structure. Tryptophan appeared to be required for enzymic activity, 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 qualitatively by thin layer chromatography. The•maltohexaose, maltoheptaose, maltoctaose, maltonanaose and maltodecaose remained in the digest mixture for sometime after the achroic point. The degree of multiple attack was 2 , as calculated by determining the ratio of the reducing value of the oligosaccharide fraction to that of polysaccharide fraction. Antisera against a-amylase isoenzyme 1, produced in rabbits by injection of a-amylase inFreund's complete adjuvant was found to be mono-specific. The inhibition of a-amylase activity by antibody and inhibitory effect of starch on the amylase-anti-amylase system were'demonstrated. The effect of anti-amylase (isoenzyme 1) globulin on amylases of diverse origin was studied by the Ouchterlony double diffusion technique. These experiments demonstrated antigenic determinants which were distinct from those present on the a-amylase of hog pancreas, Bacillus subtilis and Aspergillus oryzae. Immunoelectrophoretic analysis indicated the presence of only a single antigenic component. Quantitative pre-cipitation studies gave a typical curve with one equivalence point with an antibody to antigen ratio of 2.'31. N-Bromosuccin-imide treated a-amylase (isoenzyme 1) exhibited similar immuno-chemical behaviour to the native enzyme, but completely lost i t s catalytic activity. It is possible that catalytic and antigenic sites were distinct. Urea treated a-amylase (isoenzyme 1) did not show any precipitate with i t s specific antibody and thus,.ap-peared to have lost i t s antigenic structure. Dedicated to my parents, teachers and friends who helped, each in their own way. i TABLE OF CONTENTS Chapter Page I. INTRODUCTION . . . . . . . . . . . 1. II. REVIEW OF THE LITERATURE . . . . . . 2 Amylase . . . . - 2 Nomenclature . . • . • 2 a-Amylases and their Sources . . . 3 Non-ruminant a-Amylase . . . • 3 Ruminant a-Amylase . . . . 4 Production and Induction of a-Amylase . . . . . . 4 Physical Properties of Crystalline a-Amylase 6 Primary Structure of a-Amylases . 6 Amino Acid Analysis 6 Functional Groups 6 Non-protein Constituents and Co-factors in a-Amylase . . . . 9 a-Amylase as Metalloenzyme . . . . . . . . . 9 Secondary and Tertiary Structure of a-Amylase . . . . . 11 Quarternary Structure of a-Amylase . . . . . . . . 13 The Action Pattern of a-Amylase . . . . . . . . . 1 5 Immunochemical Study of a-Amylase • . • . . . 18 Mammalian a-Amylase 18 Microbial a-Amylase . . . . 18 v i Chapter Page III. MATERIALS AND METHODS . . . . . . . . . . . . . . 31 Chemicals • . . . . 31 Organisms . . 3 1 Maintenance of Bacteroides amylophilus Strain H-18 . . . . • . . . : . . . . . . . . 32 Growth Measurements of Bacteroides amylophilus Strain H-18 . . . . 3 2 Production and Purification of a-Amylase from Bacteroides amylophilus Strain H-18 33 Production of a-Amylase . . . . . . . . • . . 33 Purification of a-Amylase on DEAE-Sephadex A-50 and G-200 Sephadex • 33 Assay of a-Amylase • . . 34 Assay of Protease 35 Determination of Nitrogen . • 35 Determination of Protein . . . 35 Determination of Total Carbohydrate 36 Disc Gel Electrophoresis 36 Isoelectrofocusing . . . . . . . . . . . . . . 37 Charcoal-Celite Column Chromatography . . . • . • . . . 37 Paper Chromatography . . . . . . . . . . . . . 38 Thin Layer Chromatography . . . . . 39 Effect of Temperature • . ... . . . . . 3 9 Molecular Weight Determination . . . . 3 9 Calcium Content Determination . . . . . . . . . . 39 • v i i Chapter Page Amino Acid Analysis . . • 40 Immunochemical Techniques . . 40 . Production of Antibodies 40 Determination of Enzymic Inhibition • . 41 -Immunodiffusion Characteristics 41 • Protein. Determination in Antigen-Antibody Complex . . . • . . . 4 1 IV. RESULTS AND DISCUSSION . . . . . . . . . . . . . 44 Characterization of a-Amylase from Bacteroides amylophilus Strain H-18 . . ' . . • . . . . . . . 4 4 Production and Purification of a-Amylase 44 Production of a-Amylase . . . 44 Purification of a-Amylase Isoenzyme 1 54 Catalytic Properties of a-Amylase Isoenzyme 1 . . . . . . . . . . . . . . 66 Determination of Type of Amylase . . . . . . . 66 Effect of pH on a-Amylase Activity 66 Effect of pH on Enzymic Stability 66 Effect of Temperature on a-Amylase Activity . . . . • . . . . - . . . . • . . 75 Effect of Temperature on Enzymic Stability . . . . . . . . . . . . . . 75 Amino Acid Determination . . . . 8 1 Calcium Determination . . . • . . . . . . . . 8 1 v i i i Chapter Page . Effect of Chemical Reagents on Enzymic Activity . . . . . . : . . 8 3 Effect of Urea on a-Amylase Activity 83 Effect of EDTA and Metallic Ions on a-Amylase Activity . 87 Functional Groups Determination . . . . . . . 9 0 Determination of Molecular Weight 93 Determination of Isoelectric Point . . ... . . 93 The Action Patternrof a-Amylase Isoenzyme 1 . . . - . . 101 Immunochemical Studies on a-Amylase Isoenzyme 1 113 Inhibition of Enzymic Activity by Antibodies . 113 . Ouchterlony Double Diffusion Analysis 116 Immunoelectrophoretic Analysis - . 116 Quantitative Precipitation Analysis 121 Effect of N-bromosuccinimide and Urea on Antigenicity 121 The Neutralization of Amylase-Antiamylase System by Starch . . . . . . . . - . . . ... 126 V. CONCLUSIONS . . . . . . . . . . . . . . . . .135 ix Chapter Page General Properties . . • . . . . . . . . . . . . 135 Action Pattern . . . . . . . . 136 Immunochemical Properties . . . . 136 LIST OF REFERENCES : . . . . . . . . Chapter I and Chapter II 22 Chapter III . 42 Chapter IV:A . . . 96 B • . . . . . . .111 C 133 LIST OF TABLES Table Page I. ' The Sources of a-Amylase . 5 II. Physical Properties of a-Amylase .• . . . 7 III. Purification of B_. amylophilus Strain H-18 a-Amylase Isoenzyme 1 58 IV. Summary of the Optimum pH Range of a-Amylase from Various Sources 71 V. Summary of the Optimum pH Stability Range for Various a-Amylases . . . . . . • 74 VI. Optimum Temperature for Various a-Amylases . . . . . . 78 VII. Calcium Contents of Various a-Amylases . . . . . . . . 82 VIII. Effect of Reducing, Oxidizing and SH-Inactivating Agents on a-Amylase Isoenzyme 1 Activity . . . . ; . . 91 IX. Molecular Weight of Various a-Amylases 94 X. Isoelectric Points of Various a-Amylases 95 XI. Estimation of the Degree of Multiple Attack by B^. amylophilus a-Amylase Isoenzyme 1 . . • 110 XII. Precipitation reaction of B_. amylophilus a-Amylase Isoenzyme 1 with i t s Antibody . . . . . . . . . . 122 XIII. Effect of N-bromosuccinimide on Antigenicity of a-Amylase Isoenzyme 1 . . . . . . . . . . . . . . 125 XIV. Effect of Urea on Antigenicity of a-Amylase. Isoenzyme 1 . . . . . . . . . . . . . . . . 129 LIST OF FIGURES Figure Page 1. Growth curve and production of a-amylase from Bacteroides amylophilus strain H-18 . . . • . . . . 4 5 2. Linear relationship between the production of a-amylase and growth of Bacteroides amylophilus strain H-18 . . . . . . . . - 47 3. Effect of maltodextrin on the growth and production of a-amylase.from Bacteroides amylophilus strain H-18 . . . . . . . . . . . . . 49 4. Detection of 4 isoenzymes of a-amylase by disc electrophoresis . . . : . . . . : . . . . . . 52 5. Detection of 4 isoenzymes of a-amylase by electrof ocusing . 55 6. Flow sheet of methods for isolation of a-amylase isoenzyme 1 from Bacteroides amylophilus strain H-18 57 7. Chromatography.of Bacteroides amylophilus strain H-18 a-amylase on DEAE-Sephadex A-50 ,. . . . . . . . 60 8. Chromatography of Bacteroides amylophilus strain H-18 a-amylase isoenzyme 1 on Sephadex G-200 . . . . . . . . . . . . . . . . 62 9. Disc electrophoresis of a-amylase isoenzyme 1 64 10. Electrof ocusing of a-amylase isoenzyme 1 . 67. 11. Optimum pH for hydrolyzing starch . . . . . . . . . . 6 9 12. Effect of pH on the s t a b i l i t y of a-amylase . - . . . . . . 72 13. Optimum temperature for hydrolysing starch . . . . . . . 76 x i i Figure Page 14. Thermal st a b i l i t y of a-amylase 79 15. Effect of urea on a-amylase activity . . . . . . . . . . 84 16. Effect of (A) EDTA and (B) metal ions after EDTA treatment.on reactivation of a-amylase 88 17. Thin layer analysis of the digestion of amylose by a-amylase isoenzyme 1.. . . . . . . . . . 102 18. Thin layer analysis of the digestion of starch by a-amylase isoenzyme 1 104 19. Neutralisation curve of a-amylase isoenzyme 1 with antiserum . . . 114 20. A diagramatic representation of immunodiffusion precipitation reaction between B_. amylophilus a-amylase isoenzyme 1 . . . . ... . 117 21. A diagramatic representation of Immunoelectro-phoresis of j$. amylophilus a-amylase • isoenzyme 1 . . . . . . . . 119 22. Precipitation curve of a-amylase isoenzyme 1 with i t s antibody . . ... . . • . . 123 23. Precipitation curve of N-bromosuccinimide treated and native a-amylase with i t s antibody 127 24. Inhibitory effect of starch on a-amylase isoenzyme 1 and antiamylase system . , . . 131 ACKNOWLEDGEMENTS The author wishes to express his sincere thanks to Professor W. D. Kitts, Chairman, Department of Animal Science, for his supervis-ion, indispensable guidance and helpful criticism in the completion of this study. The author would also like 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 Micro-biology, 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 in enzyme preparation, immunological studies and amino acid analysis. Thanks are due to a l l those in Canada and in Pakistan whose help and encouragement were of immense importance during the course of this study. Appreciation is also expressed to Miss V. Curylo and Mrs. J. A. Shelford for typing the manuscript. Acknowledged with thanks, is the National Research Council of Canada postgraduate scholarship and the University of British Columbia Fellowship. 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 of•cellulolytic organisms and the mechanism by which cellulose is de-graded. With the recent emphasis on higher grain feeding to ruminants i t has been important to study the breakdown.of starch in the rumen. Though several species of amylolytic bacteria have been isolated from the rumen and their incidence studied under a variety of dietary treat-ments (35), the amylolytic enzymes of these organisms have not been studied in detail. It is hoped that greater knowledge of the production and mode of action of a-amylase by rumen bacteria may•facilitate better understanding of starch hydrolysis in the rumen. This is particularly significant 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 in the rumen in greater proportions than starch digesters (35). Bacteriodes amylophilus strain H-18 is a predominant starch d i -gester constituting 10 per cent of the total rumen bacterial flora (35) and secreting an active a-amylase in specific laboratory growth madium (6). Since l i t t l e is known about the a-amylase produced by B_. amylophilus, this organism was selected for the present investigation. It may be men-tioned here that with the exception of members of genera Pseudombnas and Vibrio, most organisms producing exoenzymes are Gram positive (78). 13. amylophilus,,on the other hand, deviates from this general rule in being Gram negative. CHAPTER II REVIEW OF LITERATURE A. Amylases Starch is an important source of dietary carbon and therefore i t is not surprising to find amylases widely distributed in a l l Phyla. Amylase causes the hydrolysis of amylose, amylopectin, glycogen and their degraded products. In mammals the digestion of starch is initiated by the action of salivary amylase and continued in the duodenum by the action of amylase secreted by the pancreas and.the intestine. Microbial amylases are extra-cellular, in nature and several micro-organisms continue to produce extra-cellular amylase even after the fer-mentation of starch is completed. It might be expected that the amylase produced during the growth period of the organism hydrolyzes starch; and sugar thus produced is utilized for the growth of micro-organisms. The species of genus Bacillus (82) appear, however, to deviate from this general rule, viz. , IS. stearothermophilus (112) and B^. subtilis (82) which produce extra-cellular amylase even during the stationary phase. B. Nomenclature Amylases were classified as a and 3 types by Khun (43) and Ohlsson (70). The a and 8 amylases yield 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 is their mode of attack on the substrate. a-Amylases, being endoenzymes, cleave 1—>-4 bonds located in the inner region of the substrate. Therefore, a-amylases are expected to liberate products of varying chain lengths and also rapidly decrease the viscosity and iodine staining capacity of starch during enzymatic hydrolysis. B-Amylases have been regarded as exo-amylases because they do not rapidly decrease viscosity, and iodine staining of starch during starch hydrolysis. Since 8-amylase is an exo-enzyme the penultimate bond at a non-reducing chain end is the only bond available for enzymatic hydrolysis. g-Amylase attacks in an exclusive manner and produces 8-maltose only. Although the enzyme of 13. macerans produces cyclic schardinger dextrins from starch, i t is s t i l l classified as an amylase (27). The enzyme from 13. macerans, like 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 branch linkage of glycogen.and amylopectin, thus indicating an a-amylase action pattern. Amylases also differ in their action pattern on iodine-staining polysaccharides. It is represented graphically by plotting the change in blue value against the corresponding changes in reducing value during starch or amylose hydrolysis, and various amylases follow their own characteristic curves (44). C. a-Amylases and their Sources 1. Non-ruminant a-Amylase During the last twenty years a-amylases have been isolated, 4 purified and crystallized from a variety of sources. (Table I) 2, 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 Butyrivibrio fibrisolvens ( 3 5 ) . The amylolytic enzymes of rumen bacteria have not been character-ized except for those of Streptococcus bovis ( 1 1 0 ) and Clostridium  butyricum (32). 