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Purification and characterization of proteolytic enzymes from bacteroides amylophilus H-18 Lesk, Earl Michael 1969

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PURIFICATION AND CHARACTERIZATION OF PROTEOLYTIC ENZYMES FROM BACTEROIDES AMYLOPHILUS H-18 by EARL MICHAEL LESK B . S c , U n i v e r s i t y of B r i t i s h Columbia, (1966). A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT of MICROBIOLOGY We accept t h i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h C olumbia, I a g r e e t h a 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 Study. I f u r t h e r a g r e e t h a 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 p u rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t 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 thes,is 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 . The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Department i ABSTRACT This study purposes to examine e x t r a c e l l u l a r proteases of the anaerobic rumen bacterium, Bacteroides amylophilus H-18. An enzyme was i s o l a t e d and p u r i f i e d from 29 l i t r e s of 23 hr c e l l - f r e e c u l t u r e supernatant us ing DEAE Sephadex A-50 , Sephadex G-200 and i s o e l e c t r o f o c u s i n g techniques. Although p r o t e o l y t i c a c t i v i t y i n the supernatant had a peak of a c t i v i t y at pH 6.7, there was a c t i v i t y at pH values from 4.5 to 11.5. Therefore , an attempt was made to p u r i f y the pH 6.7 a c t i v i t y and to fo l low the a c t i v i t y at other pH values as an index of p u r i t y . I t was found that separat ion of the a c t i v i t i e s at d i f f e r e n t pH values was not achieved, even though the enzyme was p u r i f i e d 1265 t imes. Ge l f i l t r a t i o n of t h i s p u r i f i e d m a t e r i a l r evea l ed the presence of two proteases , one of 60,000 and the other of 3 0,000 molecular weight. Since these enzymes were otherwise i d e n t i c a l , they could have represented the monomeric and dimeric forms of a s i n g l e p r o t e i n . I f the protease of 30,000 molecular weight was separated and resubjec ted to ge l f i l t r a t i o n , protease a c t i v i t y of molecular weight 6 0,000 reappeared. U l t r a c e n t r i f u g a t i o n of the 30,000 molecular weight protease demonstrated only one component. Therefore , i f the two forms were i n e q u i l i b r i u m , i t appeared that the dimer was the more s tab le form o f the enzyme. The p u r i f i e d protease d i d not conta in cys te ine , so that any t e r t i a r y s t ruc ture i n the enzyme could not invo lve d i s u l f i d e b r i d g e s . A l l attempts to d i s s o c i a t e the dimeric in to the monomeric form were u n s u c c e s s f u l . Examination of the i n h i b i t i o n of Na b e n z o y l - L - a r g i n i n e methyl es ter esterase and protease a c t i v i t i e s with Na tosyl-L-chloromethane revealed a complete i n h i b i t i o n of esterase a c t i v i t y at pH 8.0 but on ly a 30% i n h i b i t i o n of protease a c t i v i t y at the same pH, suggesting that more than one enzyme was respons ib le for the p r o t e o l y t i c a c t i v i t y exh ib i t ed by the p u r i f i e d enzyme. Because i t was not p o s s i b l e to achieve separat ion of p r o t e o l y t i c a c t i v i t i e s at d i f f e r e n t pH values a f t e r a 1265 times p u r i f i c a t i o n , i t must be assumed that i f there are a c t u a l l y d i f f e r e n t proteases present they must have very s i m i l a r s t r u c t u r e s . i i i TABLE OF CONTENTS Page INTRODUCTION x MATERIALS 4 METHODS 5 I . Determination of P r o t e i n ^ I I . Determination of Protease A c t i v i t y ^ I I I . Determination of Amylase ^ IV. Determination of Esterase . ^ V. I n h i b i t i o n of P r o t e o l y t i c A c t i v i t y ^ V I . U l t r a c e n t r i f ugat ion . . „ . y V I I . I s o l a t i o n of Protease g V I I I . I s o e l e c t r o f ocusing . . • g IX. Amino Acid , A n a l y s i s . . 2Q X. E f f e c t of Temperature • • 20 X I . Ge l Disc E l e c t r o p h o r e s i s 20 RESULTS AND DISCUSSION 1 3 I . Es t imat ion of Molecular Weight 23 I I . Proper t i e s of Separated Frac t ions 24 I I I . Time Course of Protease L i b e r a t i o n 24 IV. Protease P u r i f i c a t i o n 26 1. § r o w t h 16 i v Page 2. Concentration 16 3. DEAE- Chromatography. 16 4. G-200 Sephadex . . 17 5. U l t r a c e n t r i f u g a t i o n 19 6. E f f e c t of pH 19 7. Is o e l e c t r o f ocusing .^.20 8. Acrylamide Gel Electrophoresis 23 V. Protease Characterization . . . 24 1. E f f e c t of Chemical Reagents 24 2. Amino Acid Analysis 25 3. E f f e c t of Temperature 26 4. Heat S t a b i l i t y . 26 5. Act i v e S i t e I n h i b i t i o n 27 GENERAL DISCUSSION . . . 29 BIBLIOGRAPHY 33 V LIST OF TABLES Table I . Proper t i e s at var ious pH values of f r a c t i o n s from Sephadex G-200 ge l f i l t r a t i o n and from d i s so lved ammonium s u l f a t e p r e c i p i t a t e . Tab le I I . P u r i f i c a t i o n of B_. amylophilus H-18 protease . Table I I I . Amino a c i d composition of the B_. amylophilus protease. Table IV. Heat denaturat ion of enzyme sample for vary ing lengths of time at 60 C. T a b l e V. Hydro lys i s of Na Benzoy l -L-arg in ine methyl es ter (BAME) by an enzyme p r e p a r a t i o n . LIST OF FIGURES F i g . 1. Gel f i l t r a t i o n of ammonium sulphate p r e c i p i t a t e d crude enzyme. F i g . 2. C e l l dens i ty , protease and amylase a c t i v i t i e s i n the c u l t u r e supernatant p l o t t e d against hours of incubat ion . F i g . 3. Chromatography of 13. amylophilus H-18 protease on DEAE Sephadex A-50. F i g . 4. Ge l f i l t r a t i o n of a p o r t i o n of sample #5 through a Sephadex G-200 column. F i g . 5. Ge l f i l t r a t i o n of a p o r t i o n of sample #5 through a Sephadex G-200 column. F i g . 6. U l t r a c e n t r i f u g a t i o n of protease . F i g . 7. E f f e c t of pH on a c t i v i t y of p u r i f i e d protease . F i g . 8. E l e c t r o f o c u s i n g of protease (Table I I , #8) F i g . 9. D i s c g e l e l e c t r o p h o r e s i s . -3 F i g . 10. Gel f i l t r a t i o n of protease pretreated with 10>v M EBTA.(Table I I , #8). F i g . 11. Gel f i l t r a t i o n of protease pretreated with 4 M urea (Table I I , #8). F i g . 12. E f f e c t of temperature on protease a c t i v i t y . F i g . 13. Arrhenius p l o t of protease a c t i v i t y at pH 6.7. F i g . 14. Heat s t a b i l i t y of protease (Table I I , #9 B ) . F i g . 15. Heat s t a b i l i t y of protease (Table I I , #9 A ) . ACKNOWLEDGEMENTS I would f i r s t l i k e to express my s incere gra t i tude to D r . T . H . Blackburn for h i s encouragement, d i r e c t i o n and cons truc t ive c r i t i c i s m of both the research and w r i t i n g of th i s t h e s i s . I would a l so l i k e to thank D r . R . A . J . Warren f o r h i s i n t e r e s t and for e d i t i n g the t h e s i s . I would l i k e to thank D r . J . Gerwing and Mrs . Barbara M i t c h e l l for t h e i r encouragement and f o r t h e i r t e c h n i c a l a s s i s tance . Furthermore, I would l i k e to thank Mr. W i l l i a m Lynch for h i s research c o n t r i b u t i o n s . My g r a t i t u d e i s extended to my f iancee Donna Love for her pat i ence , to lerance and c r i t i c i s m during the course of my research . L a s t l y , my thanks go to Jeanette Bellamy for the typing of t h i s f i n a l manuscript . INTRODUCTION Bacteroides amylophilus H-18, a Gram-negative p r o t e o l y t i c organism, was i s o l a t e d by Blackburn and Hobson (1962) from the rumen of a sheep u s i n g s t r i c t anaerobic c o n d i t i o n s . I s o l a t i o n of such an organism was cons idered unusual , s i n c e , with the exception of members of the genera Pseudomonas and V i b r i o (Po l lock , 1962), most b a c t e r i a which produce exoenzymes are Gram-pos i t ive . Protease product ion i n B_. amylophilus H-18 i s not subject to e i t h e r product repress ion or i n d u c t i o n , nor i s a general metabol ic repress ion demonstrable (Blackburn, 1968 a ) . The presence of 0.1% (w/v) tryptose i n the medium d id decrease the lag p e r i o d , probably by s t a b i l i z i n g the va lue; however, a d d i t i o n a l n u t r i e n t s d i d not