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The partial characterization of cellulases from Cellulomonas fimi Langsford, Maureen Lynn 1983

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THE PARTIAL CHARACTERIZATION  OF CELLULASES FROM  CELLULOMONAS FIMI By  MAUREEN LYNN LANGSFORD B.Sc,  The U n i v e r s i t y o f B r i t i s h Columbia, 1981  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Department of M i c r o b i o l o g y )  We accept t h i s  t h e s i s as conforming  to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA December, 1983 © Maureen Lynn L a n g s f o r d , 1983  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree t h a t for  understood for  Library  shall  for reference  and  study.  I  f o r extensive copying of  that  h i s or  her  copying or  f i n a n c i a l gain  be  shall  g r a n t e d by  not  be  of  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  DE-6  (3/81)  of  Columbia  make  further this  thesis  head o f  this  my  It i s thesis  a l l o w e d w i t h o u t my  permission.  Department  the  representatives. publication  the  University  the  p u r p o s e s may by  the  I agree that  permission  department or  f u l f i l m e n t of  advanced degree a t  Columbia,  scholarly  in partial  written  Abstract  The  c e l l u l a s e s o f C. f i m i have been p a r t i a l l y c h a r a c t e r i z e d .  e x t r a c e l l u l a r CMCase had o p t i m a l the enzyme was not s t a b l e a t high i n a c t i v a t e d more s l o w l y  a t pH 7.0 and 52°C.  temperatures.  Since  by c a t a b o l i t e r e p r e s s i o n .  c e l l u l a s e was d e t e c t e d . than d i d a v i c e l .  l e v e l s o f c e l l u l o s e , no  CMC induced lower l e v e l s o f CMCase and fewer A n a l y s i s by non-denaturing PAGE r e v e a l e d  CMCase i n c u l t u r e supernatants c o n t i n u e d  growth f o r 9 days on CMC o r a v i c e l .  are p r o b a b l y d e r i v e d from o n l y 3 - 4  avicel-induced to increase  gels.  variation  gene p r o d u c t s .  s t o i c h i o m e t r i c d i f f e r e n c e s i n complex f o r m a t i o n ,  i n enzyme a f f i n i t y  during  The c e l l u l a s e s The  apparent m u l t i p l i c i t y o f components may be the r e s u l t o f enzymatic modification,  that  In o l d e r c u l t u r e s , the a c t i v e  components had f a s t e r m o b i l i t i e s on p o l y a c r y l a m i d e of C. f i m i  activity  When c u l t u r e s were grown on  CMC-induced c e l l u l a s e s had m o b i l i t i e s d i f f e r e n t from cellulases.  the enzyme was  CMCase was i n d u c i b l e by c e l l u l o s e and appeared  g l u c o s e o r c e l l o b i o s e i n the presence of i n d u c i n g  proteins  However,  a t lower temperatures, subsequent CMCase  was determined a t 37°C. to be r e g u l a t e d  activity  The  f o r substrate.  or  iii Table  o f Contents Page  Abstract  i i  L i s t of Tables  iv  L i s t of Figures  v  Acknowledgement  vi  Introduction  1  M a t e r i a l s and Methods  3  1.  Culture conditions  2.  Preparation  3.  Enzyme and p r o t e i n assays  4.  E l e c t r o p h o r e t i c a n a l y s i s o f c u l t u r e supernatants  .  o f c u l t u r e supernatants  Results  3 3 3  . . . .  4 5  1.  