3. Production and Induction of a-Amylase The production characteristics of a-amylase of B^. subtilis and 13. stearothermophilus have been studied by many workers which have often seemed conflicting. J3. subtilis strain N produces extra-cellular a-amylase predominantly after maximum c e l l growth has occurred (66). The a-amylase of another strain of 13. subtilis ( 1 4 ) and of B_. stearothermo- philus ( 1 1 1 ) are formed during the logarithmic phase of growth parallel-ing the increase in c e l l mass. Yoshida and Tobita (115.) reported that a-amylase is released into the medium during the stationary phase of growth in a leucine requiring mutant of B_.' s u b t i l i s . . Pseudomonas saccharophila produces inducible extra-cellular a-amylase ( 4 9 ) . Markovitz and Klein ( 4 9 , 5 0 ) , Schiff et a l . ( 8 4 ) and 5 TABLE I THE SOURCES OF a-AMYLASES Source r- Reference A. Mammalian 1. Human.saliva 25 2. Porcine pancreas. 8, 57 3. Rat pancreas 30 4. Human pancreas 20 B. Plant 1. Barley malt 22, 87 2. Sorghum malt 16 C. Bacterial 1. Bacillus subtilis 96 2. Bacillus stearothermophilus 10, 11 3. Bacillus macerans 88, 79 4. Bacillus polymyxa 80, 83 5. Pseudomonas saccharophila 51 D. Fungus 1. Aspergillus oryzae 21, 104 2. Aspergillus niger 102 j 3. Aspergillus candidus 82 6 Eisenstadt and Klein.(18,19) have presented evidence for the de novo syn-thesis and inducibility of a-amylase in P_. saccharophila. The kinetics of enzyme formation was reported to be linear and the quantity of a-amylase 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 in a stimulation of the differential rate of a-amylase production. D. Physical Properties of Crystalline a-Amylase Some of the general physical properties of a-amylase are summarized in Table II. E.- Primary Structure of the a-Amylases 1. Amino Acid.Analysis Amino acid analyses have been reported for human salivary amylase (62), porcine pancreatic amylase (9), 13. subtilis amylase (3,42), 13. stearothermophilus amylase (12), and A. oryzae amylase (1,94). The a-amylases of J3. subtilis do not contain cysteine and cystine. _B. stearothermophilus a-amylase is 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 II PHYSICAL PROPERTIES OF a-AMYLASES Properties Source B.sub-t i l i s (54,56, 96) B.stear-othermo-philus (11,12) P.Sacch-aro-phila (51) A.ory-zae (21, 104) Barley malt (61,87) Porcine pan-creas (8,57, 61) Human saliva (25,56, 58,59, 61) Per cent nitrogen 16(24) 15 — 14.9(24) 13.0 15.9(24) 17.0 3ptimum pH 6.0 5.0 5.25-5.75 4.8-5.8 (24) 4.0-5.8 (4) 6.8 6.9 Optimum pH sta-b i l i t y range 4.8-8.5 4.5-8 5.5-8.5 4.9-9.1 7.0-8.5 4.8-11 Optimum temper-ature 40° 65° 40° 35° 37° 40° Molecu^-lar weight 48,700 (23) 15,000 (47) _ 51,000 (38) 59,500 45,000 (15) _ Isoelec-t r i c point 5.4 4.8 4.2 5.7 5.2-5.6 5.2-5.6 Absorbance % A 280 mu 25.3 (24) 19.7 (24) 26(24) 26(24) Activation energy (0-12°) 15,000 12° 11,000 14,000 (0-15°) 14,400 (15-40°) 8,500 10,650 7,050 13,500 13,500 8 two techniques have been,used; (a) the e f f e c t of pH on the'Michaelis constant, Km, and the maximum v e l o c i t y , Vm and (b) chemical modification of a-amylases.• E a r l i e r work using chemical modification of an enzyme has given c o n f l i c t i n g reports regarding the p a r t i c i p a t i n g of functional groups. At le a s t part of the reason f o r t h i s discrepancy i s the fac t that the chemical reagent used had l i t t l e s e l e c t i v i t y and i s related with many side chain groups. Ono et a l . (73) investigated the e f f e c t of pH on the Km of EL s u b t i l i s a-amylase.. Their r e s u l t s indicated that the apparent.rate con-stant, K^, of th i s enzyme diminished on both.the a l k a l i n e and acid side of the optimum pH. This was ascribed to the formation of an anion and a cation which were determined to PK value of 4.2 and 7.5. These PK values along with the heat of i o n i z a t i o n indicated that the active groups involved i n the cleavage of the bonds were a carboxylate ion and an imi-dazolium ion. The apparent Michaelis constant (Km) was stable over the pH range of 3.6 to 9.4, suggesting that the side chain.groups responsible for the substrate binding must ion i z e outside the pH range studied. The p o s s i b i l i t y that t y r o s y l groups may be involved was indicated because the PK value of the phenolic hydroxyl group does not f a l l i n the range studied. Thoma et a l . (100) reported that the c a t a l y t i c groups of porcine pancreatic a-amylase were l i k e l y to be,carboxylate and imidazolium ions. The binding 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 fact that the Km changed with pH, i n d i c a t i n g that at l e a s t two groups with PK values of 5.7 and 8.7 were responsible for substrate binding. 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 catalytic activity. They also reported that p-chloromercuribenzoate, iodoacetamide and mer-curic chloride did not deactivate the a-amylase. Other work showed that sulphydryl groups were not required for enzymic activity (7). Ikenaka (36) treated A. oryzae a-amylase with dinitrobenzene sul-phonate and fluoronitrobenzene and concluded from his results that the phenolic group of tyrosine was necessary for enzymic activity. 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 in a-Amylases  The small quantity of carbohydrates present in A. oryzae a-amylase are apparently not involved in the enzymic activity (2). 4. a-Amylase as a Metalloenzyme a-Amylases so far investigated contain at least one atom of cal-cium per mole (105) which is apparently required for enzymic activity (24,105). Since no other metals could be detected in significant amount, except zinc in jB. subtilis a-amylase, i t has been suggested that a l l a -amylases have certain sites to which calcium is attached specifically (105). 13. subtilis a-amylase is quite unique because of the presence of four atoms of calcium per mole of protein. It has been suggested that the increased amount of calcium is required to maintain structural r i g i d -ity because the S-S linkage i s absent in 13. subtilis a-amylase. 10 Yamamoto and Fukumoto (114) reported partial regeneration of ca l -cium depleted subtilis a-amylase by the treatment of strontium, mag-, nesium, barium and beryllium ions. Hsui et a l , (34) have suggested that the reagent used by Yamamoto and Fukumoto (114) was not spectroscopically pure, therefore reaction might be due to the contamination of calcium in the reagent. Calcium can be removed from a-amylases by dialysis against sodium ethylenediamine tetra-acetate, by ammonium sulphate or by electrodialysis (97). The treatment of enzyme by phosphate, oxalate and citrate failed 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 irreversible denaturation of the enzyme (9,93,105). The calcium free a-amylases were highly suscep-tible to denaturation by heat, urea and acid (93) and also were attacked easily by proteolytic enzymes (93). Stein et a l . (97) and Fisher and Stein (24) have reported that enzymic activity can be regenerated by the addition of calcium to cal-cium free a-amylases. However, enzymic activity could not be revived in A. oryzae ct-amylase (97). This was thought to be due to the low isoionic point of the enzyme (pH 4.2) as compared to other a-amylases (pH 5.2 to 5.4). In calcium free .B. subtilis and hog pancreas a-amylases i n s t a b i l -ity increased as pH increased (23). Fisher et a l . (23) reported that no major structural changes occurred in calcium depleted a-amylase. The exact role of calcium in the catalytic activity of a-amylases is not known, but i t is indicated that calcium ions function in a number 11 of ways; (a) i t keeps the a-amylase molecule in compact and proper confor-mation for biological activity by forming a tight intramolecular metal chelate structure, and (b) i t protects the native enzyme against extreme pH, heat, and proteolytic 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 is not essential for porcine pancreatic,a-amylase. The results of Muss (63) showed that 1-10 mM chloride gave maximum enzymic activity for salivary 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 inhibition of enzymic activity. Walker and Whelan (108) reported similar relationships between the a c t i -vity of human salivary a-amylase and chloride ion. F. Secondary and Tertiary Structure of a-Amylase The-secondary structure of a protein is believed to be due to the folding of the polypeptide chains into a specific coiled structure. The interrelationship and arrangement of the folded polypeptide chains into specific layers of crystals are called tertiary structures of the protein. It is understood that disulfide bonds, hydrogen bonds and hydrophobic bonds maintain the secondary and tertiary structures of proteins. Since IS. subtilis a-amylase does not have disulphide linkages' i t : is expected to have different secondary and tertiary structures and behave differently towards denaturing agents. Isemura and Imanishi (40) have 12 studied carefully the conformational changes in 13. subtilis a-amylase in alkaline and urea solution. Their finding was that approximately 30 per cent of a l l the phenolic hydroxyl groups ionize freely in alkaline pH up to 11.5. The remaining groups appear to ionize irreversibly at apH of 11.5 and therefore are likely to be buried in protein molecule. Also at a high alkaline pH, the tertiary structure appears to be disrupted irreversibly due to the dissociation of hydrogen bonds between carbox-ylate groups and phenolic hydroxyl groups. However, the enzymic• activity was regenerated by dialysis after the disruption of hydrogen bonds using 8 M urea. Manning et a l . (47) reported large negative optical rotation on 15. stearothermophilus a-amylase and this was not significantly affected by 8.0 M urea, 4.0 Mguanidine, or temperature as high as 75°C. No loss of enzymic activity occurred under these conditions. It was concluded that 15. stearothermophilus a-amylase is a well hydrated molecule and has a semi-random or random c o i l in the native state (47). It was also sug-gested that secondary and tertiary 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 optical rotation and spectro-photometric absorption with changes in enzymic activity indicated that the modification in enzyme structure and activity was due to the dissoc-iation of hydrogen bonds which became disrupted at pH 10.5 by irreversible ionization of phenolic hydroxyl groups of tyrosine. The activity 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 in 8.0 M urea and found that this caused the unfolding of the linear polypeptide con-taining nine sulphydryl groups. The denaturation was reversible when the enzyme was air-oxidized after the removal of urea and thioglycolate. This regenerated preparation of a-amylase had 50 per cent.of the orig-inal activity. Toda (101) studied the effect of proteolysis on A. oryzae a -amylase and reported that modified derivatives of a-amylase had a lower maximum velocity for the hydrolysis of amylose as compared to the native enzyme. He suggested that the active site of the enzyme remained un-changed and that there.was an overall change in the molecular configura-tion by the formation of new secondary and tertiary structure. G. 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 tertiary structures. The 13. subtilis a-amylase in it s native form shows the phenomena of monomer-dimer transformation. Vallee e_t a l . (105) and Stein (92) have reported that B_. subtilis can be changed from 6S to 4S in the presence of EDTA, and both 6S and 4S forms of the enzyme were homogeneous in the ultracen-trifugation (95). Stein and Fisher (97) reported that other cation-binding agents like citrate and oxalate produce heterogeneous sedimenta-tion patterns in I3_. subtilis a-amylase. ' The addition of zinc would 14 restore the dissociated amylase molecule into the homogeneous original form. It was concluded that IL subtilis a-amylase existed in dimer form, two units of monomer being crosslinked by an atom of zinc according to Equation 1. sequestering agent (Protein-Ca )-Zn-(Protein-Ca ) 2(Protein-Ca )+Zn [ l ] x x „ x z,n The above hypothesis was confirmed by treating the monomer form of enzyme with to obtain a dimer. There was a direct correlation between the release of zinc from ^~*Zn labelled 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 that other cations,such as Mn , Ni , I | | j | | _| |_ ^ |_ j | Co and Cu gave some dimerization, while Mg , Ca , Ba and Si had no effect. A higher degree of association than dimerization was accom-++ -9 plished when the concentration of Zn was increased to 2 X 10 M. Isemura and Kakiuchi (39) studied the effect of pH on the sedimen-tation velocity of B>. subtilis a-amylase and"showed that Svedberg S de-creased from 6.23 to 4.45 as the pH was changed from 6.5 to 5.0 indicat-ing the involvement of the imidazole group in the dimerization process. Isemura and Kakiuchi'(39) further explored the possible role of imidazole groups in dimerization process by comparing the.sedimentation pattern of the native and photo-oxidized B. subtilis a-amylase in the presence of methylene blue. The sedimentation co-efficient was 6.2 to 4.45 for native and photo-oxidized amylase respectively. 