a f f ec t e i t h e r growth or protease product ion . Protease a c t i v i t y from B. amylophilus H-18 i s of i n t e r e s t as i t s func t ion i s unknown. Hobson et al-. (1968) observed that some 93% o f the c e l l n i t rogen could be accounted for by ammonia-"*"~*N disappearance i n media with and without a d d i t i o n a l i d e n t i f i e d sources of n i t r o g e n . T h i s confirmed the f ind ings of Abou Akkada and Blackburn (1963) that B_. amylophilus H-18 produced r e l a t i v e l y smal l quant i t i e s of free amino a c i d s from p r o t e i n , and t h i s was i n common with other p r o t e o l y t i c rumen b a c t e r i a which appeared to u t i l i z e ammonia i n preference to preformed amino ac ids and pept ides . The func t ion of the protease from B_. amy lo ph i H-18 may be assoc iated wi th muco-peptide synthes i s . Whitaker (1965) p u r i f i e d an a and 8 - l y t i c protease from Sorangium Sp. which hydrolyzed muco-peptides from the c e l l wa l l s of Arthrobacter g lob i formis and Micrococcus l y s o d e i k t i c u s . Another p o s s i b l e funct ion of the p r o -t e o l y t i c a c t i v i t y may be the l i b e r a t i o n of e x o c e l l u l a r amylases. B,. amylophilus H-18 has an absolute requirement for maltose or for 1 , 4 - l i n k e d glucose polymers (Blackburn and Hobson, 1962) and the protease may a s s i s t the re lease of the amylase. Blackburn (1968 b) showed that the protease l i b e r a t e d by sonic d i s r u p t i o n of log phase c e l l s of B_. amylophilus H-18 was excluded from G-200 Sephadex (exc lus ion l i m i t s 200,000 M.W.) and could not be e a s i l y p u r i f i e d . In th i s respect i t resembled the p e n i c i l l i n a s e of B a c i l l u s  l i c h e n i f o r m i s which, when l i b e r a t e d by lysozyme treatment, appeared to be bound to membrane fragments (Lampen, et a l 1968). Therefore , i t was necessary to determine whether the protease l i b e r a t e d i n the s t a t i o n a r y phase was of small molecular weight, i n d i c a t i n g p r e -f e r e n t i a l l i b e r a t i o n , or whether i t was of large molecular weight and presumably membrane or p a r t i c l e bound. I f the l a t t e r were t r u e , there would be no advantage i n attempting i t s p u r i f i c a t i o n as s i m i l a r m a t e r i a l could more convenient ly be prepared from the whole, log-phase c e l l s . Blackburn (1968 b) suggested the presence of two proteases on the b a s i s ' o f d i f f e r e n t esterase to protease r a t i o s . I t was found that M i c k l e d i s i n t e g r a t i o n of l y o p h i l i z e d c e l l s suspended i n water gave good y i e l d s of protease wi th low p - to luenesu lphony l -L-arg in ine methyl 3 ester (TAME) esterase and c a s e i n - p r e c i p i t a t i n g a c t i v i t i e s . The TAME-es terase had a pH optimum at 8.0 but the protease had a broad optimum w i t h minor peaks at pH 6.5 and 8.0. There i s a complete i n h i b i t i o n of esterase a c t i v i t y with an 8 7% i n h i b i t i o n of protease a c t i v i t y by d i - i sopropy lphosphof luor idate (DFP) (Blackburn, 1968 b ) . This i s s i m i l a r to the family of Sorangium  Sp. of s er ine proteases of Whitaker (1967) who showed the l y t i c protease to be r e a d i l y i n h i b i t e d by DFP while the l y t i c enzyme was not . Since the protease from B_. amylophilus H-18 showed a v a r i e t y of d i f f e r e n t pH optima, ion-exchange chromatography on DEAE Sephadex A-50, g e l f i l t r a t i o n through Sephadex G-200 and i s o e l e c t r o f o c u s i n g were used i n an attempt to obta in separat ion of proteases . The main purpose of t h i s pro jec t was the p u r i f i c a t i o n of pH 6.7 p r o t e o l y t i c a c t i v i t y and the study of i t s proper t i e s to determine i t s homogeneity. The s i t u a t i o n of other p r o t e o l y t i c a c t i v i t i e s being p u r i f i e d s imi l taneous ly i s c o i n c i d e n t a l . D i s u l f i d e bridges may p o s s i b l y be involved i n the maintenance of the t e r t i a r y s t r u c t u r e of the protease . I t was therefore of i n t e r e s t to,measure the cys te ine content i n the protease; P o l l o c k (1962) demonstrated a low cyste ine content i n e x t r a c e l l u l a r b a c t e r i a l p r o t e i n s . More s p e c i f i c a l l y , he found an except iona l ly low content of cys te ine ' i n a v a r i e t y of b a c t e r i a l proteases and e x t r a c e l l u l a r enzymes. 4 MATERIALS Sephadex G-200, DEAE Sephadex A-50 and SE Sephadex C-50 were purchased from Pharmacia, Uppsala , Sweden. The media const i tuents and chemicals were of reagent grade and obtained from F i s h e r S c i e n t i f i c C o . , F a i r l a w n , New Jersey; so lub le s tarch was from The B r i t i s h Drug House, Poole , England. Bovine serum albumin and Na t o s y l - L - c h l o r o -methane HC1 and Na b e n z o y l - L - a r g i n i n e methyl es ter from Calbiochem, Los Angeles , . C a l i f o r n i a . The LKB f r a c t i o n c o l l e c t o r , Uvicord (0.3 cm l i g h t path) , and the 8100 Ampholine E l e c t r o f o c u s i n g Equipment were purchased from LKB Produkter AB, Stockholm, Bromma, Sweden. Other equipment inc luded a S e r v a l l Type SS-34 and GSA centr i fuge ; a KSB:R S e r v a l l continuous flow adaptor from S e r v a l l , Norwalk, Connect icut; MSE c e n t r i f u g e , London; D i a f l o Model 50 u l t r a f i l t r a t i o n c e l l from Amicon C o . , Lexington, Mass; Spectronic 20 spectrophotometer from Bausch and Lomb, Rochester, N . Y . ; Beckman Model 120 amino a c i d analyzer and Beckman Model E a n a l y t i -c a l u l t r a c e n t r i f u g e from Beckman Instruments, Palo A l t o , C a l i f o r n i a . A Radiometer Type PHM 28 was obtained from Radiometer L t d . , Copenhagen, D enmark. METHODS Determination of P r o t e i n ; P r o t e i n concentrat ion was measured by the method of Lowry e± a l (1951) us ing bovine serum albumin (BSA) as the s tandard. Determination of Protease A c t i v i t y : The assay procedure was a m o d i f i c a t i o n of the McDonald and Chen (1965) method. The r e a c t i o n mixture contained 0.2 ml of enzyme s o l u t i o n and 1.8 ml of a 2.0% (w/v) s o l u t i o n of case in i n 0.01% merth io la te , 0.1 M phosphate b u f f e r . The pH of the case in s o l u t i o n was 6.7 . Where necessary , reac t ions were run at other pH values by making the case in up i n var ious buf fers and adjus t ing the pH with e i t h e r NaOH or HC1. The. mixture was incubated at 38 C for vary ing lengths of time, de-pending on the concentrat ion of p r o t e o l y t i c a c t i v i t y w i t h i n a given sample. At the end of the incubat ion p e r i o d , 2.0 ml of 0.72 N t r i c h -l o r o a c e t i c a c i d (TCA) were added and mixed w e l l to p r e c i p i t a t e a l l the p r o t e i n . The p r e c i p i t a t e d digest was allowed to stand at room tempera-ture for 10 min. A f t e r c e n t r i f u g i n g at 1000 xg for 10 min, 1.0 ml o f the TCA so lub le p o r t i o n was removed with a p ipe t te and added to 5.0 ml of copper s o l u t i o n . The copper s o l u t i o n was prepared by adding 1.0 ml of 0.5% CuS0 4 i n 1.0% sodium c i t r a t e to 50 ml of 1.0 N NaOH i n 2.