P r o p e r t i e s o f CMCase i n c u l t u r e supernatants  2.  The e f f e c t s o f d i f f e r e n t carbon sources on  5  c e l l u l a s e production  16  3.  A v i c e l and CMC i n d u c t i o n o f c e l l u l a s e  16  4.  E f f e c t s o f c u l t u r e age on a v i c e l - i n d u c e d c u l t u r e supernatants  24  Discussion  33  References C i t e d  38  L i s t of T a b l e s  Page Table 1.  Enzyme a c t i v i t i e s  i n C. f i m i c u l t u r e supernatants  . . . .  17  V  List  of Figures Page  F i g u r e 1.  The pH optimum o f C. f i m i CMCase  6  F i g u r e 2.  E f f e c t s o f pH and b u f f e r on the DNS r e a c t i o n  8  F i g u r e 3.  Temperature optimum o f C. f i m i c e l l u l a s e  10  F i g u r e 4.  E f f e c t o f temperature  12  F i g u r e 5.  K i n e t i c s o f heat  F i g u r e 6.  Non-denaturing  on CMCase s t a b i l i t y  i n a c t i v a t i o n o f CMCase  PAGE showing the e f f e c t s o f carbon  source on e x t r a c e l l u l a r p r o t e i n p a t t e r n s F i g u r e 7.  CMCase a c t i v i t i e s  F i g u r e 10.  - A v i c e l and CMC i n d u c t i o n o f CMCase over 9 days  25  27  P r o t e i n composition o f c u l t u r e supernatants a t different  F i g u r e 12.  22  S p e c i f i c a c t i v i t i e s o f CMCase d u r i n g growth on a v i c e l and CMC  F i g u r e 11.  20  i n supernatants from C. f i m i  c u l t u r e s grown on a v i c e l and CMC F i g u r e 9.  18  Denaturing PAGE showing the e f f e c t s o f carbon source on e x t r a c e l l u l a r p r o t e i n p a t t e r n s  F i g u r e 8.  14  stages o f growth  .29  A c t i v i t y p r o f i l e s of a v i c e l - i n d u c e d c u l t u r e supernatants a f t e r 3, 6, and 9 days o f growth  31  Acknowledgement  I would l i k e t o thank Drs. K i l b u r n , guidance  and support  throughout  M i l l e r and Warren f o r t h e i r  t h i s work.  I am e s p e c i a l l y  W. Wakarchuk f o r h i s endless p a t i e n c e and encouragement.  grateful  to  1 Introduction  The c o n v e r s i o n o f c e l l u l o s i c wastes t o g l u c o s e has been a r a p i d l y growing f i e l d abundant  o f study i n the past 20 y e a r s .  Cellulose  renewable r e s o u r c e s , and e f f i c i e n t methods are d e s i r a b l e f o r the  c o n v e r s i o n of f o r e s t r y and a g r i c u l t u r a l wastes liquid  i s one of the most  to s i n g l e c e l l  protein,  f u e l s , o r g a n i c a c i d s and o t h e r i n d u s t r i a l l y u s e f u l compounds  ( F l i c k i n g e r , 1980; an e f f i c i e n t way  Detroy and S t . J u l i a n , 1983).  t o break down c e l l u l o s e ,  Enzymatic d e g r a d a t i o n i s  i f the enzymes are a v a i l a b l e i n  mass q u a n t i t i e s . There are t h r e e types o f enzymes which form the c e l l u l a s e endoglucanase which randomly  complex:  c l e a v e s w i t h i n the c h a i n , exoglucanase which  c l e a v e s g l u c o s e or c e l l o b i o s e u n i t s from the non-reducing ends, and 6 - g l u c o s i d a s e which h y d r o l y s e s c e l l o b i o s e  (King and V e s s a l , 1969).  The  c e l l u l a s e systems produced by d i f f e r e n t micro-organisms v a r y i n t h e i r efficiency  ( e i t h e r ) because of the c a t a l y t i c p r o p e r t i e s of the enzymes  themselves or because o f the p r o p o r t i o n s o f the d i f f e r e n t enzymes w i t h i n a g i v e n system.  