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. These results further supported the hypothesis that the imidazole groups of h i s t i d y l residue are involved i n monomer and dimer transformation of B_. subtilis a-amylase through the chelating of zinc ions. Stein and Fisher (95) reported that the pure crystalline a-amylases from A. oryzae, human saliva and hog pancreas are normally present in 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 in 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 saccharo-genic/dextrinogenic quotient, which gave similar values for different amylases (33). This hypothesis was cr 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 plot of blue value against reducing value 16 gave characteristic curves for different a-amylases. The difference was possibly due to different chain lengths produced by the enzymic hydroly-sis of amylose by a-amylases (44). Subsequent studies based on paper and column chromatographic techniques have revealed that a-amylases of different origins produced low molecular weight products with molecular size distribution characteristic of individual enzymes (17,76,79,110). Robyt and French (81) reported that pancreatic and human salivary a-amylase produced very similar end products from amylose. However, the curves relating drop in blue values to the corresponding increase in the reducing values were different. In the light-of these results these authors did not accept the explanation offered by Kung et a l , (44) re-garding differences between various amylase curves relating drop in blue values to corresponding increases in the reducing value. Bird and Hopkins (5) reported another aspect of action pattern in which dif 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 valid 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 glu-cose by a-amylases through different action, patterns. The linear portion of glycogen and amylopectin essentially follows the same fate as amylose to produce maltose and glucose. In the case of amylopectin, which is a branched polymer, the limit dextrin produced by the action of salivary 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 salivary a-amylase cannot cleave certain 1—>4 bonds in 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 in terms of single chain, multichain, and multiple attack. They suggested that porcine, pancreatic, human salivary and A. oryzae a-amylases follow multiple attack patterns during amylolysis 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 different types of starches have varying degrees of susceptibility to amylases. In addition they observed no correlation between granule size and the extent of. solubilization. Simi-lar differences have been reported by Walker and Hope (109) in the sus-ceptibility of starches of different origin to amylases. Their results also indicated that porcine pancreatic and human salivary 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 in the amylose content during the i n i t i a l period of degradation of starch granules, as measured by the potentiometric titration of bound iodine. It was 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 in starch granules is in agreement with the hypothesis of Ulmann (103). I. Immunochemical Study of a-Amylase 1. 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 different 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 for determining d i f -ferences and similarities among amylases of various species and also of' a given species. 2. • Microbial a-Amylase Wada (107) demonstrated that when crystalline Taka a-amylase was injected into rabbits the antibody was formed against the enzyme. He also studied the serological properties of the anti-sera produced and found only a single homogenous antibody,. but this antibody could not i n -hibit enzyme activity completely. He further observed that starch and starch hydrolysates inhibited the amylase-antiamylase reaction. It was found that anti-Taka-amylase antibody specifically inhibited the activity 19 of a-amylase from Aspergillus species. On the other hand, a-amylase activity 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 -hibit the reaction between Taka-a-amylase and i t s antibody. Nomura and Wada (67) obtained antibodies by injecting crystalline —' subtilis a-amylase into rabbits. Antiserum produced in rabbits by injection of crystalline amylase neutralized the enzymic activity to about 90 per cent. A competitive inhibition 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. subtilis 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 anti-bacterial a-amylase gave almost identical precipitation curves when treated with bacterial a-amylase and modified N-bromosuccinimide-bacterial a-amylase. In.addition, by.using the agar-gel immunodiffusion technique, they observed a single sharp precipitation line between N-bromosuccinimide-bacterial a-amylase and anti-bacterial a-amylase, and the precipitation line fused together with the. line between bacterial a-amylase and anti-bacterial 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 activity was due to oxidation of one tryptophan residue. These results indicated that the catalytic site of bacterial a-amylase might be different from that of the antigenic site. Onoue ej: a l . (75) prepared purified antibodies against 13. subtilis 20 a-amylase and the purified antibodies neutralized the a-amylase activity completely. The antibody in 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) in which they reported that 10 per cent enzymic activity remained after antibody treatment. Onoue et a l . (75) also demonstrated that the neutralizing a b i l i t y of papain treated anti-body was 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 interaction of amylase and starch by steric hindrance and therefore would be expected to decrease when the molecular size of antibody is reduced. Okada,et a l . (71) re-ported that photo-oxidized a-amylase of .13. subtilis did not form a pre-cipitate with I3_. subtilis a-amylase antibody. It was further demonstra-ted that ih the presence of calcium, photo-oxidized 13. subtilis 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 activity of both Taka-amylase A and p-phenylazobenzyl-Taka-amylase A was inhibited up to the same degree by anti-TakaTamylase A and by anti-p-phenylazobenzyl-Taka-amylase 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 activity of Taka-amylase A was partially inhibited by anti-Taka-amylase A and anti-p-phenylazo-benzyl-Taka-amylase A was ineffective to inhibit the enzyme activity. Moreover, the maltosidase activity of p-phenylazobenzyl-Taka-amylase A 21 was not neutralized by anti-Taka-amylase A or anti-p-phenylazobenzyl-Taka-amylase A. Since the neutralizing ab i l i t y of the antibody depends on the molecular size of the substrate (starch, phenyl maltoside) i t was sugges-ted that the antibody inhibited enzymic activity by steric hindrance (72). Sirisinha and Allen (90) used immunochemical methods to study the structure of Aspergillus a-amylase. Urea treated native enzyme under various conditions resulted in a preparation which gave a reaction partly identical with the non-treated enzyme during immunodiffusion analysis. Quantitative precipitation curves with urea treated enzyme preparation indicated that only a partial loss of immunochemical reactivity occurred even with prolonged treatment. The appearance of several bands of pre-cipitation with urea treated enzyme preparation suggested that various intermediate states exist between the fully unfolded structure of protein and the native protein (90). Immunochemical changes were also observed with enzyme preparation treated with EDTA alone or in combination with .1 M mercaptoethanol. Sirisinha and Allen (91) reported marked differences regarding im-munochemical behaviour between urea treated and oxidized a-amylase from A. oryzae. Although oxidized a-amylase would precipitate the same amount of antibody, the efficiency of oxidized enzyme decreased per unit weight. On the other hand, urea treated a-amylase would precipitate only a cer-tain portion of antibody from a specific antiserum. These authors also suggested that antigenic sites are not involved with the catalytic activ-ity and the decreased activity shown by enzyme antibody complex is due to the steric hindrance caused by attachment of antibody with respect to the catalytic center. REFERENCES I, . II 1. Akabori, S., T. Ikenda, H. Hanafusa and Y. Okada. 1954. Studies on taka-amylase A. II. Amino acid composition of taka-amylase A. J. Biochem. Tokyo. 41:803. 2. B. Maruo, M. 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M. , E.- A. Stein, H. Neurath and E. H. Stein. 1959. The amino acid composition of a-amylase from Bacillus s u b t i l i s . J. Biol. Chem. 234:556. 43. Kuhn, R. 1925. Der wirkungsmechanismus der amylasen; ein beitrag zum knofiguration sproblem der starke. Liebigs Ann. 443:1. 44. Kung., J. F. , V. M. Hanrahan and M. L. Caldwell. 1953. A compari-son of the action of several alpha amylases upon a linear fraction from corn starch. J. Amer. Chem. Soc. 75:4438. 45.. Leach, H. W. and T. J. Schoch. 1961. Structure of the starch granules. II. Action of various amylases on granular starches. Cereal Chem. 38:34. 46. L i t t l e , J. E. and H. L. Caldwell. 1942; A study of the action of pancreatic amylase. J. Biol. Chem. 142:585. 47. Manning, G. C., L. L. Campbell and R. J. Foster. 1961. Thermo-stable a-amylase of Bacillus stearothermophilus. J. Biol. Chem. 236:2958. 48. Mansour, T. G., E. Bueding and A. B. Stavitsky. 1954. The effect of a specific antiserum on the activities of lactic dehydro-genase of mammalian muscle and of Schistosoma mansoni. Brit. J. Pharmacol. 9:182. 49. Markovitz, A. and H. P. Klein. 1955. Some aspects of induced biosynthesis of a-amylase of P_. saccharophilia. J. Bacterol. 70:641. 50. . . 1955. On the study of carbon for the i n -duced biosynthesis of a-amylase in P_. saccharophilia. J. Bacterol. 70:649. 51. , and E. H. Fisher. 1956. Purification, crystals lization,.and properties of the a-amylase of Pseudomonas  saccharophila. Biochem. Biophys. Acta. 19:267. 26 52. McGeachin, R. L. and.J. M. Reynolds. 1959. Difference in mam-malian amylases demonstrated by enzyme inhibition with specific antisera. J. Biol. Chem.. 234:1456. 53. . 1968. Multiple molecular forms of amylase. Ann. N. Y. Acad. Sci. 151:208. 54. Menzi, R., A. Stein and E. H. Fisher. 1957. Proprietes de deux a-amylase de 13. s u b t i l i s . Sur les enzymes amylolytiques. Helv. Chim. Acta. 40:534. 55. Meyer, K. H. and P. Bernfeld. 1941. Recherches sur l'amidon XIV. La reaction coloree a l'iode de l'amidon et du glycogene. Helv. Chim. Acta. 24:389, 56. , M. Fuld and P. Bernfeld. 1947. Purification et c r i s t a l -lisation de 1'a-amylase de bacterie. Experentia. 3:411. 57. , E. H. Fisher and P. Bernfeld. 1947. Sur les enzymes amylolytiques (1). L'isolement de 1'a-amylase de pancreas'. Helv. Chim. Acta. 30:64. 58. , . 1948. Sur les enzymes amylolytiques. Isolement e t . c r i s t a l l i s a t i o n de 1'a-amylase de salive humaine. Helv. Chim. Acta. 31:2158. 59. , , A. Staub and P. Bernfeld. 1948. Proprietes de 1'a-amylase de salive humaine c r i s t a l l i s e e . Helv. Chim. Acta. 31:2165. 60. and W. F. Gonon. 1951. La degradation de l'amylose par les a-amylases. Helv. Chim. Acta. 34:294. 61. . 1952; The past and present of starch chemistry. Experentia. 8:405. 62. Muus,.J. 1954. The amino acid composition of human salivary amylase. J. Amer. Chem. Soc. 76:5163. 63. , F. P. Brockett and C. C. Connelly. 1956. The effect of various ions on the stability of crystalline salivary amylase in solution.-Arch. Biophys. 65:268. 64. - Myrback, K. 1926. Uber verbindungen einiger enzyme mit inak-. tiverenden stoffen. II. J. Physiol. Chem. 159:1. 65. Nisselbaum, J. A. and 0. Bodansky. 1960. Reaction of human tissue la c t i c dehydrogenases with antisera to human heart and liver l a c t i c dehydrogenases. J. Biol. Chem. 236:401. 27 66. Nomura, M. , J.- -Hosoda and H. Yoshikawa. 1958. Studies on amylase - formation by Bacillus, s u b t i l i s . VI .• The mechanism of amylase excretion arid cellular structure of Bacillus s u b t i l i s . J. Biochem. Tokyo. 45:737. 67. , 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 Bacillus s u b t i l i s . J. Biochem. 45:629. 68. Nordin, P. and D. French. 1958. I. Phenyl-flavazole deriva-tives of starch dextrins. J. Amer. Chem. Soc;. 80:1445. 69. and Y. S. Kim. 1960. The reaction of.amylases with starch granules. J. Amer. Chem. Soc. 82:4604. 70. Ohlsson, E. 1930. Uber die beiden komponenten der malzdiastase, besonders mit rucksicht auf die mutarotation der bei der hydro-lyse der starke gebildeten product. J. Physiol. Chem. 189:17. 71. Okada, Y., S. Nakashima and Y.-Yamamura. 1963. Relationship be-tween immunological memory and structure of bacterial a-amylase. J. Biochem. 54:99. 72. , Y. Matsuoka, T. Yagura, T. Kenda and Y. Yamamura. 1964. Immunochemical study of taka-amylase A and Phenylazobenzoyl taka-amylase A. J. Biochem. 55:446. 73. Ono, S.j.K. Hiromi and Y. Yoshikawa. 1958. Kinetics of hydro-l y t i c reaction catalyzed by crystalline bacterial a-amylase. Bull. Chem. Soc. Japan. 31:957. 74. Onoue, K., Y. Okada and Y. Yamamura. 1962. Modification of bac-t e r i a l a-amylase with N-brobosuccinimide. J. Biochem. 51:443. 75. , , S. Nakashima, K. Shimada and Y. Yamamura. 1963. Studies on enzyme-antienzyme .system. I. Immunochemical studies on Bacillus subtilis a-amylase. J. Biochem. 53:472. 76. Pazur, J. H., D. French and D. Knapp. 1950. Mechanisms of s a l i -vary amylase action. Proc. Iowa Acad. Sci. 57:203. 77. Pollock, M. R. 1962. "Exoenzyme." In.the Bacteria. Ed. by I. C. Gunsalus and R. Y. Stanier. Vol. 4. Acad. Press. New York and London. 78. Roberts, P. J. -P., and W. J.. Whelan. 1960. The mechanism of carbohydrase action.. V. Action of human salivary a-amylase on amylopectin and glycogen. Biochem. J. 76:246. 28 79. Robyt, J. F. and D..French. 1963. Action pattern and specificity of an amylase from Bacillus s u b t i l i s . Arch. Biochem. Biophys. 100:451. 80. • 1964. Purification and action pattern of an amylase from Bacillus polymyxa. Arch. Biochem. Biophys. 104:338; 81. . 1967. Multiple attack hypothesis of c t-amylase action: action of porcine, pancreatic, human salivary and Aspergillus oryzae a-amylase. Arch. Biochem. Biophys. 122:8. 82. . and J. W. Whealn. 1968. "The a-amylase." In starch and ists derivatives. Ed. by J. A. Radley. Fourth Edition. Chapman and Hall Ltd., 11 New Fetter Lane, London EC4. 83. Rose, D. 1948. The amylase of Bacillus polymyxa. Arch. Biochem. 16:349. 84. Schiff, J. A., J. M. Eisenstadt and H. P. Klein. 1959. a-amylase formation in growing and non-growing cells of P_, saccharophila. J. Bacteriol. 78:124. 85. Schlamowitz, M. and 0. Bodansky. 