-0% Na?C0o. A f t e r 15 min incubat ion at 38 C, 0.5 ml of 1.0 N F o l i n 6 Reagent was added and mixed i n immediately us ing a Vortex-Genie mixer. The tubes were then incubated at 38 C for 30 min and the ex t inc t ions were read at 700 my on a Spectronic 20. The e x t i n c t i o n at zero time (where TCA was added before incubat ion) was subtracted from the e x t i n c t i o n of the t e s t and the correc ted value compared with a standard BSA curve (100 ug BSA g i v i n g an E^QQ of 0 .32) . A u n i t of p r o t e o l y t i c a c t i v i t y was def ined as the amount of enzyme which under standard condi t ions would s o l u b i l i z e the equiva lent of 1.0 ug BSA i n one min. There was a l i n e a r r e l a t i o n s h i p between the time of incubat ion and the amount of case in s o l u b i l i z e d . S p e c i f i c a c t i v i t y was defined as un i t s of protease a c t i v i t y per m i l l i g r a m of p r o t e i n . Determinat ion of Amylase A c t i v i t y ; One ml of enzyme was added to 1.0 ml of a 1.0% (w/v) s o l u t i o n of s o l u b l e s t a r c h i n 0.2 M Tris(hydroxymethyl)aminomethane, 0.1 M malate. The pH of the s tarch s o l u t i o n was 6 .9 . A f t e r 30 min incubat ion at 38 C, 2 .0 ml of stopper reagent were added. The stopper reagent was an a l k a l i n e s o l u t i o n of d i n i t r o s a l i c y l i c a c i d . A f t e r the stopper reagent was added to the enzyme mixture , i t was b o i l e d for 5 min cooled and d i l u t e d with 20 ml of water. The ex t inc t ions were read at 540 mu on a Spec tron ic 20. The e x t i n c t i o n of the c o n t r o l , which had the stopper reagent added immediately before i n c u b a t i o n , was subtracted from the e x t i n c t i o n of the tes t sample and t h i s corrected value was compared to a standard maltose curve (1.0 mg maltose per ml gave an of 0 .220) . A u n i t of amylase a c t i v i t y was defined as the amount of enzyme which under standard condit ions would s o l u b i l i z e the equivalent of 1.0 m i l l i g r a m maltose i n one min. Determinat ion of Esterase A c t i v i t y : Esterase a c t i v i t y was measured by the method of Schwert et a l (1948) by using a Radiometer Type PHM 28, Radiometer L t d . , Copenhagen, Denmark. The r e a c t i o n mixture contained 0.4 ml of 0.1 M es ter subs tra te , 0 .5 ml water, and 0.1 ml enzyme preparat ion and was incubated at 25 C under N ^ . The pH of the mixture was maintained by t i t r a t i o n with 0.1 N NaOH from a 0.5 ml micrometer s y r i n g e . I n h i b i t i o n of P r o t e o l y t i c A c t i v i t y with Na tosyl-L-chlofomethane(TLCM): A 0.1 ml sample of enzyme preparat ion was incubated wi th 0.9 ml -2 o f 1 x 10 M TLCM at 25 C for one h r . Then protease a c t i v i t y was assayed a t pH 5 .0 , 6 .7 , 8.0 and 11.0. Esterase a c t i v i t y of the treated enzyme was assayed at pH values 5.0 to 11.0. U l t r a c e n t r i f u g a t i o n : The enzyme sample was run i n a synthet ic boundary c e l l at 56,000 8 rev/min i n 0.1 M phosphate buf fer (pH 7.0); bar angle , 60 "•; p r o t e i n c o n c e n t r a t i o n , 0.3 mg/ml; p i c t u r e s were taken 5 min a f t e r a t t a i n i n g f u l l speed at 4 min i n t e r v a l s . Bacteroides amylophilus H-18 was i s o l a t e d by Blackburn and Hobson (1962) and was maintained as described by Blackburn (1964 a ) . The p r e p a r a t i o n and composition of the media were a l so descr ibed by Blackburn (1968 a ) . The basa l medium contained (g /1) : K 2 H P 0 4 , 0.45; K H T 0 4 , 0.45; (NH ) SO , 0.9; NaCl , 0.9; MgS0,.7 H O , 0.09; CaCl , 2 < _ ' T* 2 • 4 ^ 2 2 -0.09; r e s a z u r i n , 0.001; L-eys te ine HC1, 0.5; NaHC0 3 > 5.0; maltose, 3 .0 ; and t ryptose , 3 .0 . I s o l a t i o n of Proteases from B_. amylophilus H-18. A 32 l i t r e s t a i n l e s s s t e e l mi lk can conta in ing 29 l i t r e s of growth medium (Blackburn, 1968 a) was inocu la ted with a one l i t r e l og phase c u l t u r e of B_. amylophilus H-18 and was incubated anaerob ica l l y under C O 2 f or 23 hr at 38 C . Then the can and i t s contents were cooled r a p i d l y with a water hose and the c e l l s removed by continuous flow c e n t r i f u g a t i o n at 8700 xg at 4 C using a S e r v a l l c e n t r i f u g e . The opt imal pH for the attachment of the protease to the i o n -exchanger was found to be pH 5 .5 . Fortunate ly i t was not necessary to adjust the pH of the supernatant as the 23 hr c u l t u r e of B_. amylophi l i s H-18 had a pH of 5 .5 . DEAE Sephadex A-50 (0.2 g dry weight/100 ml) was added batchwise to the c u l t u r e supernatant and CO 9 was bubbled through the suspension overnight at 4 C to ensure good mixing. A f t e r a l lowing the suspension to s e t t l e , the supernatant was poured o f f and the DEAE c o l l e c t e d on a s i n t e r e d glass f i l t e r . The DEAE was suspended i n 500 ml of 0.1 M N a C l , the suspension mixed w e l l and then cen tr i fuged . The supernatant was poured o f f and s t o r e d . The DEAE was extracted i n t h i s way a t o t a l of s i x t imes. The f i r s t f i v e ex trac t ions ( t o t a l volume 2500 ml) were pooled and d i a l y z e d against 0.05 M phosphate buf fer (pH 7 .0) . A DEAE Sephadex A-50 column (5.0 cm x 100 cm) was e q u i l i b r a t e d with 0.05 M phosphate buf fer (pH 7.0) and the 2500 ml of d i a l y z e d p r o -tease passed through i t . The enzyme was e luted with a l i n e a r gradient of 0.2 M to 1.0 M NaCl i n the same buf fer and the f r a c t i o n s were tested f o r p r o t e o l y t i c a c t i v i t y at pH 6.7. The ac t ive f r a c t i o n s were pooled and d i a l y z e d against 0.05 M phosphate buf fer (pH 7 .0) . The s o l u t i o n was concentrated to 70 ml with a D i a f l o u l t r a f i l t r a t i o n c e l l , (40 p . s . i . ) , ( exc lus ion l i m i t s 10,000 M.W. ) . The concentrated s o l u t i o n was passed through a Sephadex G-200 column (5.0 cm x 90 cm) which had been e q u i l i b r a t e d with 0.05 M phosphate buf fer (pH 7 .0) . The protease was e luted with the same b u f f e r , f r a c t i o n s were c o l l e c t e d and the d i s t r i b u t i o n of p r o t e i n was e s tab l i shed by 280 my absorbance readings . I s o e l e c t r o f o c u s i n g of the Protease: Low molecular weight ampholines were used of the range pH 3.0 to 6.0 (8%) and pH 3.0 to 5.0 (40%). Both the ampholines and the protease were randomly d i s t r i b u t e d throughout the column before a p p l i c a t i o n of the p o t e n t i a l grad ient . For d e t a i l e d i n s t r u c t i o n s r e f e r to LKB 8100 Ampholine I n s t r u c t i o n Manual. Amino A c i d A n a l y s i s of the Protease: A protease sample was hydrolyzed under vacuum with 6 N HC1 for 18 h r at 105 C . Approximately 0.3 mg of p r o t e i n hydrolysate was analyzed u s i n g the Beckman amino a c i d ana lyzer . E f f e c t of Temperature on Protease A c t i v i t y : Protease a c t i v i t y at var ious temperatures was determined us ing 2.0% case in as the subs tra te . Samples of 2.0% case in i n 0.1 M phosphate b u f f e r (pH 6.7) were he ld at temperatures ranging from 30 C to 75 C i n a thermal gradient apparatus. The enzyme preparat ion was d i l u t e d 1/50 i n 0.05 M phosphate buf fer (pH 7.0) and 0.2 ml samples added to the preheated case in . The react ions were incubated for 45 min. G e l D i sc E l e c t r o p h o r e s i s : Ge l d i s c e l ec t rophores i s was c a r r i e d out i n a standard ge l 47%) which was stacked at pH 8.9 and run at pH 9.5 according to the method o u t l i n e d i n the Canalco Model 6 system i n s t r u c t i o n bookle t , Canal I n d u s t r i a l C o . , Bethesda, Md. The enzyme samples were run i n d u p l i c a t e . A f t e r e l e c t r o -phores i s one ge l was immediately s ta ined for p r o t e i n with a n i l i n e b l a c k , whi le the d u p l i c a t e ge l was used to assay for var ious a c t i v i t i e s . Protease a c t i v i t y and amylase a c t i v i t y were measured by l a y i n g the gels on a f l a t surface on an agar medium conta in ing an appropriate subs tra te . The enzymes could then d i f f u s e from the bands i n the g e l in to the agar 1ayer . Protease A c t i v i t y a t : pH 4.0 - An underlay of 0.8 M acetate buf fer (pH 35) with 2.0% agar was made i n a p e t r i d i s h . 