M o l e c u l a r c l o n i n g would a l l o w the c o n s t r u c t i o n o f s t r a i n s  producing o p t i m a l p r o p o r t i o n s o f the enzymes w i t h i n a system or enzymes o r i g i n a t i n g from more than one organism.  Recently, t h i s laboratory  has  r e p o r t e d the c l o n i n g of c e l l u l a s e s from the b a c t e r i u m Cellulomonas f i m i (Whittle et a l . ,  1982;  G i l k e s e t a l . , manuscript s u b m i t t e d ) .  The  s u c c e s s f u l r e c o n s t r u c t i o n by m o l e c u l a r g e n e t i c s of the e n t i r e C. c e l l u l a s e system depends on an u n d e r s t a n d i n g o f the s t r u c t u r a l involved.  A l t h o u g h the g e n e t i c s of C. f i m i  fimi  genes  i s e s s e n t i a l l y unknown,  2 characterization number o f genes.  o f the enzymes i n the system should i n d i c a t e the c r i t i c a l Here, the b a s i c  constituents,  properties  of C. f i n d ' s e x t r a c e l l u l a r c e l l u l a s e s are r e p o r t e d .  and r e g u l a t i o n  3 M a t e r i a l s and Methods  1.  Culture conditions. Cellulomonas  f i m i ATCC 484 was  Leatherwood, 1976) 0.5  grown i n a b a s a l medium (Stewart  c o n s i s t i n g of 1 g NaNO^, 1 g K^HPO^, 0.5  g D i f c o y e a s t e x t r a c t , 0.5  ( e i t h e r a v i c e l , CMC  g MgSO^H^O and 1 g carbon  or g l u c o s e ) i n 1 1 d i s t i l l e d H 0, 2  were grown on a r o t a r y shaker at approximately 200 media c o n t a i n e d 11 g D i f c o bacto-agar  RPM  g  and  KC1,  source  pH 7.0.  Cultures  at 30°C.  Solid  and 1 g g l u c o s e .  I n o c u l a were made from a s i n g l e c o l o n y p i c k e d i n t o b a s a l medium c o n t a i n i n g g l u c o s e , grown o v e r n i g h t to l a t e l o g phase, then d i l u t e d fold  into 1 l i t r e  2.  Preparation of c u l t u r e C e l l s and  of medium. supernatants.  i n s o l u b l e c e l l u l o s e were removed by c e n t r i f u g a t i o n f o r 20  minutes at 10,000 X g and 4°C. 0.02%  i n Na^N  Supernatants filters,  3.  and 0.3  mM  The  supernatants were decanted  in phenylmethylsulfonylfluoride  were f i l t e r e d twice with s u c t i o n through  and made  (PMSF).  2 Whatman g f / c  then c o n c e n t r a t e d 50-100 f o l d by u l t r a f i l t r a t i o n through  Amicon PM10  100  membrane i n an Amicon p r e s s u r e  Enzyme and p r o t e i n Cellulolytic  an  cell.  assays.  a c t i v i t y was  determined  c o l o r i m e t r i c a l l y by a s s a y i n g f o r  the p r o d u c t i o n o f r e d u c i n g groups from c a r b o x y m e t h y l c e l l u l o s e (CMC). r e a c t i o n mixtures c o n t a i n e d 0.5 and 0.25 cells,  ml 4% CMC  (w/v)  ml c u l t u r e supernatant or whole c u l t u r e  c e l l u l o s e and c u l t u r e medium) d i l u t e d  i n 50 mM  P°^i  pH  The  6.8,  (a suspension o f whole  i n 50 mM  PO  , pH 6.8.  