1959. Tissue sources of human serum alkaline phosphatase as determined by immunochemical procedures. J. Biol. Chem. 234:1433. 86. . 1954. Specificity of dog intestinal phosphatase anti-serum. J. Biol. Chem. 206:369. 87. Schwimmer, S. and A. K. Balls. 1949. Isolation and properties of crystalline a-amylase from germinated barley. J. Biol. Chem. 179:1063. 88. . and J. A. Garibaldi. 1952. Further studies on the pro-duction, .purification and properties of ..the Scharadinger dex-trinogenase of macerans. Cereal Chem. 29:108. 89. . 1953. Evidence for the purity of Schardinger dextrin-ogeriase. Arch. Biochem. Biophys. 43:108. 90. Sirisinha, S. and P. Z. Allen. 1965. Immunochemical studies on a-amylase. I.. Effect of denaturing agents and.proteolytic enzymes on the immunochemical reactivity of a-amylase from Aspergillus oryzae. Arch. Biochem. Biophys. 112:137. 91. . 1965. Immunochemical studies on a-amylase. II. Examination of immunochemical and enzymic activities of native and modified a-amylase from Aspergillus oryzae. Arch. Biochem. Biophys. 112:149. 29 92. Stein, E. A. 1957. Structure of s u b t i l i s a-amylase. Federa-t i o n Proc. 16:254. 93. and.E. H. Fischer. 1958. The resistance of a-amylase to-wards p r o t e o l y t i c attack. J . B i o l . Chem. 232:867. 94. , J . M. Junge and E. H. Fisher. 1960. The amino acid com-p o s i t i o n of a-amylase from A s p e r g i l l u s oryzae. J . B i o l . Chem. 235:371. 95. and E..H. Fischer. 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. Acta, 39:287. 96. ' . 1961. a-Amylase from B a c i l l u s s u b t i l i s . Biochemical Preparation. 8:34. 97. , J . Hsui and E. H. Fisher. 1964. Alpha amylase as c a l -cium-metalloenzymes. I. Preparation of calcium-free apoamy-lases by chelation and e l e c t r o d i a l y s i s . Biochemistry. 3:56. 98. Takagi, T. and H. Toda. 1960. Studies on the amphoteric proper-t i e s of taka-amylase A. I. Ionization of phenolyic hydroxyl groups. J . Biochem. Tokyo.8:781. 99. . 1962. Studies on the denaturation of taka-amylase A and on i t s r e v e r s i b i l i t y . J . Biochem. Tokyo. 52:16. 100. Thoma, J. A., J . Wakim and L. Stewart. 1963. Comparison of the ac t i v e s i t e s of alpha and beta amylase. Biochem. Biophys. Res. Comm. 12:350. 101. Toda, H. 1963. Enzymatic modification of phenylazobenzoyl-taka amylase A. J . Biochem. Tokyo. 53:425. 102. Tsuchiya, H. M. , J-. Corman and H. J . Koepsell. 1950. Production of mold amylases i n submerged culture. I I . Factors a f f e c t i n g the production of alpha-amylase and maltase by c e r t a i n A s p e r g i l l i . Cereal Chem. 27:322. 103. Ulmann, M. 1957. Bestimmung der chemischen natur der h i l l l e cines gerguollenen starkekornes. K o l l o i d . Z. 150:128. 104. 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 dextrinase. Cereal Chem. 28:18. 105. V a l l e e , B. L., E. A. Stein, W. N. Summerwell and.E. H. Fischer. 1959. Metal content of a-amylases of various o r i g i n s . J . B i o l . Chem. 234:2901. 30 106. Wada, T. and M. Nomura. 1958. An immunochemical study of micro-b i a l amylase (1). J. Biochem. 45:639. 107. . . 1959. An immunochemical study of microbial amylase (11) J. Biochem. 46:239. 108. Walker, G. J. and W. J. Whelan. 1960. The mechanism of carbo-hydrase action. VII. Stages in the salivary a-amylosis of amylose, amylopectin and glycogen. Biochem. J.. 76:257. 109. and P. M. Hope. 1963. The action of some a-amylases on starch granules. Biochem. J. 86:452. 110. -. - •;• 1965. The c e l l bound a-amylase of Streptococcus bovis. Biochem. J. 94:289. 111. Welker, N. E. and L. L. Campbell. 1963. Effect of carbon source on formation of a-amylase by Bacillus stearothermophilus. J. Bact; 86:681. 112. . 1963. Induction of a-amylase of Bacillus 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 bacterial a-amylase reactivated with various alkaline earth metals. Bull. 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 precur-sor. Biochem. Biophys. Acta. 37:513. CHAPTER III MATERIALS AND METHODS A. Chemicals The sources from which the substrates and chemicals were ob-tained are as follows: starch (British Drug House, Poole, England), amylose (Stein-Hall and Co., New York, U.S.A.), maltose, technical and reagent grade (Fisher Scientific Co., New Jersey, U.S.A.), bovine serum albumin (Calbiochem, Los Angeles, California, U.S.A.), casein hydroly-sate, 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 Allie d Chemical Company Canada, Ltd., Vancouver, B.C., and Fisher Scientific Co. Ltd., Vancouver, B.C. DEAE Sephadex A-50 and Sephadex G-200 were purchased from Pharmacia, Uppsala, Sweden. B. Organism The organism used in this investigation was Bacteroides amylo- philus strain H-18, kindly supplied by Dr. T. H. Blackburn, Department of Microbiology, University of British Columbia, Vancouver 8, B.C., Canada. Blackburn and Hobson (2) isolated this strain from the rumen of sheep. 32 C. Maintenance of Bacteroides amylophilus Strain H-18 The complete chemically defined basal medium used during this investigation was that developed by Hungate (8). This medium contained (g/1): K2HP04, 0.45; KH2P04, 0.45; (NH^SO^ 0.9; NaCl, 0.9; HgS04, 0.09; CaCl^, 0.09; resazurin, .001; L-cysteine hydrochloride, 0.5. The resazurin and mineral solutions, or any additions to the medium were placed in a screw capped bottle 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 fifteen minutes at 120°C and on removal of the bottle from the autoclave the cap was immediately screwed tight. Fifty ml. of 1 per cent (w/v) L-cysteine hydrochloride and f i f t y ml. of 10 per cent (w/v) sodium bicarbonate sol-ution were steam autoclaved separately at 100°C for fifteen minutes and then added to the remaining medium under a stream of CO2. The f i n a l pH of the medium was 6.7. A l l the dispensing of the medium and incubation of the culture was done under oxygen-free CO^ as described by Blackburn (3). Stock cultures were maintained on nutrient agar slopes which contained in addition to the basal medium 2 per cent agar and 0.5 per cent each of maltose and casein. The cultures were stored at 4°C and transfers were made each week to fresh slopes by stab innoculation under an atmosphere of CO^. A l l cultures were grown at 38°C. D. Growth Measurements of Bacteroides  amylophilus Strain H-18 The growth of _. amylophilus was measured in a Bausch and Lomb 33 Spectronic 20 Colorimeter (Bausch and Lomb, Rochester, New York, U.S.A.) at 660 nm. E. Production and Purification of a-Amylase from Bacteroides amylophilus Strain H-18 The amylase was a by-product of protease purification undertaken by Lesk (9) who kindly denoted the fraction 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 steel milk can and inoculated with 1 l i t r e of log phase culture of .B. amylophilus. After anaerobic incubation for 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 Servall Centrifuge (Servall type SS-34, equipped with a KSB:R Servall continuous flow adopter from Servall, Norwalk, Connecticut). The super-natant had a pH of 5.5 which was the optimum for the attachment to DEAE Sephadex A-50 for purification. 2. Purification 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 for twelve hours at 4°C to ensure proper mixing. The DEAE suspension was allowed to settle; the supernatant decanted and the DEAE collected on a sintered glass f i l t e r . The DEAE was mixed thoroughly in 500 ml. of 1 M 34 NaCl and centrifuged. The supernatant was then decanted and stored. This procedure was repeated six times. The f i r s t five 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 linear gradient of 0.2 M to 1.0 M NaCl in phosphate buffer (pH 7.0). The fractions were tested for a-amylase activity and three fractions having enzymic activity were collected. Volumes of 538 ml, ,500 ml and 840 ml were collected for fraction I, II, and III respectively. Each fraction was dialysed against 0.05 M phos-phate buffer (pH 7.0) overnight at 4°C. Fraction III was concentrated to 40 ml with a Diaflo U l t r a - f i l t r a t i o n Cell (Diaflo Model 50 Ultra-f i l t r a t i o n Cell from Amicon Co., Lexington, Mass., U.S.A.) equipped with a Pm - 10 f i l t e r (exclusion limit;10,000 MW) under a pressure of 40 p.s.i. The concentrated a-amylase fraction III was further purified 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 activity was obtained in a single peak and the enzyme solution was concentrated by pressure dialysis to 45.0 ml. F. Assay of a-Amylase • The a-amylase activity in the sample was assayed by determining the amount of reducing sugars produced 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 in an equal volume of 2 per cent (w/v) soluble starch or amylose buffered to pH 6.7 with 0.2 M Tris and 0.1 M maleate. Unless otherwise indicated the time of incubation was fifteen minutes at 44°C. When i t was desired to calculate the degree of multiple attack, the a-amylase activity was determined by the Nelson Copper method (16). A unit of a-amylase activity was defined as the amount of enzyme that would produce the equivalent of 1.0 mg. maltose in 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 activity 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 in 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 Miller (13). K. Disc Gel Electrophoresis Disc gel electrophoresis apparatus was constructed by Mr. R. J. Hudson and Mr. James A. Shelford, i n the Department of Animal Science Laboratory, University of British Columbia, following the procedure of Davis (4). Disc electrophoresis chemical r e f i l l pack containing standard 7 per cent acrylamide gel 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 in a standard gel (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. The enzyme samples were run in duplicate. After gel electrophoresis for two hours (5 mA per column) one gel was stained immediately for protein with Amido black 10B. The second gel.was used to detect a-amylase activity by lay-ering the gel.on starch coated glass slides. 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 in 2 M TRIS-Maleate buffer at pH 6.7. After being superimposed with the gel, the slides were placed in petri dishes and.incubated at 40°C for 15 minutes. After the incubation the gels were removed and the slides dipped momentarily in Lugol's iodine solution to 37 stain the unhydrolysed starch. The clear band (S) indicating hydrolysis of starch was visible against the blue stain produced by the starch. L. Isoelectrofocusing The LKB fraction collector with Uvicord (0.3 on light 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 in 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, capacity 440 ml. Low molecular weight ampholines in the range pH 3.0 to 10.0 (4 per.cent).were used. M. Charcoal-Celite Column Chromatography The technique was essentially the same as described by Whistler and Duro (22) using a charcoal-celite column to isolate and detect oligo-saccharides found in technical grade Maltose. A 50 per cent solution of the sugar was autoclaved for 20 minutes at 40 lbs. (p.s.i.) and filtered to remove the precipitate. A chromatographic column (4.5 x 50 cm.) was f i l l e d to a height of 40 cm. with charcoal-celite mixture. The column was washed with 1.5 l i t e r s of 0.1 N HCl to remove basic ash; acid was removed by washing ex-haustively with d i s t i l l e d water. 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 din i t r o s a l i c y l i c acid method. Qualitative detection of various sugars and oligosaccharides was done by paper chromatography. The water frac-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 - , tetra- and penta-saccharides 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 lids (Research Specialties Co., New Jersey, U.S.A.). This technique was essentially 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 sufficient to separate the f i r s t seven members of homologous series of malto-oligosac-charide. The spots on the paper chromatograms were detected by spraying with aniline phosphate reagent (6) and.heating at 115°C for 20 minutes. These spots were used as markers for sectioning the remaining portions of the chromatograms. The individual sugars were extracted from the paper and concentrated in vacuo. They were further dried with acetone ' and washed with a small amount of n- butanol. The syrup was dissolved 39 in 50 ml. of water and freeze-dried. The freeze-dried fractions were stored in 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 for the separation of oligosaccharides by TLC was developed (20) using the solvent systems, isopropanol-t^O-ethyl-acetate (2:1:2 v/v) or 1-propanol-nitromethane-water (5:2:3) as reported by Huber ___1. (7). P. Effect of Temperature The effect of temperature on a-amylase activity under various treatment was studied in 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 accord-ing 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 in the SP 90 spectrophotometer manual. S. Amino Acid Analysis The amino acid analysis of the purified a-amylase was done on a Phoenix Amino Acid Micro-Analyser (Model M-7800). The enzyme preparation was f i r s t dialysed against deionized water for 48 hours at 5°C. Hydrol-ysis was performed in hydrolysis tubes containing 1.0 mg.of protein in 1.0 ml. of deionized water and 1.0 ml. of concentrated HCl. The hydrol-ysis tubes were put in an oven set at 110°± 1°C for a period of 24 hours. Hydrochloric acid was removed by repeated evaporation under reduced pres-sure by a rotary evaporator. The residue was dissolved in 1.0 ml. of sodium citrate buffer pH 2.2 and 0.95 ml. was f i n a l l y applied to the column for amino acid analysis. A Piez-Morris (19) accelerated buffer system was used for elution 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 in rabbits. Each of the four rabbits received, by subcutaneous injection over a course of three weeks, a total of 4.0 mg. of protein in complete Freund's adjuvant. 41 The animals were bled two weeks after the last injection to provide immune serum. The-antisera was inactivated by heating and i t was stored in frozen state (15). 