1.0% casein plus 1.0% agar were mixed and added as an o v e r l a y . pH 6.0 - Glass s l i d e s were layered with 1.0% case in made up i n 0.2 M phosphate b u f f e r (pH 6.0) plus 1.0% agar. pH 11.0-! Glass s l i d e s were layered with 1.0 case in plus 1.0% agar made up i n 0.8 M sodium carbonate. Amylase A c t i v i t y : Glass s l i d e s were layered with 1.0% s tarch plus 1.0% agar made up with 0.2 M phosphate buf fer at pH 6.0 on ly . The d u p l i c a t e gels were placed on the var ious s l i d e s and p l a t e at 37 C for 30 min. The gels were then removed and a 12% H g C ^ , 16.5% H C 1 - s o l u t i o n was app l i ed to the case in s l i d e s and p l a t e . The s tarch l i d e s were f looded with L u g o l ' s i o d i n e . The r e s u l t s of the case in nd s tarch s l i d e s were compared to the p r o t e i n s ta ined acrylamide ge l RESULTS AND DISCUSSION E s t i m a t i o n of Molecular Weight of Crude Protease The c e l l - f r e e protease from a 48 hr c u l t u r e supernatant of B_. amylophilus H-18 was p r e c i p i t a t e d by sa tura t ing the s o l u t i o n with ammonium s u l f a t e . The s o l u t i o n was allowed to stand i n the co ld (4 C) for 12 hr and the p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g i n g at 8700 xg for 40 min. The p r e c i p i t a t e was d i s so lved i n 0.05 M phosphate b u f f e r , pH 7.0 and brought up to a volume of 3.0 ml . F igure 1 shows the e l u t i o n p a t t e r n obtained upon f i l t r a t i o n of the r e d i s s o l v e d p r e c i p i t a t e through a Sephadex G-200 column (2.5 cm x 45 cm) which was equipped with an upward flow adaptor. The column was e q u i l i b r a t e d and the protease e luted with 0.05 M phosphate b u f f e r , pH 7.0. The p r o t e o l y t i c a c t i v i t y appeared i n four peaks. Peak A was i n the v o i d volume region and the molecular weight was estimated to be greater than 1,000,000 (Andrews, 1961). The protease i n peak B had a molecular weight of approximately 130,000 and was of high s p e c i f i c a c t i v i t y . Peaks C and D had higher p r o t e i n concentrat ions but the molecular weight of the protease was l e s s than 100,000, suggesting that the protease was no longer cova lent ly attached to membrane fragments as i n the s i t u a t i o n found by Blackburn (1968 b ) . Gel f i l t r a t i o n of ammonium sulphate p r e c i p i t a t e d crude enzyme through Sephadex G-200 (2.5 cm x 45 cm). E luent : 0.05 M phosphate b u f f e r , pH 7.0. Sample load: 6 mg of p r o t e i n i n 3.0 ml +, Blue dextran from a separate r u n . 14 P r o p e r t i e s of the Separated F r a c t i o n s from Gel F i l t r a t i o n ; : Table I summarizes the proper t i e s of protease a c t i v i t y at var ious pH values of the peaks A to D obtained from ge l f i l t r a t i o n using Sephadex G-200. The r a t i o s E „ „ „ : E i n d i c a t e d that the amounts 280 260 of n u c l e i c a c i d present i n peaks A , B, C and D were 3.6%, 3.8%, 5.0% and 4.2% r e s p e c t i v e l y (Layne, 1957). This was al.lower n u c l e i c a c i d content compared to the 6.0% n u c l e i c a c i d found by Blackburn (1968 b ) , suggesting that l i t t l e c e l l l y s i s had taken p l a c e . I f only a s i n g l e protease was present , the r a t i o o f . a c t i v i t i e s at the var ious pH values i n each f r a c t i o n should have been the same. Although the a c t i v i t y was greatest at pH 6.7 i n a l l four f r a c t i o n s , the r a t i o s of a c t i v i t i e s at var ious pH values d i d d i f f e r from f r a c t i o n to f r a c t i o n . However, enzyme complexes may not have the same pH p r o f i l e as an enzyme free i n s o l u t i o n . Peak B showed a p u r i f i c a t i o n as high as e ight times i n the case of the pH 6.7 va lue . I t therefore appeared that Sephadex G-200 ge l f i l t r a t i o n would be a u s e f u l step i n the p u r i f i c a t i o n of protease from 15. amylophilus H-18. Time Course of Protease L i b e r a t i o n : P r e l i m i n a r y experiments showed that p r o t e o l y t i c enzymes, a c t i v e over a wide pH range, were l i b e r a t e d in to the c u l t u r e supernatant. F r a c t i o n T o t a l E 2 o n : E 9 f i n P r o t e i n Protease A c t i v i t y S p e c i f i c A c t i v i t y P u r i f i c a t i o n M o l . Volume (Ug/ml) U n i t s / m l Wt. (Ratio) pH pH pH 5.0 6.7 8.0 11.0 5.0 6.7 8.0 11.0 5.0 6.7 8.0 11.0 A . 20 ml 0.965 2.88 0.38 2.12 - 0.10 132 736 - 347 1.13 6.5 - 3.37 10 6 (1.8) (10) - (0.47) B. 2 5 m l 0.945 2.88 0.65 2.60 0.54 0.71 226 900 187 247 1.92 8.0 3.2 2.40 130,000 (2.5) (10) (2.0)(2.7) C. 3 0 m l 0.875 28.8 2.36 4 . 5 7 1 . 4 7 0.98 82 162 51 34 - 1.4 - - 20,000 (1.15) (10) (0.9)(1.1) D. 35 ml 0.900 31.4 0.32 2.83 0.27 0.32 10 9.3 8.6 10 - - 5,000 Disso lved ( N H 4 ) 2 S 0 4 3.0 ml 0.915 2000 235 226 117 206 117 113 58 103 P r e c i p i t a t e Table 1. Proper t i e s at various pH values of f r a c t i o n s from Sephadex G-200 g e l f i l t r a t i o n and from d i s so lved ammonium s u l f a t e p r e c i p i t a t e . Since i t seemed probable that no s i n g l e enzyme could be re spons ib l e , the time course of the l i b e r a t i o n of protease a c t i v i t y at pH 5.0 , 6 . 7 , 8.0 and 11.0 was examined i n the hope that the pH 6.7 a c t i v i t y might be l i b e r a t e d at a d i f f e r e n t time from the other a c t i v i t i e s . The c h a r a c t e r i s t i c sharp f a l l i n c e l l dens i ty once the maximum was reached, d i d not co inc ide with a l i b e r a t i o n of protease a c t i v i t y ( F i g . 2 ) . However, on prolonged i n c u b a t i o n , the propor t ion of c e l l - f r e e protease increased and t h i s and the sharp decrease i n cu l ture densi ty at the end of l ogar i thmic growth i s c h a r a c t e r i s t i c of many rumen b a c t e r i a (Bryant and Robinson, 1961). This substant iated the data ( F i g . 1) that the c e l l - f r e e protease was not attached to d i s i n t e g r a t e d c e l l p a r t i c l e s . Protease , a c t i v e at a l l the pH values examined, was l i b e r a t e d p r o g r e s s i v e l y throughout the logar i thmic and s ta t ionary phases, reaching a maximum 12 hr a f t e r the c u l t u r e had reached maximum c e l l dens i ty ( F i g . 2 ) . F l u c t u a t i o n s i n protease a c t i v i t y occurred but these f l u c t u a t i o n s were co inc ident for protease a c t i v i t y over the pH range, which suggested that one enzyme might be respons ib le for a l l the a c t i v i t y which was observed. The time course of amylase l i b e r a t i o n was a l so fol lowed and was seen to reach a peak a c t i v i t y much e a r l i e r , only 5 hr a f t e r maximum c e l l dens i ty ( F i g . 2 ) . Th i s a c t i v i t y a l so f luc tuated subsequently, but not i n ' t h e same pat tern as the protease a c t i v i t y . F i g u r e 2. C e l l dens i ty ( 0 - 0 ) , protease a c t i v i t y at pH 5.0 (A - A ) , 6.7 (X - X ) , 8.0 (G - B ) , 11.0 ( • - O ) and amylase (9 - fi) i n the c u l t u r e supernatant are p l o t t e d against hours of i n c u b a t i o n . Protease P u r i f i c a t i o n : The procedure i s descr ibed i n the Methods and the r e s u l t s are summarized i n Table II. Growth: A good y i e l d of protease was obtained i n the 23 hr c u l t u r e supernatant . This represented 80% of the t o t a l protease ( c e l l -bound and c e l l - f r e e ) which had reached a maximum at 13 h r . A sample of the supernatant was d i a l y z e d thoroughly against 0.05 M phosphate b u f f e r , pH 7.0, to remove the cyste ine and tryptose peptides so that the p r o t e i n content could be determined a c c u r a t e l y . Concentrat ion: The concentrat ion step was s u c c e s s f u l l y accomplished by b inding the protease to DEAE-Sephadex. The e lu ted , d i a l y z e d protease represented 75% of the s t a r t i n g m a t e r i a l . Although t h i s step gave only a 4 - f o l d p u r i f i c a t i o n , i t d i d reduce the volume of enzyme s o l u t i o n from 29 to 2.6 l i t r e s . DEAE-Chromatography: Further p u r i f i c a t i o n (3- fo ld) and concentrat ion (2,600 ml to Table I I . P u r i f i c a t i o n of I5_. amylophilus H-18 protease . Recoveries are c a l c u l a t e d as i f a l l m a t e r i a l was used. Sample pH.of Volume Uni ts T o t a l P r o t e i n Sp. A c t . X Pur % assay /ml Uni ts (mg/ml) recov. I. 23 hr sup. 5.0 29 1 7.2 209,000 0.317 22.7 1 100 6.7 29 1 9.4 270,000 0.317 29.7 1 100 8.0 29 1 8.0 232,000 0.317 25.4 1 100 11.0 29 1 8.5 246,000 0.317 27.0 1 100 2. DEAE e l u t i o n 6.7 2600 ml 78.0 202,000 0.618 126 4 75 3. DEAE column #57-84 F i g . 3 6.7 500 ml 270 135,000 0.725 373 126 50 4. DEAE column #85-89 F i g . 3 6.7 90 ml 62 5,580 1.010 61.5 2.8 2.0 5. # 3 cone. 5.0 71 ml 4800 340,000 4.0 1200 53.3 116 6.7 71- ml 4670 330,000 4.0 1165 40.7 126 8.0 71 ml 3260 228,000 4.0 815 32.1 84 11.0 71 ml 4300 308,000 4.0 1070 39.6 125 6. 40 ml #5 through Seph-adex G-200 6.7 230 ml 420 96,500 0.056 7440 251 62.i #69-87 F i g . 4 7. 31 ml #5 through Seph-adex G-200 A . #71-81 6.7 138 ml 284 39,200 0.056 5040 170 31.6 B. #83-93 6.7 138 ml 112 15,300 0.058 1930 65 12.3 #6 cone. 5.0 35.5 ml 3700 131,000 0.330 11,200 494 107.0 6.7 35.5 ml 3900 138,000 0.330 11,900 404 89.1 8.0 35.5 ml 2770 98,000 0.330 8,400 286 83.0 11.0 35.5 ml 3800 135,000 0.330 11,500 425 108.0 #7 cone 5.0 14.0 ml 5600 78,500 0.340 16,500 725 85.0 6.7 14.0 ml 5370 75,000 0.340 16,100 545 62.7 A . 