The  4 r e a c t i o n s were i n c u b a t e d a t 37°C f o r 30 minutes, a d d i t i o n o f 0.8 ml DNS reagent  (Miller  e t a l . , 1960) and 50 ug Sigma  Standard G l u c o s e , then steamed f o r 15 minutes. c o o l e d t o room temperature,  the absorbance  Unicam SP800 spectrophotometer. was  t e r m i n a t e d by t h e  When the r e a c t i o n s had  a t 550 nm was determined  The number o f r e d u c i n g groups  with a  produced  e s t i m a t e d from a g l u c o s e s t a n d a r d curve and expressed as ug g l u c o s e  e q u i v a l e n t s produced  min ^ ml ^ enzyme.  P r o t e i n was determined Sigma lysozyme as s t a n d a r d . were b o i l e d f i r s t  by the method o f Lowry e t a l . (1951) u s i n g When measuring  f o r 5 min i n 1% SDS.  total culture protein,  The c o r r e s p o n d i n g c o n t r o l  samples reaction  a l s o c o n t a i n e d 1% SDS. 4.  Electrophoretic analysis of culture  supernatants.  C o n c e n t r a t e d c u l t u r e supernatants were e l e c t r o p h o r e s e d i n 1.5 mm t h i c k p o l y a c r y l a m i d e s l a b g e l s , e i t h e r 6% g e l s under conditions  non-denaturing  ( J o v i n e t a l . , 1964) or 10% g e l s c o n t a i n i n g sodium d o d e c y l  s u l p h a t e (SDS) (Laemmli,  1970).  Gels were s t a i n e d f o r p r o t e i n w i t h  Coomassie Blue ( B i o r a d ) o r w i t h s i l v e r by the method o f T s a i and F r a s c h (1982).  Protein profiles  on a Helena Quick Scan. d e t e c t e d as f o l l o w s :  were o b t a i n e d by scanning Coomassie s t a i n e d g e l s Cellulase activity  i n a non-denaturing  a l a n e was e x c i s e d from the g e l and c u t i n t o 2 mm  s l i c e s ; each s l i c e was i n c u b a t e d with 0.5 ml 50 mM phosphate, 48 hours  pH 6.8, f o r  a t 4°C; t h e e l u a t e s were assayed f o r CMCase a c t i v i t y as  d e s c r i b e d above.  g e l was  5 Results  1.  P r o p e r t i e s of CMCase  i n c u l t u r e supernatants.  The pH and temperature determined f o r the CMCase f o r 3 days w i t h a v i c e l .  optima and temperature  s t a b i l i t y were  i n c o n c e n t r a t e d supernatant from a c u l t u r e grown The pH optimum was 7.0 ( F i g . 1 ) .  were some e f f e c t s o f pH and b u f f e r on the DNS r e a c t i o n absorbance phosphate  ( F i g . 2).  The  a t 550 nm was enhanced by the a c e t a t e b u f f e r compared w i t h the buffer.  both b u f f e r s .  The absorbance  a l s o i n c r e a s e d w i t h i n c r e a s i n g pH i n  T a k i n g these e f f e c t s  CMCase remained  i n t o c o n s i d e r a t i o n , the o p t i m a l pH f o r  pH 7.0.  The o p t i m a l temperature (Fig. 3).  However t h e r e  f o r the CMCase r e a c t i o n was 52-55°C  However, the enzymes were not s t a b l e at high temperatures  to i n c u b a t i o n w i t h s u b s t r a t e ( F i g . 4 ) . temperatures  prior  A f t e r h e a t i n g f o r 15 minutes a t  l e s s than 50°C, 95% of the o r i g i n a l a c t i v i t y was  retained.  A f t e r h e a t i n g a t 58°C f o r 15 minutes, the enzymes were almost c o m p l e t e l y inactivated. 90 minutes  A c t i v i t y was l o s t more s l o w l y a t lower temperatures:  at 37°C over 30% o f the o r i g i n a l a c t i v i t y was l o s t ;  after  a t 49°C  50% o f the a c t i v i t y was l o s t and at 55°C 75% of the a c t i v i t y was  lost  (Fig. 5). The r e s u l t s  i n d i c a t e d t h a t h y d r o l y s i s of s u b s t r a t e and i n a c t i v a t i o n o  of CMCase both o c c u r r e d much f a s t e r at h i g h e r temperatures. the r a t e o f i n a c t i v a t i o n exceeded  Above 60 C  the r a t e of h y d r o l y s i s , and the enzyme  was denatured c o m p l e t e l y b e f o r e i t c o u l d h y d r o l y z e the c e l l u l o s e .  