2. Determination of Enzymic Inhibition The determination of the percentage of the inhibited activity of a-amylase was done according to the method of McGeachin and Reynolds (12). Homologous normal rabbit serum was included as a control in a l l experiments. Control with homologous normal rabbit serum showed no i n -hibition of a-amylase activity. The 3 to 5 dinitro s a l i c y l i c acid method was used to measure the a-amylase activity. 3. Immunodiffusion Characteristics Antigenic relationships were studied by Immunoelectrophoresis and double diffusion in agar gel (17). 4. Protein Determination in Antigen-Antibody Complex Protein of antigen-antibody precipitate was determined according to the method of Lowry et a l . , using bovine albumin as standard (11). REFERENCES III 1. Andrews, P. 1965. The gel f i l t r a t i o n behaviour of proteins re-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 isolation of proteolytic 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 II. Method and applica-tion to human serum proteins. Ann. N.Y. Acad. Sci. 121:404. 5. Fisher^ E.- and E. A. Stein. 1961. a-Amylase from human saliva. Biochem. Preparation. 8:27. 6. Frahn, J. L. and J. A. Mills. 1959. Paper ionophoresis of carbo-nydrates. I. Procedures, and results for four electrolytes. 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 megalo-saccharides with mixed support and multiple irrigation. 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. Purification and characterization of Proteolytic enzymes from _. amylophilus Strain H-18. M.Sc- Thesis. Univer-sity of British Columbia, Vancouver, B.C. 10. Jeanes, A., C. W. Wise and R. J. Dimlee. 1951. Improved techniques in 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. Biol. Chem. 193:265. 43 12. McGeachin, R. J. and J. M. Reynolds. 1959. Differences i n mammal-ian amylases demonstrated by enzyme inhibition with specific antisera. J. Biol. Chem. 234:1456. 13. Miller, G. L. 1960. Micro-column chromatographic method for anal-ysis 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 Bacillus-subtilis. V. Immunochemical studies of amylase pro-duced by Bacillus-subtilis. J. Biochem. 45:629. 16. Nelson, N, 1944. A photometric adaptation of the Somogyi method for determination of glucose. J. Biol. Chem. 153:375. 17. Ouchterlony, 0. 1968. "The techniques of double diffusion in two dimensions, and Immunoelectrophoresis." In Handbook of Immunodiffusion and Immunoelectrophoresis. Ann. Arbor Science Publishers, Michigan, 48106. 18. Pazur, J. and D. French. 1952. The action of transglucosidase of Aspergillus oryzae onmaltose. J. Biol. 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. Kitts. 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 Hall Ltd. , London, EC4. 22. whistler, R. L. and D. F. Durso. 1950; Chromatographic separation of sugars on charcoal. J. Amer. Chem. Soc. 72:677. \ CHAPTER IV RESULTS AND DISCUSSION A. Characterization of a-Amylase from Bac- teroides amylophilus Strain H-18 1. Production a'nd Purification of a-Amylase a. Production of a-Amylase Figure 1 shows that a-amylase was produced extracellularly during the logarithmic, and stationary phase of growth by 13. amylophilus. These results are in agreement with the findings of Lesk (20). In this regard ]3. amylophilus is similar to .B. stearothermophilus which starts producing a-amylase during the logarithmic.period of growth (51) , but.it is di f f e r -ent from B. subtilis which produces a-amylase during the stationary phase of growth (36). The growth curve (Figure 1) of 13. amylophilus is characteristic 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 mid-logarithmic growth phase the' amount of a-amylase liberated was linear (Figure 2). Since a-amylase did not contain cysteine (see IV:A:2:f) and its production began during the logarithmic phase.of growth, i t has the characteristic features of other extracellular enzymes (41). The effect of maltodextrins on a-amylase formation is shown in 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 activity released into the culture Supernatant. O - - O Percentage of maximum a-Amylase activity in culture Supernatant. a-Amylase activity in Supernatant (units/ml) to o to 46 LO O LO Ul Ul Ul o c t-i to O to Ui LO O Lo Ul to O O ON o 00 o Percentage of Maximum Activity (32.5 units/ml) J I J o o — J 4> 00 O.D,660 47 Figure 2 Linear relationship between the production of a-Amylase and growth of Bacteroides  amylophilus Strain H-18. a -Amylase (units/ml) t-1 1—1 NJ NJ Ul O Ul O Ul O 49 Figure 3 Effect of Maltodextrin on the growth and production of a-Amylase from Bacteroides  amylophilus Strain H-18. Maltose o—o Maltotriose A - — A Maltotetraose O—O Maltopentaose a-Amylase (units/ml) 50 had just entered the logarithmic phas.e, there was an increase in the production of amylase. However when maltodextrins were replaced by glu-cose, sucrose, and cellobiose, there was no change in the amount of amylase produced. These results, are essentially in agreement with the findings of Blackburn (2) and Hungate (19) in which the a 1-4 linked glucose polymers were the only carbohydrate substrates metabolized by B_. amylophilus. Under the experimental conditions used I5_. amylophilus cells appeared to be permeable to maltose and maltotriose. After incubation for 6 hours with maltodextrins, maltose was the only sugar detected extra-cellular ly by thin layer chromatography, and glucose was never found. It was not investigated to find i f I3_. amylophilus takes up other malto-oligosaccharides directly like Micrococcus Sp 40 (53) or hydrolyses them to maltose or maltotriose. These results indicate that 1$. amylophilus cells are permeable to maltose and maltotriose and therefore the maltose and maltotriose up-take systems in this micro-organism are constitutive like Micrococcus Sp 40 (53) but unlike that of E_. cold (52) which is adaptive. The results in 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 iso-enzymes of a-amylase (Figure 4) accompanied by 12 other bands of protein were detected by disc electrophoresis. Four peaks of activity were also 52 Figure 4 Detection of 4 Isoenzyme of a-Amylase by disc electrophoresis. Illustration A. The- separation of 4 iso-enzyme of a-Amylase by disc electrophoresis on acrylamide gel. The direction of migra-tion was from the top of the figure. Illustration B. The starch slide after i n -cubation at 38°C for 15 minutes with acry-lamide gel. After incubation the unhydrolysed starch was stained with Lugol's iodine. The clear bands indicating hydrolysis of starch was visible against the blue stain produced by the starch and iodine complex. Stained acrylamide gel A Amylase B Amylase D Amylase A Amylase C Stained slide with 1% starch B 54 detected by iso-electrofocusing (Figure 5). Isoelectric points as de-termined by electrofocusing were pH 3.7, 4.5, 5.9 and 8.0. The iso-enzymes were named 1, 2, 3 and 4 with respect to their increasing iso-electric points (Figure 5). The reasons why this organism produces four a-amylases is not . clear at the present time, but possibilities may be suggested. Since they have different isoelectric points they should have different amino acid compositions. It has been reported recently that different a-, amylases have different a f f i n i t i e s towards various starches (5,28). Evidently .B. amylophilus is a very versatile organism and may control secretion of different isoenzymes depending upon the nature of starch in the diet of the animal. b. Purification of a-Amylase Isoenzyme 1 The methods used for purification of a-amylase isoenzyme 1 are summarized in Figure 6 and the results are presented in Table III. The stepwise purification process was conducted as follows: Step 1 - A good quantity of a-amylase was obtained by growing the culture for 23 hours. 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 concentrated by DEAE-Sephadex batch operation. This step reduced the volume of the enzyme sol-ution from 29000 ml to 2540 ml, and gave a 5 fold purifica-tion. 55 Figure 5 Detection of 4 isoenzymes of a-Amylase (x—x) by electrofocusing with superimposed pH curve (.—.). The pi values of separated components are obtained by taking the pH of the corres-ponding fraction at the maximum activity. The pi value of the components were 3.7, 4.5, 5.9 and 8.2. The isoenzymes were named 1, 2, 3 and 4 with respect to their increasing iso-electric points. The figures above the enzyme activity peaks give the pi values of a-Amylase isoenzymes. Relative activity (%) 56 < 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 slurry with 1.0 M NaCl on a Buchner ;funnel 2540.0 ml Dialysis overnight•against 0.05 M phosphate buffer, pH 7.0 2600.0 ml DEAE-Sephadex column chromatography. Enzyme was eluted with a linear gradient of 0.2 M to 1.0 M NaCl in 0.05 M phosphate buffer, pH 7.0 Fraction I 27-56 (Fig. 7)"* 538.0 ml Fraction II 57-84 (Fig.7)* 500.0 ml This indicates Fraction number in Figure VII . Fraction III 85-131 (Fig.7)* a-amylase Isoenzyme 1. Total volume collected, 840.0 ml. It was re-duced by pressure dia-lysis to 40 ml. Sephadex G-200 column chromatography. Total volume collected, 760.0 ml. It was re-duced by pressure d i -alysis to 45 ml. Figure 6 Flow sheet of methods for the isolation of a-amylase isoenzyme 1 from Bacteroides  amylophilus Strain H-18. 58 TABLE III PURIFICATION OF a-AMYLASE ISOENZYME 1 FROM B. AMYLOPHILUS STRAIN H-18 Pro-cedure Volume ml Concen-tration units/ ml Total Units (xlO-3) Protein mg/ml Specific Activity (units/ mg pro-tein Yield % P u r i f i -cation 24 hour superr-natant 29000.0 4.0 116.00 0.30 13.3 100.00 1 DEAE-Sephadex batch opera-tion 2540.0 40.0 101.60 0.60 66.6 87.5 5.00 Dialysis against P0 4 buffer 2600.00 35.0 91.00 0.50 70.0 78.4 5.26 DEAE-Sephadex Column Fraction No. I l l 840 75.0 65.52 0.15 550.00 56.00 41.35 . Sephadex G-200 Column Fraction and Pressure Dialysis 45 1000 45.00 0.7 1428.5 38.8 107.4 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 activity. Since the amount of protein present decreased from 0.60 mg to 0.50 mg per ml, the decrease in enzymic activity may.be due to denatur-ation of the a-amylase protein. Step 4 - The enzymic preparation was fractionated by DEAE-Sephadex chromatography using a linear sodium gradient (Figure 7). At this stage the volume of enzyme solution was reduced to 840 ml and purification obtained was 41 fold. The solution was further concentrated to 40.00 ml by pressure dialysis. Step 5 - The enzyme solution obtained in Step 4 was subjected to gel f i l t r a t i o n on Sephadex G-200. An elution profile of a-amylase activity is shown in Figure 8. A l l of a-amylase activity, was obtained in a single peak. At this stage the recovery was 38.8 per cent and purification obtained was 107- fold. This enzyme preparation was free from protease activity. The enzyme solution was concentrated by pressure dialysis 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 electro-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 linear NaCl gradient (0.2 to 0.75 M) in phosphate buffer (0.05 M, pH 7.0). a-Amylase activity (Units/ml) NaCl (M) 62 Figure 8 Chromatography of Bacteroides amylophilus Strain H-18 a-amylase isoenzyme 1 on Seph-adex G-200. Enzyme was eluted with phos-phate- buffer (0.05 M, pH 7.0). 700, cn vO 600 500 400 •300 200 100 / / / 50 100 150 200 250 300 350 400 - z 1 450 Fraction Number (5.0 ml/Fraction) 64 Figure 9 Disc electrophoresis of a-Amylase Isoenzyme 1. Illustration A. Stained disc gel. Illustration B. Densiometric tracing. 65 + td 66 peak was obtained (Figure 10). Therefore-it is concluded from these results that a-amylase isoenzyme 1 is a single homogeneous protein. 2. Catalytic Properties of a-Amylase Isoenzyme 1 a. Determination of Type of Amylase To determine whether the amylase in question is 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 is of the a-type. b. Effect of pH on.a-Amylase Activity The pH profiles of the activity of a-amylase are shown in Figure 11. The pH of the maximum enzymic activity was found to be 6.7 at 44°C. The optimum pH values for a-amylases from other sources as reported in the literature are in the acid region between 4.5 and 7.0. These re-sults are summarized in Table IV. c. Effect of pH on Enzymic Stability As seen in Figure 12, the a-amylase is stable in the pH range of 6.2 to 7.6. The stable pH i s narrow on both acidic and alkaline side. The pH s t a b i l i t i e s of a-amylases from other sources are reviewed in 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 activity peak gives the pi value of a-Amylase.Isoenzyme 1. pH Gradient 69 Figure 11 Optimum pH for hydrolyzing starch. The enzymatic a c t i v i t y was determined at 44°C for 15 minutes a f t e r incubation with starch at respective pH values. Relative Activity (%) 71 TABLE IV SUMMARY OF THE OPTIMUM pH RANGE OF a-AMYLASE FROM VARIOUS SOURCES Source of a-amylases Optimum References pH range Porcine pancreas 6.8 4,31,34 Monkey small intestine 6.8 . 