8.0 14.0 ml 4600 64,500 0.340 13,500 455 85.0 11.0 14.0 ml 5350 75,300 0.340 15,700 580 69.4 #7 cone. 5.0 11.5 ml 2600 30,000 0.330 7,900 348 32.1 6.7 11.5 ml 2580 30,800 0.330 7,800 265 24.8 B. 8.0 11.5 ml 2150 24,800 0.330 6,500 256 24.1 11.0 11.5 ml 2500 28,800 0.330 7,600 281 26.6 E l e c t r o -focus ing Peak I 6.7 1.9 ml 3760 7,140 0.19 37,600 1265 27.7 Peak I I 6.7 0.9 ml 704 633 0.87 7,270 245 2.7 500 ml) were achieved by adsorpt ion on a DEAE-Sephadex column with subsequent 'e lut ion by a sodium c h l o r i d e gradient ( F i g . 3 ) . In t h i s s tep , 67% of the protease a c t i v i t y was recovered i n a s i n g l e peak (Table I I , #3), with only 3% of low s p e c i f i c a c t i v i t y protease i n the t r a i l i n g shoulder of t h i s peak. Concentrat ion of the pooled protease l ed to an apparent increase i n a c t i v i t y (244%) and a decrease i n t o t a l p r o t e i n , r e s u l t i n g i n a 3 . 0 - f o l d p u r i f i c a t i o n . At th i s po in t the protease a c t i v i t i e s at pH 6.7 and pH 8.0 had both been p u r i f i e d 40 - fo ld o v e r a l l . Nei ther had the protease a c t i v i t i e s at pH 5.0 and 11.0 been separated from the pH 6.7 protease . I t appeared, there-f o r e , that a s i n g l e enzyme was respons ib le for the protease a c t i -v i t i e s over the range pH 5.0 to 11.0. A po int of cons iderable i n t e r e s t was the f a i l u r e of the p u r i f i c a t i o n steps employed to t h i s po int to separate the amylase from the protease . G-200 Sephadex:; .' . Pre l iminary experiments (Table I ) , i n d i c a t e d ge l f i l t r a t i o n to be a u s e f u l method i n r e s o l v i n g the protease a c t i v i t y at d i f f e r e n t pH values which were used i n the assays. Some of the concentrated enzyme s o l u t i o n obtained a f t e r the DEAE-Sephadex step was passed through a G-200 Sephadex column (Table I I , #6). Protease a c t i v i t y was e luted i n a s i n g l e peak, termed peak A , Chromatography of B_. amylophilus H-18 protease on DEAE Sephadex A-50 . E luent : 0.05 M phosphate b u f f e r , pH 7.0; a l i n e a r gradient of NaCl (0.2 to 0.75 M) was app l i ed f o r the e l u t i o n of p r o t e i n . with a 36.7% recovery and a 6 . 4 - f o l d p u r i f i c a t i o n . When the pooled f r a c t i o n s were concentrated again , there was an apparent increase i n p r o t e o l y t i c a c t i v i t y , r e s u l t i n g i n a 143% recovery and a 1 . 6 - f o l d increase i n a c t i v i t y (Table I I , #8). The p r o -tease was c a l c u l a t e d (Andrews, 1964) to have a molecular weight i n the 60,000 range ( F i g . 4 ) . The Sephadex G-200 column was r e -packed to improve i t s flow c h a r a c t e r i s t i c s . When the remainder of the concentrated enzyme s o l u t i o n obtained a f t e r the DEAE Sephadex step was passed through t h i s column, the protease a c t i v i t y was e luted i n two peaks ( F i g . 5 ) . The enzyme i n one peak ( F i g . 5*.;B) had a molecular weight of approximately 60,000. The enzyme i n the second peak ( F i g . 5, C) had a molecular weight of approximately 27,000. Of the enzyme app l i ed to the column, 27% appeared i n peak B, with a 4 . 3 - f o l d p u r i f i c a t i o n , and 10.4% appeared i n peak C with a 1 . 7 - f o l d p u r i -f i c a t i o n . When the pooled f r a c t i o n s were concentrated again there was an apparent increase i n p r o t e o l y t i c a c t i v i t y : 3 .2-f o l d and 4 . 0 - f o l d for B and C r e s p e c t i v e l y , with recover ies of 191% and 201% (Table I I , #9). The sum of the t o t a l a c t i v i t y i n peaks A , B and C represented a 90% recovery of the p r o t e o l y t i c a c t i v i t y present i n the o r i g i n a l 29 l i t r e s of cu l ture medium. The p o s s i b i l i t y was recognized that the protease i n peak B might be a dimer of that i n peak C. Corroborat ive evidence for th i s was found i n the fac t that both peaks contained protease F i g u r e . 4. Ge l f i l t r a t i o n of a p o r t i o n of sample #5 (Table II) through a Sephadex G-200 column (5.0 cm x 90 cm). E luent : 0.05 M phosphate b u f f e r , pH 7.0. Sample Volume U n i t s / m l T o t a l P r o t e i n S p . A c t . T o t a l X Pur % (Table II) Uni ts (mg/ml) P r o t e i n (mg/ml) Recov #5 app l i ed 40 ml 4670 263,000 2.25 1165 90.0 A #69-85 (#6) 230 ml 420 96,500 0.056 7440 13.0 6.4 36.7 A cone. (#8) 35.5 ml 3900 138,000 0.33 11900 11.7 1.6 143.0 ©— © pH6.7 PROTEASE ACTIVITY (UNITS/ml) * 8 T « pH 6.7 P R O T E A S E ACTIVITY(UNITS/ml) q8i a c t i v e i n the range pH 5.0 to 11.0 and thus very s i m i l a r i n p r o p e r t i e s , although d i f f e r e n t i n s i z e . When g e l f i l t r a t i o n of peak C ( F i g . 5) was repeated using Sephadex G-200, the protease appeared i n the 60,000 rather than the 30,000 molecular weight range where i t was prev ious ly i s o -l a t e d . This r e i n f o r c e d the hypothesis that the 60,000 and 30,000 molecular weight proteases were i n e q u i l i b r i u m with the dimeric form predominating. U l t r a c e n t r i f u g a t i o n : U l t r a c e n t r i f u g a t i o n of peak C ( F i g . 5) revealed only one homogenous peak ( F i g . 6 ) . Since the repeated run of peak C on Sephadex G-200 gave only 60,000 molecular weight protease-, i t i s reasonably safe to assume that the s i n g l e peak obtained from the u l t r a c e n t r i f u g a t i o n of peak C i s the 60,000 molecular weight protease . E f f e c t of pH on Protease A c t i v i t y : I t was considered unusual that protease a c t i v i t y at a l l pH values from 5.0 to 11.0 should remain together despi te the cons iderable degree of p u r i f i c a t i o n . Blackburn (1968 b) showed a c t i v i t y over a wide range and Blackburn's unpublisheed r e s u l t s D Figure 6. The enzyme sample was run i n a synthet ic boundary c e l l at 56,000 rev/min i n 0.1 M phosphate buf fer (pH 7 .0) . P i c tures were taken 5 min a f t er a t t a i n i n g f u l l speed at 4 min i n t e r v a l s . 03 indicated proteolytic activity over a pH range from 4.0 to 11.0. It was assumed that the peaks of activity 5.0, 6.7, 8.0 and 11.0 were due to different proteases. However, this may not be the case, since Hofsten et a l (1965) characterized a protease from an Arthrobacter which demonstrated proteolytic activity from pH 4.0 to 11.0 and Matsubara et a l (1958)crystallized a protease from Bacillus subtilis N which was active from pH 5.5 to 9.5. The protease i n peak A was active over the pH range from pH 4.5 to 11.5 (Fig. 7). There were major peaks of activity at pH 5.0 and 11.5, with minor peaks at pH 8.0 and possibly 9.5. Thus, unlike the single broad peak of activity obtained by Matsubara et a l (1958), there were distinct peaks of activities at either ends of the pH activity range. Since the curve of protease activity against pH (Fig. 7) for the purified protease showed pH optima differing from those of the starting material, the possibility again had to be con-sidered that more than one protease was present. If so, the fact that the act i v i t i e s had not been separated from each other at this point i n the purification indicated that they might be closely related in structure. Isoelectrofocusing: Preliminary evidence from DEAE-Sephadex absorption studies E f f e c t of pH on a c t i v i t y of p u r i f i e d protease (Table II #8). React ion mixture contained, (0.2 m l ) , enzyme and (1.8 m l ) , 2% case in (pH 4.5 to 12 .0) . 20a 2000f co z 1800 1600 1400 1200 > i= 1000f y $ 800f < uJ o or 600f 400-200r 4.5 50 55 6.0 65 7.0 7.5 8.0 8.5 9.0 9.5 10.010.511.0 11.512.0125 pH suggested that the protease was an a c i d i c p r o t e i n with an i s o e l e c t r i c po int below- pH 5.0 . Attempts to bind the protease to the c a t i o n exchanger, SE-Sephadex, C-50, at pH 4.2 r e s u l t e d i n loss of protease a c t i v i t y , although the protease i t s e l f was s tab le at low pH va lues . Therefore , I soe lec tro fucus ing was used to p u r i f y the protease fur ther and to separte the proteases ac t ive at d i f f e r e n t pH va lues . The s t a r t i n g m a t e r i a l i n the e l ec t ro focus ing experiment was protease which appeared i n the 60,000 molecular weight range a f t e r g e l f i l t r a t i o n with Sephadex G-200 (Table I I , #8). P r o t e o l y t i c a c t i v i t y i n the f r a c t i o n s obtained from the e l ec t ro focus ing experiment appeared i n two major regions ( F i g . 8 ) . The p r o -t e o l y t i c a c t i v i t y at pH 5.0 , 6 .7 , 8.0 and 11.0 a l l gave the same p r o f i l e i n the two major peaks. The enzyme i n peak I had an i s o e l e c t r i c po int at pH 4 .3 , that i n peak I I an i s o e l e c t r i c po int at pH 7.95. D i sc g e l e l ec trophores i s of concentrated peak I enzyme showed the presence of proteases but no amylase, even though the s t a r t i n g m a t e r i a l contained amaylase. The fac t that only one band of a c t i v e protease was seen a f t er d i s c ge l e l e c t r o -phoresis . could be accounted for as the length of ge l used was too short to obta in the r e s o l u t i o n required to observe two p r o t e o l y t i c components. Therefore i t was s t i l l p o s s i b l e that the dimeric and monomeric forms ex i s ted i n peak I . Disc ge l e l ec trophores i s of peak II enzyme revealed very many p r o t e i n s . I t was l i k e l y that Figure 8. Protease p r o f i l e from an e l ec tro focus ing experiment with a superimposed pH curve (pH 3.0 to 6 .0) . Symbols: Protease a c t i v i t y at pH 5.0 (0 - 0) , 6.7 ( 0 - 0 ) , 8.0 (A - A) and 11.0 ( - ) . Sample Volume U n i t s / m l T o t a l P r o t e i n S p . A c t . X Pur % (Table II) Units T o t a l Recov. (mg/ml) #8 appl i ed 5.8 ml 3900 22,700 1.91 11,900 Peak I (#53-63) 1.9 ml 3760 7,140 0.19 37,600 3.2 31 Peak II (#84-92) 0.9 ml 704 633 0.87 7,270 0.6 3 - , 6 5 JL 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 9 0 95 FRACTIONS (1.0ml) peak I I contained degradation products of protease molecules , some of which s t i l l r e ta ined t h e i r p r o t e o l y t i c a c t i v i t y . The contents of the tubes i n each peak were pooled, d i a l y z e d to remove ampholines and then concentrated. The p r o t e i n contents of these concentrated so lut ions were very low and could not be determined by the Lowry Method without using a l l the m a t e r i a l . Consequently, the p r o t e i n contents were c a l c u l a t e d from the recovery of E absorbing m a t e r i a l and the known p r o t e i n 2o0 my contents of the sample run i n the e l ec t ro focus ing apparatus (Table I I , #8). For the s o l u t i o n peak I there was a 3 . 2 - f o l d p u r i f i c a t i o n with a 32% recovery of pH 6.7 protease . In t h i s experiment the p o t e n t i a l gradient was app l i ed for 48 h r , a f t e r which time there was v i s i b l e p r e c i p i t a t i o n of p r o t e i n w i t h i n the e l e c t r o f o c u s i n g column. The experiment was repeated us ing a narrow range of ampholites (pH 3.0 to 5.0) and a 24 hr grad ient . In t h i s case, the i s o e l e c t r i c point of the enzyme i n peak I was at pH 4.25. However, a p p l i c a t i o n of the p o t e n t i a l gradient for 24 hr was i n s u f f i c i e n t time to concentrate the protease and so the peak of a c t i v i t y was more d i f f u s e than i n the 48 hr run . The p u r i f i e d protease (Table I I , #8) had now been separated in to two components by both ge l f i l t r a t i o n and by e l e c t r o f o c u s i n g . However, s ince the protease pH a c t i v i t y p r o f i l e s were i d e n t i c a l a f t e r each of these s teps , i t was s t i l l pos s ib l e that there was only a s i n g l e protease or perhaps a fami ly of very c l o s e l y r e l a t e d proteases , as was prev ious ly suggested. Acrylamide Gel E l e c t r o p h o r e s i s : Acrylamide ge l e l ec trophores i s ( F i g . 9) of the p u r i f i e d protease preparat ion (Table I I , #8) tended to confirm the data from the g e l f i l t r a t i o n : that two proteases were present which were presumably being separated on the bas i s of s i z e or charge. Therefore , ' i t was a n t i c i p a t e d that samples #9 A and #9 B (Table II) conta in ing proteases with molecular weights of 60,000 and 30,000 r e s p e c t i v e l y , would give d i f f e r e n t g e l e l e c t r o p h o r e t i c p a t t e r n s . In fac t they both gave the same protease pa t t ern as that shown i n F i g . 9, the only d i f f erence being the absence of three amylase bands from the 30,000 molecular weight f r a c t i o n . Each of the protease bands showed p r o t e o l y t i c a c t i v i t y from pH 4.0 to 11.5. Thus, there was no q u a l i t a t i v e d i f f erence between t h e i r a c t i v i t i e s ; q u a n t i t a t i v e l y protease band #1 contained more p r o t e i n and more p r o t e o l y t i c enzyme a c t i v i t y . A poss ib l e explanation, for these r e s u l t s i s that the protease i s an e q u i l i b r i u m mixture of a monomeric and dimeric form, the dimeric form (protease band #1, F i g . 9) predominating. Disc ge l e l ec trophores i s of a sample from e l e c t r o f o c u s i n g peak I a l so showed the protease p r o t e i n band #1 but the amylase bands were mis s ing . Al though, i t was c l e a r that amylase a c t i v i t y could be separated from protease a c t i v i t y , the 23al ;Acrylamide .Gel * ' • v.i(stained) >/';" 1% Casein 1% Starch mi Amylase ' Amylase. • 2 Protease 1 Amylase ;'3' Protease 2 i/nknowfi' p r o t e i n pH 4.0- . • pH.6.0 •. pH 11.5 : :v;;c,:4;:: \;'fA,V::..A .»•  •.'.r.,Pi1gure'*'9"iV:"- V.,r it- :^  M n •'JVA.tVi'. I lie '.' pH ,6.0-Represfehta'tiori of X, acrylamide e l ec trophores i s gel'-'with • stained; p r o t e i n bands. B, case in s l i d e s at various' pH values;.-after incubat ion at" 37 :C for 30 min.'with acrylamide . ge l s . -is of. a s tarch s l i d e pH 6:0, a f t e r s i m i l a r t r e a t -ment.-.'The unhydrolyzed casein,was' p r e c i p i t a t e d with HgC^' and the unhydrolyzed stairch was complexed with L u g o l ' s • i o d i n e . • . - . ' <• •' '• • •''. fac t that i t was unresolved by ge l f i l t r a t i o n from the protease whi le the l a t t e r was p u r i f i e d 404-fo ld (Table I I , #6) i n d i c a t e d that the two types of enzymes were very s i m i l a r s t r u c t u r a l l y . Protease C h a r a c t e r i z a t i o n : E f f e c t of Chemical Reagents on the Protease: I f the protease i n sample #8 (Table II) was a dimer, i t should have been p o s s i b l e to d i s s o c i a t e i t chemical ly in to the 30,000 molecular weight form protease , s ince sample #5 (Table II) was separable i n t o two components of d i f f e r i n g molecular weight by means of ge l f i l t r a t i o n . The low concentrat ion of p r o t e i n i n sample #8 (Table II) precluded the use of the Lowry Method for the detec t ion of protease p r o t e i n i n the f r a c t i o n s obtained from ge l f i l t r a t i o n . Therefore , the chemical techniques used i n an attempt to d i s s o c i a t e the d imeric form of the protease would have to be such that the p r o t e o l y t i c a c t i v i t y of the enzyme was not de-stroyed during prolonged exposure to the chemicals . To t h i s end, 10 M EDTA and 4 M urea were used as agents to break bonds which may have been b ind ing the dimeric form of the proteases together (Figures 10 and 11). In both cases, the protease appeared only i n the 60,000 molecular weight reg ion a f t e r ge l V = 54.5ml o V = 115.2 ml e V /V = 2.12 e o Molecular Weight = 60,000 Figure 10. Gel f i l t r a t i o n of(Table II, #8) which had been pre-treated with 10~3 M EDTA. Assay mixture contained 0.2 ml of each fraction and 1.8 ml, 2.0% casein, pH 6.7. t Blue dextran. V = 51.2 ml o V = 99.2 ml e V /V = 1.94 e o Molecular Weight = 60,000 Figure 1 1 . Ge l f i l t r a t i o n of (Table I I , #8) which had been p r e -treated with 4 M urea . Assay mixture contained 0.2 ml of each f r a c t i o n and 1.8 ml , 2.0% case in , pH 6 .7 . + Blue dextran. f i l t r a t i o n wi th Sephadex G-200. Treatment of sample #8 (Table II) wi th sodium dodecyl sulphate 10 ^ M concentrat ion , completely i n a c t i v a t e d the protease making i t impossible to loca te a f t e r g e l f i l t r a t i o n . Amino A c i d A n a l y s i s : Amino a c i d ana lys i s of the protease (Table I I , #8) from B_. amylophilus H-18 revealed a complete absence of cys te ine , so that d i s u l f i d e bridges could not be involved i n the maintenance of t e r t i a r y s t r u c t u r e and enzymic a c t i v i t y of the protease molecule The amino a c i d composition reported i n Table I I I i s compatible wi th the molecular weight obtained using Sephadex G-200. That i s , 27,000 and 60,000 molecular weight protease . That no s u l f h y d r y l groups were present was confirmed by the f a i l u r e of p-chloromercuribenzoate to i n a c t i v a t e or even react with the enzyme. Hofsten et a l (1965) reported h a l f - c y s t e i n e to be absent from the ox id ized form of protease p u r i f i e d from an Arthrobacter Sp P o l l o c k (1962) demonstrated a low cyste ine content to be a common feature of e x t r a c e l l u l a r b a c t e r i a l p r o t e i n s , i n c l u d i n g a v a r i e t y of proteases and e x t r a c e l l u l a r enzymes. Table I I I . Amino a c i d composition of the _B. amylophilus H-18 protease (Table I I , #8). 