43 Human saliva 6.9 32,34 Aspergillus oryzae 4.8-5.8 12 Bacillus subtilis 6.0 29 Pseudomonas saccharophila 5.25-5.75 27 Bacillus stearothermophilus 5.0 6,7 4.5-6.5 37 Streptococcus bovis 4.6-6.1 17 Clostridium butyricum 5.5-6.5 17 B. polymyxa 6.2-7.5 42 Bacteroides amylophilus 6.7 S.U.R. 72 Figure 12 E f f e c t of pH on the s t a b i l i t y of a-amylase. The buffer 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 buffer solutions 0.2 ml of 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 mixture was kept at 37°C for 24 hours. Af t e r adjusting the pH to 6.7, the f i n a l volume was made up to 4.0 ml. The enzymic a c t i v i t y was de-termined before and a f t e r treatment and the percentage of the a c t i v i t y which remained was calculated. % of Residual Activity 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 sta b i l i t y range References Barley malt 4.9-9.1 34 Porcine pancreas 7.0-8.5 4,34 Human saliva 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 subtilis 4.8-8.5 30.46 Bacteroides amylophilus 6.2-7.6 S.U.R. 75 d. Effect of Temperature on a-Amylase Activity The temperature profiles of the activity of a-amylase isoenzyme 1 are shown in Figure 13. Temperature•for the maximum activity is 44°C as compared to a-amylases for other sources (Table VI). e. Effect of Temperature on Enzymic Stability  Figure 14 (curve B) illustrates that a-amylase isoenzyme 1 re-tained 100 per cent of i t s original activity after heat treatment up to 42°C for 15 minutes. In this regard thermophilic a-amylase from B. stearothermophilus retained 100 per cent of i t s original activity at 65°C for 15 minutes and the mesophilic a-amylase from B_. subtilis main-tained 100 per cent of i t s original activity at 43°C for 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 origins have been shown to contain calcium which is essential i n the catalytic activity and stabilization of the enzyme molecule (12,18,22,50). There-fore the effect of calcium ions on the st a b i l i t y of a-amylase isoenzyme 1 was studied for comparative purposes. In the presence of 0.02 M CaC^, 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 is 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 susceptibility of a-amylase to temperature was caused by the non-availability 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 in 0.02 M Tris-maleate buffer (pH 6.7). The-reaction was carried out at various temperatures for 10 minutes. Relative Activity (%) 77 78 TABLE VI OPTIMUM TEMPERATURE FOR VARIOUS a-AMYLASES Source of a-amylase Optimum Temperature References Barley malt 35°C 3 4 Porcine pancreas 37°C 4,31,34 Human saliva 40 °C 1 3 , 3 2 , 3 3 , 3 4 Aspergillus oryzae 40°C 10,49 Bacillus subtilis 40°C 2 9 , 3 0 , 4 6 43°-58°C 37 Bacillus stearothermophilus 65°C . 6,7 65°-73°C 37 Streptocuccus bovis ,; 48°C 17 Clostridum butyricum 48°C 17 Bacteroides amylophilus 44°C S.U.R. 79 Figure 14 Thermal st 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 activity was determined and percentage of the activity which remained was calculated with respect to each treatment. The various treatments were' as follows: Curve A. 0.02 M CaCl 2 in 0.02 M Tris-maleate buffer, pH 6.7. Curve B. 0.02 M Tris-maleate buffer, pH 6.7. Curve C. Enzyme was dialysed against 0.22 M EDTA in Tris-maleate buffer, pH 6.7, 20°C. % of Residual Activity 80 81 been reported by EDTA treatment for human saliva, hog pancreas, B. sub-t i l i s and A. oryzae a-amylases (50), _. stearothermophilus a-amylase (38) and a-amylase from monkey small intestine (43 ) . f. Amino Acid Determination The amino acid analysis of a-amylase isoenzyme 1 was principally 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 in maintaining the enzymic activity and tertiary structure of a-amylase molecules. The absence of sulphydryl groups is in agreement with the finding that p-chloromercuribenzoate did not inactivate the enzyme (IV:A:2). It is also interesting to note that protease from B. amylo- philus H-18 is completely void of cysteine and cystine (20). In this regard a-amylase isoenzyme 1 is comparable with _. subtilis a-amylase which does not contain cystine (25). Bacterial a-amylase from B_. stear-othermophilus does contain cysteine, but completely lacks tryptophan (6 ) . Pollock (41) has reported the absence of disulphide linkages as a characteristic feature of various bacterial 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 activity when calcium 82 TABLE VII CALCIUM CONTENTS OF VARIOUS a-AMYLASES Source of a-amylase Amount of Calcium Present* References Human saliva 1-2 50 Hog pancreas 1-2 50 A. oryzae 2-3 50 B. subtilis 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, indicat-ing a functional, role, of calcium (50) . The findings of Hsiu.et al_. • (18) , are similar for 13. subtilis a-amylase and human salivary a-amylase. By spectrophotometric analysis i t was found that purified a-amylase isoenzyme 1 from B. amylophilus contains 3 gram-atom of calcium per mole of a-amylase. This value is in 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 original activity and the calcium content at this stage was 0.8 gram atom per mole of a-amylase. When I3_; subtilis a-amylase was dialysed against 0.01 M EDTA for 50 hours, the enzyme retained 40 per cent of i t s original activity and calcium content after dialysis was 0.4 gram-atom per mole of enzyme (50) . These results indicate that while isoenzyme 1 binds calcium more strongly than 13. subtilis a-amylase, i t is less stable at low calcium levels than I3_. subtilis a-amylase. h. Effect of Chemical Reagents on Enzymic Activity  (i) Effect of Urea on a-Amylase Activity The effect of urea on a-amylase activity is shown in Figure 15. The results (Curve A) indicate the remaining enzymic activity at d i f f e r -ent concentrations of urea. In 8.0 M urea the activity was completely inhibited. After removal of urea by dialysis against Tris-maleate buffer of pH 6.7, partial regeneration of the enzyme was obtained (Curve B, Figure 15). After incubation in 8.0 M urea (pH 8.5) at 30°C for 30 min-utes, 13. subtilis a-amylase lost .40 per cent of i t s original activity, 84 Figure 15 Effect of urea on a-amylase activity. Curve A. Various concentrations of urea as indicated were added to standard assay mix-ture and the percentage of the activity re-maining was calculated. Curve B. Solutions of enzymes (0.03 mg/ml), containing various concentrations of urea were kept at 37°C for 16 hours at pH 7.0. After dialysis in cold against 0.02 M Tris-maleate buffer (pH 6.7) for 24 hours, the. percentage of the remaining activity was calculated. 86 whereas B. stearothermophilus a-amylase lost 10 per cent of the original activity (38). Imanishi et a l . (23) recovered about:80 per cent of the original enzymic activity of _. subtilis a-amylase after treating with 8.0 M urea containing EDTA. Maximum recovery of a-amylase isoenzyme 1 was 68 per cent in 1.0 M urea. It 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 for 16 hours, which was higher than that used by other workers as noted above. _. aeruginosa protease was completely inhibited in a solution of 8.0 M urea, after treatment-at 37°C for 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 in inactivation which was appar-ently irreversible (43). Fukushi e_ a l . (14) , also recovered 60 to 90 per cent of denatured B_. subtilis a-amylase activity at pH 8.5 in 8.0 M urea at room temperature. These workers further reported that changes in optical rotatory dispersion and spectral shift 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 is followed by a slower intra-molecular rearrangement to a characteristic native structure responsible for a native and biologically active protein. Activity obtained was re-ported to be due to partial regeneration of enzyme molecules having the same specific activity as that of native a-amylase (14). The failure of regeneration may be due to the chemical binding of cyanate which is 87 present in urea with, amino groups to.yield carbamyl derivatives (8,44). The other possible causes of irreversible inactivation of denatured a-amylase reported by others may be due to incorrect refolding and inter-molecular aggregation (23) , and partially due to proteolytic degradation of the unfolded enzyme molecule by proteolytic contaminants (23,45). Re-generation of the enzymic activity also depends on the pH value and ionic strength of the solution and concentration of enzyme (23). ( i i ) Effect of EDTA and Metallic Ions on a-Amylase Activity  As seen in Figure 16, treatment with EDTA reduced the enzymic activity to 30 per cent of the original activity. By the addition of various metals the enzymic activity was regenerated (Figure 16, B). Treatment with calcium restored the enzymic activity completely while magnesium reactivated the enzymic activity up to 90 per cent. It may be noted that dialysis had no effect on the a-amylase isoenzyme activity. The activity remained constant during the period of dialysis. Treatment with EDTA removed the calcium from the various a-amy-lases, thus decreasing their activity (18,50). Addition of calcium re-sulted i n the restoration of enzymic activity (18,48). In this regard the results presented in this thesis are similar to the findings of other workers (18,38,46). The evidence has been presented that the ca l -cium atom is necessary in maintaining the catalytically active conforma-tion of the amylase molecule (18). The role of calcium becomes important particularly in .B. subtilis a-amylase which lacks intramolecular disul-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) metal ions 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 dialysed against 0.02 M EDTA i n 0.02 M tris-maleate b u f f e r (pH 7.0) at 5°C for 100 hours. EDTA was removed by further d i a l y s i s for 24 hours against various metal solutions as indicated i n 0.02 M t r i s -maleate buffer (pH 6.7) and the enzyme a c t i v -i t y was determined. % of the A c t i v i t y Remained 89 t o O O ON o oo o t-c c /—\ bd g • X) C O tn Mi r-1 t o M l fD S •~J (D fo • H r t H o fD M I o (S3 g W o 1-3 > Reactivation (%) t o o o O N o oo o c "7 o • o o n 4> r o n • 01 o O 4S M g ho 3 O n *~ M g g • CM O M g ISJ 90 active conformation of protein may be maintained by certain intramolecu-lar non-covalent linkages rather than usual disulphide bridges (18). Actually the non-existence of disulphide linkage appears to be character-, i s t i c of various bacterial exoenzymes (41). The role.of calcium in maintaining enzymic activity has also been suggested in several other bacterial exoenzymes, viz. proteinase (16) and ribonuclease (18). In this regard a-amylase isoenzyme 1 is an exo-enzyme, lacks disulphide linkage and free sulfhydryl groups and requires calcium for i t s activity. Imanishi (22) has reported that removal of calcium does not cause detec-table changes in protein conformation and suggested that calcium ions may be located on.the surface of the protein. It appears, therefore^ that calcium forms a tight metal-chelate structure with the protein molecule to maintain a proper configuration for biological activity (18,50). Takagi and Iseumura (48) also indicated the role of calcium ions in refolding the reduced taka-a-amylase A. As noted in Figure 16 (B), the treatment with cobalt, zinc and magnesium could not regenerate the enzymic activity completely. The reason may be that certain metals could not form-a correct metal chelate structure with protein, thus re-sulting in unstable or incomplete secondary and tertiary structures, which did not have complete biological activity. i . Functional Groups Determination The results on the effect of reducing, oxidizing and SH-inactivat-ing agents are presented in Table VIII. The a-amylase activity, remained unaffected after the treatment with reducing agents, viz . , 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) Residual Activity (%) Cysteine 5 x 10"3 100.00 Sodium cyanide 5 x IO - 3 100.00 Sodium thioglycolate 5 x IO - 3 102.00 Mercaptoethanol 5. x IO - 3 100.00 p-Chloromercuribenzoate 5 x IO - 3 98.00 Monoiodoacetic acid 5 x IO - 3 92.0 Potassium permanganate IO"3 0 N-Bromosuccinimide 5 x IO - 3 0 Solutions of enzyme (6 units/ml) containing various concen-r trations of different reagents at pH 7.0 are kept for one hour at 37°C. The percentage of the activity which re-mained after the treatment was calculated. 92 Further the enzymic activity is not inactivated by specific SH-inactivating agents such as p-chloromerciiribetizoate and monoiodoacetic acid. These results indicate the noninvolvement of sulfhydryl groups in enzymic reaction mechanisms, and are in agreement with the finding that a-amylase isoenzyme 1 did not contain cystine. The slight inhibition may be due to the reaction with methionine, serine or imidazole-group of histidine. Since the inhibition observed was slight these.amino acids may be located near the active center. It is to be noted that enzymic activity was completely lost by ox-idizing agents viz., potassium permanganate and N-bromosuccinimide. Although potassium permanganate is a non-specific oxidizing agent, N-bromosuccinimide is more specific with controlled conditions in i t s reaction with tryptophan. (39). Okada et^.a_. (39) used N-bromosuccinimide to oxidize tryptophan and suggested that i t was involved ih the active center of ._. subtilis ar-amylase. It has been reported by Sugae (47) that B. subtilis a-amylase lost i t s activity when an azo-group.was intro-duced into the a-amylase molecule. He suggested that a peptide group in the neighbourhood of a particular tyrosine residue which was modified by the azo group was closely related with the activity of bacterial ct-. amylase. In general, the active center includes, besides the catalytic site, the grouping conforming to the substrate specific for the enzyme (40); the two groups are sufficiently close to each.other. It is possible that the active center of bacterial a-amylase may have two groups,.try-ptophan (39) and tyrosine (47). Yamato (54) indicated that.the trypto-phan and tyrosyl group a-amylase obtained from B, amyloliquefaciens Fukumoto was essential for i t s enzymic activity. Ikenda (21) reported that in case of A. oryzae, the phenolic group of tryosine was essential for enzymic activity. The activity of a-amylase isoenzyme was lost with the treatment.of N-bromosuccinimide (Table VIII). It is suggested from this result that tryptophan is essential for the catalytic a b i l i t y of a-amylase isoenzyme 1. j . Determination of Molecular Weight The estimated molecular weight for a-amylase isoenzyme i s 45,000. The'molecular weight of various a-amylases are given in Table IX. k. Determination of Isoelectric Point Isoelectric point as determined by electrofocusing technique was pH 3.7. Isoelectric points of other a-amylases are also in the acidic region as indicated in 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 Isoelectric points References Barley malt 5.7 34 Porcine pancreas 5.2-5.6 4,31,34 Human saliva 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 bacterial amylase. I. Amino acid composition of crystal-line bacterial amylase from B_. subtilis N. J. Biochem. 43:741. 2. Blackburn, T. H. 1968; Protease production by Bacteroides amylo-philus strain H-18. J. Gen. Microbiol. 51:27. 3. Bryant, M. P. and I. W. Robinson. 1961. Some nutritional require-ments of genus Ruminococcus. Apply Microbiol. 9:91. 4. Caldwell, M. L., M. Adams, J. F. Kung and G. C. Toralballa. 1952. Crystalline pancreatic amylase. II. Improved method for i t s preparation from hog pancreas glands and additional studies of it s properties. J. Amer. Chem. Soc. 74:4033. 5. Clary, J. Jj G. E. Mitchell, Jr. 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. III. Amino acid composition. J. Biol. Chem. 236:2962. 7. and P. D. Cleveland. 1961. Thermostable a-amylase of Bacillus stearothermophilus. Crystallization and some general properties. J. Biol..Chem. 236:2952. 8. Cole, R. D. 1961. On the transformation of insulin in concentrated solution of urea. J. Biol. Chem. 236:2670. 9. Danielsson,. C.' E. 1947. Molecular weight of a-amylase; Nature. 160:899. 10. Fischer, F. H. and R. DeMontmollin. 1951. Purification et crys-ta 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. • 11. , W. N. Summerwell, J. M. Junge and E. A. Stein. 1958. Pro-ceedings of Symposium VIII, IVth International Congress of Bio-chemistry, Vienna, Pergamon Press. 97 12. 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. 13. . 1964 a-Amylase from human saliva. Biochemi-cal Preparation. 8:27.-14. Fukushi, T., A. Imanishi and T. Isemura. 1968. Changes in enzyma-t i c activity and conformation during regeneration of native bacterial amylase from denatured form. J. Biochem. 63:409. 15. Hagihara, B. 1954. Crystalline bacterial amylase and proteinase. Ann. Rep. Sci. 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. II. Calcium and catalytic activity. Biochem. 3:61. 19. Hungate, R. E. - 1966. "The rumen bacteria." In the rumen and i t s microbes. By R. E. Hungate. Academic Press. New York and London. 20. Lesk, E. M. 1969. Purification and characterization of proteolytic enzymes from _. amylophilus strain H-18. M.Sc. Thesis. Univer-sity of British Columbia, Vancouver, B.C. 21. Ikenda, T. 1959. Chemical modification on taka-amylase A. II. Phenylazobenzoylation of taka-amylase A. J. Biochem. 46:297. 22. Imanishi, A. 1966. Calcium binding by bacterial a-amylase. J. Biochem. 60:381. 23. , K. Kakiuchi and T. Isemura. 1963. Molecular stability and reversibility of denaturation of _. subtilis a-amylase. II. Regeneration of urea denatured enzyme by removal or dilution pf urea. J. Biochem. 54:89. 24. Isemura, T. andS. Fujita. 1957. Physicochemical studies on taka-amylase A. I. - Size and shape determination by the measurement of sedimentation, diffusion coefficient and viscosity. 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 Bacillus stearothermophilus. II. Physical proper-ties and molecular weight. J. Biol. Chem. 236:2958. 27. Markovitz, A., H. P. Klein and E. H. Fisher. 1956. Purification, crystallization, 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 pancreatitis. I. Effect of substrate variation. Clin. 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. Purification 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. . , 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. 32. , , A. Stauband P. Bernfeld. 1948. Proprietes de l'a-amylase de salive 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 salive humaine. Helv. Chim. Acta. 31:2158. 34. . 1952. The'past and present of starch chemistry. Exper-entia. 8:405. 35. Morihara, K. 1963. Pseudomonas aeruginosa proteinase. I. Puri-fication 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 concentra-tion of polyethylene glycol on amylase formation by Bacillus  s u b t i l i s . J. Biochem. 43:143. 37. Ogasaharaj K. , A. Imanishi and T. Isemura. 19.70. Studies orither-mophilic a-amylase from Bacillus stearothermophilus. I. Some' general and physico-chemical properties of thermophilic a-amylase. J. Biochem. 67:65. 99 38. Ogasahara; K. , A. Imanishi and T. Isemura. 1970. Studies on ther-mophilic a-amylase.from Bacillus stearothermophilus. II. Ther-mal st 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. II. N-bromosuccinimide modified bacterial 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. Interna-tional 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. Vol. 4. New York and London. Academic Press, Inc. 42. Rose, D. 1948. The amylase of Bacillus polymyxa. Arch. Biochem. 16:349. 43. Seetharam, B., N. Swaminathan and A. N. Radhakrishna. 1969. Puri-fication and properties of a-amylase from monkey small intestine. Indi. J. Biochem. 6:51. 44. Stark, G. R., W. H. Stein and S. Moore. 1960. Reaction of the cyanate present in aqueous urea with amino acids and protein. J. Biol. Chem. 235:3177. 45. Stein, E.A., and E. H. Fisher. 1958. The resistance of a-amylase towards proteolytic attack. J. Biol. Chem. 232:867. 46. . 1961. a-Amylase from Bacillus s u b t i l i s . Biochemical preparation. 8:34. 47. Sugae, K. I960; . Studies on bacterial a-amylase. V. Chemical modification of-bacterial 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 re-generation of reduced Taka-amylase A. J. Biochem. 57:89. 49. Underkofler, L. A. and D. K. Roy. 1951. Crystallization of fungal alpha-amylase and limit 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 origins. J. Biol. Chem. 234:2901. 51. Welker, N. E. and L. L. Campbell. 1963. Effect of carbon source on formation of a-amylase by Bacillus stearothermophilus. J. Bact. 86:861. 100 52. Wiesmeyer, H. and M. Cohn. 1960. The characterization of the path-way of maltose u t i l i z a t i o n by Escherichia Coli. III. A des-cription of the concentrating mechanism. Biochem. Biophys. Acta. 39:440. 53. Williams,. P. J. and J. J. McDonald. 1966. Permeability of a.micro-coccal c e l l to maltose and some related sugars. J. Canad. Microbiol. 12:1213. 54. Yamamoto. T. 1955. Studies on sensitive groups of.crystalline bacterial a-amylase. Bull. Agr. Chem. Soc. Japan. 19:121. 55. . 1955. Studies on the a-amylase destroying enzyme. Part I. Occurrence and some properties of the enzyme. Bull. 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 qualitatively by thin layer chromatography. The importance of the iodine-staining characteristics is reported in terms of possible mechanisms for a-amylase action. The differences and similarities of a-amylase isoenzyme 1.action are discussed with respect to other a-amylases. The progress of hydrolysis of amylose and starch is shown chromatographically in Figures 17 and 18. Figure 17 represents hydrol-ysis beyond the achroic point and this was reached by about thirty min-utes. In Figure 18 hydrolysis was also extended beyond the achroic point. The reference samples in each case were obtained by partial acid hydrolysis of amylose. : Due to the following two observations, the spots were regarded as linear oligosaccharides of maltose series: I. The experimental spots from enzymatic digests had a corres-ponding Rf with regard to reference sample (3). II. The introduction of branch point (4) retards the mobility i n such a way that linear oligosaccharides are separated from branched oligosaccharides of equal D.P. For example, a branched dextrin of four glucose units f a l l s at a point inter-mediate in distance between maltopentaose arid maltotetraose These experimental results suggest that significant 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 remain-ing columns contained.the products of the pro-gressive hydrolysis of Amylose taken from the digestion mixture at various time intervals as indicated.on the chromatograms. The chromato-gram 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 30 3 5 u 0 Tine (Minute) 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 progres-sive hydrolysis of Amylose taken from the digestion mixture at various time intervals as indicated on the chromatogram. The chromato-gram 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 pro-ducts of amylose and starch were the same, and could not be differeritia-ted from one another. It is suggested from these results that the pro-duction of higher malto-dextrin is a characteristic pattern of B. amylophilus a-amylase isoenzyme 1. Figures 17 and 18 show the complete maltodextrin spectrum from D.P. 2 to 14. The early products in the digest are maltodextrin mixtures from D.P. 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. Maltodextrin of D.P. 12 to 14 started disappearing after the achroic point, and corres-pondingly the intensity 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 characteristic maltodextrin spectrum produced by other a-amylases. B_. subtilis 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 malto-triose, 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 well (13). Hanrahn and Caldwell (7) reported that with high concentra-tion of enzyme and.if a sufficient 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 is similar to that of human salivary a-amylase (14). The results of enzymatic hydrolysis shown in Figures 17 and 18 indicate that only certain series of maltodextrin could be separated by thin layer chroma-tography, as reducing spots were present at the point of application on the chromatogram. In the present, investigation the changes in the polysaccharide-iodine 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 polymeriza-tion of straight chains of the polymer (16), a decrease in absorption during progressive enzymatic hydrolysis has been taken as a decrease in the average degree of polymerization of the substrate. During the enzy-matic hydrolysis of amylose and starch, a decline in 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. It is suggested that in a qualitative way the dextrinizing action of JB; amylophilus a-amylase isoenzyme 1 is comparable to salivary a-amylase; It i s generally regarded that the mode,of attack of various a-amylases on substrate is random (2,10), and the enzyme-product complex 108 dissociates i t s e l f after single catalytic events. It is 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 is supported by a rapid decrease in iodine colour and viscosity (6), but with the availability of chromatographic results as reported above i t was apparent that such a group of highly characteristic malto-oligosaccharides and malto-dextrins could not be produced through random attack (1). This problem has been resolved (5). 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 in the proximity of the f i r s t . The specific nature of this multiple attack on substrate could be determined by enzymes with the production of characteristic series of malto-saccharides (1,3). The action pattern of _. amylophilus a-amylase isoenzyme 1 is in line with this hypothesis, and the concept of multiple attack at the site of encounter explains the data presented for this a-amylase. Kung et a l . (9) reported that variation in the curves relating blue value to the increase in the reducing values were due to the d i f f e r -ent a-amylases degrading amylose in different chain lengths. But chroma^ tographic results have shown that.different a-amylases produced different types of digestion products and these maltodextrins are characteristic of individual enzymes, as mentioned above. Robyt and French (15) reported that porcine pancreatic a-amylase and human salivary a-amylase digest produced very identical results but their blue value-reducing value curves were very different. 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 different a-amylases by determining the ratio of the reducing value of the oligosaccharide fraction to that of the polysaccharide fraction. 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) is 2 at optimum pH 6.7 and temperature 44°C (Table XI). 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* %BV1 3 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 ratio of the total reducing value to the reducing value of 67% ethanol polysaccharide precipitate. " * , * • • • • Average r determination. Degree of multiple attack was determined by the method of Robyt and French (15). The definitions 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) of the iodine complex of the digest at zerotime and at t times of hydrolysis. 2. RV_ = Total reducing value is expressed as mg of apparent maltose/ml digest. 3. RVp = Reducing value of 67% ethanol precipitate in terms of mg of apparent maltose/ml of digest. 4. Average degree of polymerization of 67% ethanol precipitate determined by the quotient: [Total carbohydrate (mg/ml)/apparent maltose (mg/ml)] x 2.: 5. Quotient of the total reducing value divided by the reducing value of the 67% ethanol precipitate (RV^RVp) . 6. Degree of multiple attack (r-1) = (3.06-1) = 2.06. 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. 1965. a-Amylosis of starch. Staerke. 17:219. 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 irrigation. Anal. Chem. 40:207. 9. Kung, J. T., V. M. Hanrahan and M. L. Caldwell. 1953. A compari-son of the action of several alpha amylases upon a linear fraction from corn starch. J. Amer. Chem. Soc.. 75:5548. 10. Myrback, K. 1948. Products of the enzymic degradation of starch and glycogen. Advance in Carbohydrate Chem. 3:251. 11. Rahman, Sh. Saif-^ur-, C. R. Krishnamurti and W. D. Kitts. 1968. Separation of Cello-saccharides by thin layer chromatography. J. Chromat. 38:400. 12. Robyt, J. and D. French. .1963. Action pattern and specificity 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. Purification and action pattern of an amylase from Bacillus polymyxa. Arch. Biochem. Biophys. 104:338. 14. . 1962. Action pattern of some alpha-type amylases. Ph.D. thesis. Iowa State University. 15. . and D. French. 1967. Multiple attack hypothesis of ct-amylase action. Action of porcine, pancreatic, human salivary and Aspergillus oryzae. Arch. Biochem. Biophys. 122:8. 16. Swanson, M. A. 1948. Studies on the structure of polysaccharides IV. Relation of iodine colour to structure. J. Biol. Chem. 172:825. 