0.3 mg of protease p r o t e i n was analyzed. The molar r a t i o was adjusted to give a molecular weight of 30,000 by having methionine represent 3 res idues . Amino A c i d Residue umoles Ugram Molar Molecular Weight of Rat io Amino Acids x Number of Residues Lys ine H i s t i d i n e A r g i n i n e A s p a r t i c A c i d Threonine Serine Glutamic A c i d P r o l i n e Glyc ine Alan ine H a l f - c y s t i n e V a l i n e Methionine I so leuc ine Leucine Tyros ine Phenylalanine 0.24428 0.07301 0.17281 0.34727. 0.18901 0.13958 0.33579 0.24909 0.32026 0.20847 0.17615 0.03546 0.09903 0.12732 0.04940 0.07219 35.70 11.33 30.10 44.22 22.53 14.67 61.65 28.67 24.04 18.15 20.64 5.30 13.00 16.70 9.00 12.00 20 6 14 30 15 11 27 20 26 17 14 3 8 10 4 4 2923 886 2488 3802 1702 1201 4983 2302 1931 1527 1673 426 1124 1349 719 944 T o t a l 367ug 29,980 E f f e c t of Temperature on Protease A c t i v i t y : The optimum temperature for a c t i v i t y of the p u r i f i e d protease (Table I I , 7/8), was between 60 C and 65 C ( F i g . 12 ) . Th i s i s s i m i l a r to the proteases-of Pseudomonas aeruginosa (Morihara, 1963) and Mucor p u s i l l u s (Somkuti and Babel , 1968). An Arrhenius p l o t showed a l ogar i thmic increase i n enzyme a c t i v i t y from 30 C to 50 C ( F i g . 13) . Above 50 C there was an i r r e v e r s i b l e i n -a c t i v a t i o n of the enzyme. Heat S t a b i l i t y of the Protease: Since the 60,000 molecular weight protease appeared to predominate (Table I I , #9), i t was pos s ib l e that t h i s was the more s tab le of the two forms of the enzyme. Therefore , i t was u s e f u l to study the heat s t a b i l i t y of the two forms of the enzyme ( i . e . Samples #9 A and #9 B. from Table I I , for the 60,000 and 30,000 molecular weight forms, r e s p e c t i v e l y ) . An i n d i c a t i o n of the presence of m u l t i p l e proteases might be seen i f there were vary ing degrees of i n a c t i v a t i o n of p r o t e o l y t i c a c t i v i t y assayed at d i f f e r e n t pH va lues . The heat s t a b i l i t i e s of samples #9 A and #9 B (Table II) were examined under var ious condi t ions ( F i g . 14 and 15). Pretreatment of the protease samples with -2 10 M EDTA d i d not make them more suscept ib le to heat denaturat ion Figure 12. Effect of temperature on protease activity. Assay mixtures contained 0.3 mg of enzyme per ml and 2.0 mg of casein (pH 6.7) per ml. 26a I \ \ \ I » i L i ; i i i i i 1 1 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 T E M P E R A T U R E C ° ' Figure 13. Arrhenius p l o t of protease a c t i v i t y at pH 6.7. 26b Figure 14. Heat s t a b i l i t y of (Table I I , #9 B) . Enzyme p r o t e i n was exposed to temperatures ind ica ted for .15 min. Symbols: Calcium I O " 3 M added to EDTA 10~ M treated enzyme, (© - 8 ) , EDTA treated enzyme, (• - B ), and untreated enzyme, (A - A ) . 26c E oo 3500 3000 > h-U < L U C O < LU o or o_ r x CD IE CL 2500 2000 1500 1000 500 30 35 40 45 50 55 60 65 70 75 TEMPERATURE C° Figure 15. Heat stability of (Table II, #9A). Enzyme protein was exposed to temperatures Indicated for 15 min. ? Symbols: Calcium 10~3 M added to EDTA 10~ M treated enzyme, (9-9), EDTA treated enzyme, ( • - fil ) , and untreated enzyme, (A - A). 27 but ra ther i t gave an increase i n p r o t e o l y t i c a c t i v i t y of 8.0 to 10%. Thi s phenomenon could p o s s i b l y be caused by the c h e l a t i o n of i n h i b i t o r y metal ions by EDTA. The observat ion that c h e l a t i n g agents d i d not i n h i b i t protease a c t i v i t y suggested that the protease was not a metalloenzyme. In t h i s respect , the enzyme of B_. amylophilus H-18 resembles the proteases of A s p e r g i l l u s oryzae (Buguish, 1963), Paecilomyces v a r i o t ; (Sawada, 1964) and Rhizopus ch inens i s : (Fukumoto et a l 1967). Blackburn (1965) treated extracts of protease der ived from d i s i n t e g r a t e d c e l l s of ]3. -2 amylophilus H-18 with 10 M EDTA and observed an 8.0% increase -3 i n a c t i v i t y of the protease . A d d i t i o n of calc ium ions (10 M) increased by 10% the a c t i v i t y of both the EDTA treated and untreated protease at lower temperatures. However, the temperature of i r -r e v e r s i b l e denaturat ion of sample #8 (Table II) was equal for a l l four d i f f e r e n t pH values of protease a c t i v i t y , which were not the r e s u l t s to be expected i f there were a number of proteases present , (Table I V ) . A c t i v e S i t e I n h i b i t i o n of the Protease: The esterase and protease a c t i v i t i e s were compared i n an e f f o r t to determine whether one or more enzymes were producing the p r o t e o l y t i c a c t i v i t y found between pH 4.5 and 11.5. Blackburn (1968 b) noted a change i n the r a t i o of esterase a c t i v i t y : protease a c t i v i t y between var ious extracts obtained from d i s i n t i g r a t e d c e l l s 27a. Tab le IV. Heat denaturat ion of enzyme sample #8 (Table II) for vary ing lengths of time at 60 C. Percentage of A c t i v i t y Remaining Incubation Time at 60 C. pH 5.0 pH 6.0 pH 8.0 pH 11.0 Zero Time 100 100 100 100 10 minutes 100 91 82 84 20 minutes 82.0% 82.0% 79% 78.5% of B_. amylophilus H-18. Esterase a c t i v i t y i n Sample #8 (Table II) against Na B e n z o y l - L - a r g i n i n e methyl es ter (BAME) was found _3 to have a s i n g l e optimum at pH 8.0 and TLCM at 10 M concentrat ion completely i n h i b i t e d t h i s a c t i v i t y (Table V ) . Blackburn (1968 b) showed that a cons iderable propor t ion of the t o t a l protease a c t i v i t y may be due to an enzyme with a t r y p s i n - l i k e s p e c i f i c i t y and protease and esterase a c t i v i t i e s were i n h i b i t e d by d i - i sopropy l -phospho-f l u o r i d a t e , a ser ine-protease i n h i b i t o r . A number of b a c t e r i a l proteases with a t r y p s i n - l i k e a c t i v i t y have been described such as a s t r e p t o c o c c a l protease (Mycek et al ,1952) and a s e m i - p u r i f i e d protease from B a c i l l u s l i c h e n i f o r m i s ( H a l l . e t a l , 1966). However, ne i ther the esterase nor the protease were i n h i b i t e d by soy-bean t r y p s i n i n h i b i t o r , and i f the main p r o t e o l y t i c a c t i v i t y i s not due to a t r y p s i n - l i k e enzyme then ne i ther i s i t l i k e chymotrypsin nor pepsin as i t d i d not a t tach substrates s p e c i f i c f o r these enzymes (Blackburn, 1968 b ) . -3 Protease a c t i v i t y was not completely i n h i b i t e d by 10 M TLCM. Un l ike the esterase a c t i v i t y which was i n h i b i t e d 100% at pH 8.0 , the protease was i n h i b i t e d at pH 5.0, 6 .7 , 8.0 and 11.0 , 20%, 29%, 30% and 48% r e s p e c t i v e l y . I t was evident from the d i f f erence i n the extent of i n h i b i t i o n between the;.pH 5.0 and 11.0 a c t i v i t i e s that there may be d i f f e r e n t enzymes i n v o l v e d . The i n h i b i t i o n of the protease a lso i n d i c a t e s that the ac t ive s i t e i n the protease molecule contained a ser ine res idue s ince TLCM binds s p e c i f i c a l l y to s e r i n e . V . Hydro lys i s of Na B e n z o y l - L - a r g i n i n e methyl es ter (BAME) by an enzyme preparat ion (sample #8, Table I I ) . pH umole/mg prote in /min umole/mg pro te in /min (BAME) (BAME) TLCM IO""3 M added 6.0 • -6.5 10.0 7.0 16.6 7.5 19.2 8.0 23.0 8.5 17.0 ,9 .1 13.4 9.6 5.1 10.0 GENERAL DISCUSSION The protease which was l i b e r a t e d i n cu l tures conta in ing l a t e s t a t i o n a r y phase c e l l s of 13. amylophilus H-18 was shown to be of r e l a t i v e l y low molecular weight. I t s p u r i f i c a t i o n was undertaken i n a n t i c i p a t i o n that i t could be more r e a d i l y p u r i f i e d than the p a r t i c l e -bound protease which Blackburn (1968 b) l i b e r a t e d from d i s i n t e g r a t e d l o g phase c e l l s of t h i s organism. The r e l a t i v e l y weak p r o t e o l y t i c a c t i v i t y found i n the c u l t u r e supernatant made i t necessary to process l a r g e volumes. F o r t u n a t e l y , the p r o t e o l y t i c a c t i v i t y could be removed from the supernatant by absorbing i t to DEAE-Sephadex. The a c t i v i t y cou ld then be e luted from the washed absorbent i n a concentrated and p u r i f i e d form. Blackburn (1968 b) demonstrated two pH optima for B_. amylophilus H-18 protease and suggested that two or more proteases might be present . I n t h i s present work, i t was e s tab l i shed that the c u l t u r e supernatant possessed p r o t e o l y t i c a c t i v i t y over a wide pH range (pH 4.0 to 11.0) w i t h peaks of a c t i v i t y at pH 5 .0 , 6 .7 , 8.0 and 11.0. Aga in , t h i s suggested the presence of s evera l d i f f e r e n t proteases , and the i n i t i a l o b j e c t i v e was to p u r i f y the pH 6.7 protease . I t was found at each s tep i n p u r i f i c a t i o n , by DEAE-Sephadex chromatography, ge l f i l t r a t i o n and i s o e l e c t r i c focus ing that the protease a c t i v i t i e s at the d i f f e r e n t pH va lues , were not separated from each other nor d i d the r a t i o of t h e i r a c t i v i t i e s change s i g n i f i c a n t l y . These r e s u l t s favoured the idea that a l l the p r o t e o l y t i c a c t i v i t y , over . the pH range examined was due to a s i n g l e protease . There were only two minor pieces of evidence against t h i s hypothes i s . In pre l iminary experiments, some f r a c t i o n s obtained from ge l f i l t r a t i o n of an ammonium s u l f a t e p r e c i p i t a t e d protease showed d i f f e r i n g r a t i o s of p r o t e o l y t i c a c t i v i t i e s at pH 5 .0 , 6 .7 , 8.0 and 11.0. I n a d d i t i o n , the a c t i v i t i e s at these pH values were i n h i b i t e d to d i f f e r e n t ex tents , by treatment of the p u r i f i e d enzyme with TLCM. In s p i t e of t h i s , g e l e l ec trophores i s of p u r i f i e d protease f r a c t i o n s f a i l e d to reso lve those a c t i v i t i e s . In other words, a band showing the pH 6.7 p r o t e o l y t i c a c t i v i t y a l so gave peaks of a c t i v i t y a t pH 5.0 or pH 11.0. The p r o t e o l y t i c a c t i v i t i e s at pH 5 .0 , 6 .7 , 8.0 and 11.0 were i n a c t i v a t e d to the same extent by heat treatment. The i n t e r e s t i n g observat ion was made that ge l f i l t r a t i o n could r e s o l v e the p r o t e o l y t i c a c t i v i t y in to two f r a c t i o n s having molecular weights of approximately 30,000 or 60,000. Since the pH range of both p r o t e o l y t i c f r a c t i o n s was the same i t seemed poss ib l e that the 60,000 molecular weight species was a dimer of the 30,000 molecular weight s p e c i e s . This was substant ia ted by a demonstration that the 30,000 molecular weight f r a c t i o n on rerunning through the g e l , gave a peak c h a r a c t e r i s t i c of the 60,000 molecular weight protease . Further evidence that the two molecular weight species ex i s ted i n e q u i l i b r i u m was given by the fac t that ge l e l ec trophores i s always demonstrated two c h a r a c t e r i s t i c protease bands. The only exception to t h i s was where poor r e s o l u t i o n was a t t a i n e d , through the use of i n s u f f i c i e n t l y long columns of acrylamide g e l . I t i s proposed that the dimer was more s t a b l e than the monomer as the 60,000 molecular weight species p r e -dominated on ge l f i l t r a t i o n and the upper band on acrylamide ge l e l ec trophores i s was always greater i n p r o t e i n and protease a c t i v i t y . Nei ther EDTA - nor 4.0 M urea treatment of the p u r i f i e d protease r e s u l t e d i n a predominance of the 30,000 molecular weight spec ies . The condi t ions for s p l i t t i n g the dimer, i f indeed that i s what the 6 0,000 molecular weight m a t e r i a l represented, remained unknown. More d r a s t i c methods of e f f e c t i n g a separat ion could not be used to the r e s u l t i n g i n a c t i v a t i o n of the protease . Amino a c i d ana lys i s of the protease revealed a complete absence o f c y s t e i n e . I t has been shown for severa l other e x t r a c e l l u l a r proteases p u r i f i e d from b a c t e r i a that d i su lph ide bridges are not i n v o l v e d i i i mainta ining t e r t i a r y s t r u c t u r e . The temperature optimum for protease a c t i v i t y was between 60 C and 65 C, i n s p i t e of the temperature of i n a c t i v a t i o n of the enzyme be ing only 50 C . Thi s could have been due to the case in used as sub-s t r a t e p r o t e c t i n g the enzyme from denaturat ion during assay. Desmazeand and Hermier (1968), working with a protease from Micrococcus c a s e b l y t i c u s , and Ryden and Hofsten (1968) who were -3 examining a protease from S e r r a t i a i n d i c a , found that EDTA at 10 M concentra t ion i n h i b i t e d p r o t e o l y t i c a c t i v i t y . When the protease -3 from 13. amylophilus H-18 was treated with 10 M EDTA a 8 - 10% increase i n p r o t e o l y t i c a c t i v i t y was observed. The heat s t a b i l i t y of the enzyme was not a f fec ted by a d d i t i o n of EDTA. Thi s suggested that the protease was not a metalloenzyme. Although the a d d i t i o n of Ca in-creased , the a c t i v i t y of the protease was not dependant upon t h i s i on for i t s a c t i v i t y . Although the proteases produced by var ious species I | o f Micrococcus were reported not to requ ire Ca for a c t i v i t y , they were a c t i v a t e d by th i s i o n ( C o l b e r t , 1957 and Gomer, 1950). The p u r i f i e d protease s t i l l showed esterase a c t i v i t y , which was maximal at pH 8.0 . I t was s i g n i f i c a n t that although esterase a c t i v i t y -3 was i n h i b i t e d completely by 10 M TLCM, protease a c t i v i t y was not completely i n h i b i t e d under these c o n d i t i o n s . This observat ion again suggested the p o s s i b i l i t y of more than one protease being present , even i n h i g h l y p u r i f i e d p r e p a r a t i o n s . 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The Lowry modi f i ca t ion of the F o l i n reagent f o r determination of prote inase a c t i v i t y . A n a l . Biochem. 10: 175-177. Mor ihara , K. (1963). Pseudomonas aeruginosa prote inase . I . P u r i f i c a -t i o n and general p r o p e r t i e s . Biochim. Biophys. Acta 7_3: 113-124. Mycek, M . J . , E l l i o t t , S .D. and Fruton , J . S . (1952). The s p e c i f i c i t y of a c r y s t a l l i n e S treptococca l prote inase . J . B i o l . Chem. 197: 637-640. P o l l o c k , M.R. and Richmond, M.A. (1962). Low cyste ine content of b a c t e r i a l e x t r a c e l l u l a r .proteins: I t s pos s ib l e p h y s i o l o g i c a l s i g n i f i c a n c e . Nature 194: 446-449. Ryden, A . and Hofsten, B. (1968). Some proper t i e s of the e x t r a c e l l u l a r prote inase and the ce l l -bound peptidase of S e r r a t i a . Acta Chem. Scand. 22: 2803-2308. Sawada, J . (1964). Studies on the a c i d protease of Paecilomyces v a r i o t i B a i n i e r TPR-220. I I . Some enzymic proper t i e s of the c r y s t a l l i n e protease . A g r . B i o l . Chem. (Tokyo) 28: 348-355. Somkuti, G . A . and Babel , F . J . (1968). P u r i f i c a t i o n and Proper t i e s of Mucor p u s i l l u s a c i d protease . J . B a c t e r i o l . 95: 1407-1414. Spackman, D . H . , S t e i n , N . H . and Moore, S. (1958). Automatic recording apparatus for use i n the Chromatography of amino a c i d s . A n a l . Chem. 30: 1190-1206. Whitaker, D.R. (1965 a ) . I s o l a t i o n . a n d enzymatic proper t i e s of the a and 3 l y t i c proteases i n Sorangium Sp. Can. J . Biochem. 43: 1935-1954. Whitaker, D .R. and Jurasch , L . (1965 b ) . A comparison of some p h y s i c a l proper t i e s of the a and 3 proteases . Can. J . Biochem. 43: 1955-59. 

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