113 C. Immunochemical Studies on a-Amylase•Isoenzyme 1 1. Inhibition of Enzymic Activity by Antibody The results of enzymic inhibition by antibody are essentially similar to those of bacterial and mold a-amylases (5,13). Normal control serum did not inhibit the enzymic activity unlike Nomura and Wada (5) who obtained slight inhibition of the enzymic activity. The time course study of the inhibition of the a-amylase activity by antibody disclosed that neutralisation was complete in 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 activity was determined and percentage of remaining activity was calcu-lated. The results are presented in Figure 19. The curve was linear u n t i l 84 per cent of the enzymatic activity was neutralized, that i s , the quantity of enzyme neutralized was proportional to that antibody added in the region of antigen excess. A small amount of residual activity (16 per cent) remained in the presence of excess antibody. This might be explained either by reversible dissociation of anti-body-enzyme complex or that the complex exhibits amylase activity (13). Cinader (3) has also reported.that enzyme-antibody complexes themselves have some residual activity.~" Cinader and Lafferty (2) have.established the presence of three types of antibodies to biologically active antigen. These antibodies can affect the activity of enzyme in three different ways: antibody may combine and inhibit; antibody may combine with anti-gen but not inhibit i t s activity; and antibody may combine, not inhibit, and may interfere in the combination with inhibiting 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 original antiserum, a-amylase activity was determined after the incubation of 1 hour at 37°C. 116 2. Ouchterlony Double-Diffusion Analysis Results of double diffusion in agar gel are shown in 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 precipitation line 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. su 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 distinct from those present on the a-amylase of hog pancreas, _B. subtilis 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 antigenically distinct. These results are in 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). It was also noted that _. subtilis 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 immunodif-fusion precipitation reaction between _. amylophilus a-amylase isoenzyme 1 and anti-amylase and anti-amylase antiserum of var-ious origins. Well 1 - _. amylophilus a-amylase iso-enzyme 1. Well 2 - Anti-_. amylophilus a-amylase isoenzyme 1 antiserum. Well 3 - Anti-B. subtilis a-amylase anti-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 Immunoelec-trophoresis of 13. amylophilus a-amylase isoenzyme 1. Medium: 2 per cent agar in pH 8.2, 1=0.033 Veronal buffer. Antigen: 5 mg/ml added in central well. Electrophoresis was carried out at 5 mA per slide for 2 hours. Bromophenol blue was used as tracking dye. After electrophor-esis antiserum was added in troughs 1 and 2. Precipitation line between a-amylase. isoenzyme 1 and i t s antiserum was observed. 120 4 -I 121 4. Quantitative Precipitation Analysis The precipitation reaction was studied quantitatively and a typi-cal curve with one equivalence point was obtained. These results are presented in Table XII and Figure 22. The molar ratio range of antibody to antigen in precipitates at equivalence and in the antibody excess re-gion were found to be between 1.8 and 2.31. Other reported values re-garding the molar ratio between 13. subtilis a-amylase and i t s antibody are 2.16 (7); 1.9 (12) and for Taka-amylase A 2.58 (8), 2.6, 2.8, 4.0 (10). At the point of equivalence tests of the supernatant solution indi-cated well defined zones of antibody excess, equivalence and antigen excess (Table XII). The quantity of precipitate decreased in the anti-gen excess zone. During Immunoelectrophoresis at pH 8.2 and agar gel diffusion, a single precipitate line formed between I3_. amylophilus a-amylase isoenzyme 1 and i t s antibody. It is 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 activity (Table XIII). This result was probably due to the oxidation of tryptophan residues at the catalytic site of the enzyme molecule (6). Immunochemical analysis was performed to detect molecular configuration differences between native and NBS-treated enzyme. The quantitative precipitation curve with the NBS-modified enzyme preparation was found to be comparable to that given by native enzyme in the area of 122 TABLE XII PRECIPITATION REACTION OF B. AMYLOPHILUS a-AMYLASE ISOENZYME 1 WITH ITS ANTIBODY Antigen added ug Total pre-cipitate ug Presence of Ag+AB in supernatant Ag AB Antibody pre-cipitate ug Molar ratio AB mole AG mole 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 in 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 pre-cipitate was determined. a-Amylase activity was measured in the supernatant. Molecular ratio was calculated from the following molecu-lar weights: rabbit antibody (AB) 165,000 and a-amylase isoenzyme 1 (AG) 45,000. 123 Figure 22 Precipitation curve of a-amylase isoenzyme 1 with i t s antibody. The condition of the reaction as described in Table XII. 125 TABLE XIII EFFECT OF N-BROMOSUCCINIMIDE ON ACTIVITY OF ct-AMYLASE ISOENZYME 1 Reagent Treatment Length of treatment Residual activity Molar ratio minutes % NBS/enzyme NBS 6 15 8 NBS 7 15 0 a-Amylase (1 mg/ml) in 0.05 M sodium acetate buffer (pH 6.0) was treated with NBS. The reaction was termi-nated with Na2S0^ solution and enzymic activity was determined. 126 antibody excess and in the equivalence zone (Figure 23). It is suggested that catalytic and antigen sites are distinct. Similar are the results with B. subtilis a-amylase (6), A. oryzae a-amylase (11) and bovine ribo-nuclease (1). Treatment of a-amylase with urea (8 M) completely inactivated the catalytic 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 activity and antigenic.characters were destroyed completely. The effect of urea is due to an unfolding of protein molecules bringing about the loss of biological activity (9). Comparable immunochemical re-sults have been reported with urea treated phage lysozyme (4). It i s interesting to note that both phage lysozyme (4) and .B. amylophilus a-amylase isoenzyme 1 do not have disulphide linkage, whether this com-plete loss of antigenic character is related to the absence of disulphide bonds is not known (10). It is interesting that Aspergillus a-amylase, which contains disulphide linkages, has been shown to retain part of i t s immunochemical reactivity following urea treatment (10). The retention of a certain proportion of i t s antigenic reactivity may be related to disulphide linkages which probably stabilize 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 for 50 minutes at 37°C. After this, 5 per cent starch solution was added in the incubation mix-ture and amylase activity was determined by the iodine reaction. The 127 Figure 23 Precipitation curve of N-bromosuccinimide treated and native a-amylase with i t s anti-body. Reaction condition as reported in Table XII. x—x. NBS treated enzyme. Native enzyme. 1000 100 200 300 400 500 Antigen added (ug) TABLE XIV EFFECT OF UREA ON. ACTIVITY OF a-AMYLASE ISOENZYME 1 Reagent Treatment M Length of treatment H Residual activity Urea Urea 6 8 24 24 1.0 0 a-Amylase (1 mg/ml) was treated with urea at 37°C. After treatment the sample was dialysed against 0.02 M tri s buffer (pH 6.7) for 24 hours and the enzymic activity was determined. 130 percentage of starch hydrolysis was decreased due to the presence of antibody (Figure 24, curve C), and the rate was constant for 150 minutes at least. In the next experiment enzyme was added to 5 per cent starch solution and after 10 minutes of incubation at 37°C, the same amount of antibody was added. As shown in 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 inhibitory effect of antibody appeared (Figure 24, curve B). Therefore, i t appears that the products of starch digestion are not as effective as starch to neutralize the amylase-antiamylase system. Wada and Nomura (12), and Onoue et a l . (7) have also reported that starch did interfere with amylase-antiamylase system. Antibody interference may be due to steric hindrance or due to conformational change in the enzyme molecule (2). In the present experiment i t is possible that both factors may be involved, as previous experiments with NBS indicated that anti-genic and catalytic sites of enzyme molecules are different. 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 units). 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 units). 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 incu-bated 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 incuba-tion 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. 50 100 150 Time (minutes) REFERENCES IV:C 1. Brown, Ray K. 1963. Immunological studies of bovine ribonuclease derivatives. Ann. N.Y. Acad. Sci. 103:754. 2. Cinader, B. and K. J. Lafferty. 1963. Antibody as inhibitor of ribonuclease: the role of steric hindrance, aggregate forma-tion, and specificity. Ann. N.Y. Acad. Sci. 103:653. 3. . 1967. "Antibodies to enzymes - a discussion of the mechanism of inhibition and activation." In the proceedings of the 2nd meeting of the Federation of European Biochemical Societies, Vienna, April 21-24, 1965. Ed. by B. Cinader. Pergamon Press, Toronto. 4. Merigan, Thomas C. and William J. Dreyer.. 1963. Studies on the antigenic combining sites in Bacteriophage lysozymes. Ann. N.Y. Acad. Sci. 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 pro-duced by Bacillus s u b t i l i s . J. Biochem. 45:629. 6. Onoue, K., Y. Okada and Y. Yamamura. 1968. Modification of bac-t 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 subtilis 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.phenylazo-benzoyl taka-amylase A. J. Biochem. 55:446. 9. Schachman, H. K. 1963. Considerations on the tertiary structure of protein., Cold Spring Harbour Symp. Quant. Biol., 28:409. 10. Sirishinha, S. and Peter Z. Allen. 1965. Immunochemical studies on a-amylase. I. Effect of denaturing agents and proteolytic enzymes on the immunochemical reactivity of a-amylase from A. oryzae.- Arch. Biochem. Biophys. 112:137. 11. 1965. Immunochemical studies on a-amylase. 134 II. Examination of immunochemical and enzymatic activities of native and modified a-amylase from Aspergillus oryzae.• Arch. Biochem. Biophys. 112:149. 12. Wada, T. and M. Nomura. 1958. An immunochemical study of microbial amylase (1). J. Biochem. 45:639. 13. , . 1959. An immunochemical study of microbial amylase II. J. Biochem. 46:329. CHAPTER V CONCLUSIONS Bacteroides amylophilus strain H-18 produces four isoenzymes of a-amylase, as detected by disc electrophoresis and electrofocusing. Iso-electric 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 i n -creasing isoelectric points. a-Amylase isoenzyme 1 was purified and some of i t s physico-chemical properties were examined and summarized in a subsequent section. A. General Properties 1. The optimum pH was 6.7 and exhibited a.narrow stable range of 6.2 to 7.6. 2. Its optimum temperature was 44°C and thermal stability range was 0 to 42°C. Since optimum temperature was not within the stability range, i t was possible that substrate (starch) protects the enzyme from heat, denaturation during assay. 3. The thermal s t a b i l i t y of the enzyme was affected, by EDTA treatment, due to non-availability of chelated calcium. Treatment by calcium protected the enzyme from heat denaturation.• 4. Oxidizing agents but not reducing agents and SH-reagents inactivated the enzymic activity. Enzyme was susceptible to urea treatment. 5. ' Amino acid analysis indicated the absence of cysteine, therefore, disulphide linkages are not involved in maintaining the tertiary 136 structure. • Tryptophan appeared to be essential for catalytic activity. 6. The estimated molecular weight was 45,000. 7; a-Amylase isoenzyme 1 was found to contain 3 gram atoms of calcium per mole. Various other metals tested could not replace the calcium in regenerating the maximum activity. 8. The isoelectric point was found to be pH 3.7. B. Action Pattern 1. The products of enzymatic hydrolysis of starch and amylose were mal-tohexaose, maltoheptaose, maltoctaose, maltonanaose and maltodecaose at the achroic point and sometime after i t , as revealed by thin lay-er chromatography. 2. Maltose was the smallest disaccharide detected. Since glucose was never found in-these experiments, i t appeared that a-amylase isoen-zyme 1 does not hydrolyse maltotriose and maltose. 3. The degree of multiple attack under the optimum conditions of tem-perature and pH was 2, as calculated by the ratio of the reducing value of the oligosaccharide fraction to that of the polysaccharide fraction. C. Immunochemical Properties 1. Antisera against a-amylase isoenzyme l.was found to be monospecific and a small amount of residual activity remained in the presence of excess of antibodies. 137 2. The inhibitory effect of starch on the amylase-antiamylase system was demonstrated. 3. The effect of anti-amylase (isoenzyme 1) globulin on amylase of var-ious origins was studied by Ouchterlony double-diffusion, and the results indicated that antigenic determinants of a-amylase isoenzyme 1 were distinct from those present on a-amylase of hog pancreas, Bacillus subtilis and Aspergillus oryzae. 4. Immunoelectrophoretic analysis revealed the presence of only a single antigenic component. 5. The precipitation reaction was studied quantitatively and a typical curve with one equivalence point was obtained. 6. The molar ratio ranges of antibody to antigen in precipitates at equivalence and in the antibody excess zone were found to be between 1.8 and 2.31. 7. N-Bromosuccinimide treated a-amylase.(isoenzyme 1) had no enzymic activity, but exhibited comparable immunochemical behaviour to native enzyme. It is possible that antigenic and catalytic sites are dis-tinct. 8. Urea treatment destroyed the a b i l i t y of the enzyme to precipitate with i t s specific antibody. 

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