6  F i g u r e 1.  The pH optimum o f C. f i m i CMCase.  Concentrated supernatant was  prepared from a 3 day c u l t u r e grown w i t h 0.1% a v i c e l . i n the a p p r o p r i a t e b u f f e r t o 4% (w/v),  CMC was d i s s o l v e d  and the f i n a l pH was determined.  b u f f e r o f the same pH was used to d i l u t e the enzyme.  A c o n s t a n t amount o f  enzyme was added t o each r e a c t i o n and assayed f o r CMCase as d e s c r i b e d i n MATERIALS AND METHODS. (x—x)  CMCase was expressed as ug g l u c o s e m i n  0.2 M a c e t a t e b u f f e r ;  A  (o^-o) 0.05 M phosphate b u f f e r .  - 1  .  8  Figure  2.  E f f e c t s o f pH and b u f f e r on the DNS  reaction.  Standard g l u c o s e was prepared i n v a r i o u s  buffers.  added to the DNS reagent.  were steamed, and the absorbance  was read a t 550 nm. buffer.  The r e a c t i o n s  ( x — x ) 0.2 M a c e t a t e b u f f e r ;  100 ug g l u c o s e were  ( o — o ) 0.05 M phosphate  9  10  F i g u r e 3.  Temperature optimum o f C. f i m i CMCase.  Concentrated  supernatant was d i l u t e d  i n SO mM phosphate,  at v a r i o u s temperatures  f o r 30 minutes as d e s c r i b e d i n MATERIALS  METHODS.  pH 6.8 and assayed f o r CMCase  CMCase was expressed as ug g l u c o s e min  \  AND  Temp °C  12  F i g u r e 4.  E f f e c t o f temperature  supernatant was d i l u t e d v a r i o u s temperatures  on CMCase s t a b i l i t y .  i n 50 mM phosphate,  f o r 15 minutes.  Concentrated  pH 6.8, and i n c u b a t e d at  The enzyme then was c o o l e d on i c e ,  and an a l i q u o t was assayed f o r CMCase a c t i v i t y .  A c t i v i t y was expressed as  a p e r c e n t o f the a c t i v i t y i n the sample i n c u b a t e d a t 0°C.  13  —i o O  1  1  1  o CO  o CO  o ^  AJIAIPV  ———to t\l  |DUi6uo %  14  F i g u r e 5.  K i n e t i c s o f heat  supernatant was d i l u t e d 37°, 49° and 55°C. i c e bath.  i n 50 mM phosphate,  pH 6.8 and i n c u b a t e d a t 0 ° ,  A l l samples were assayed  f o r CMCase a t 37°C as d e s c r i b e d i n  A c t i v i t y was expressed as a percentage  i n the sample i n c u b a t e d a t 0°C.  90 minutes  (x—x).  o f the  A c t i v i t y remaining a f t e r 10  minutes ( o — o ) ; 20 minutes ( o — o ) ; 30 minutes (A—A); (A—A);  Concentrated  A t v a r i o u s times, an a l i q u o t was removed t o an  MATERIALS AND METHODS. activity  i n a c t i v a t i o n o f CMCase.  45 minutes  15  20]  0  20  40  Temp °C  60  80  16 2.  The  e f f e c t s of d i f f e r e n t carbon  C. f i m i was  sources on c e l l u l a s e p r o d u c t i o n .  grown on c e l l u l o s e with or without high g l u c o s e or  c e l l o b i o s e to determine  i f c e l l u l a s e p r o d u c t i o n was  catabolite repression.  CMCase a c t i v i t y was  supernatants  (Table 1).  measured i n the  culture  Concentrated supernatants were a l s o a n a l y z e d by  polyacrylamide gel electrophoresis or  r e g u l a t e d by  (PAGE), e i t h e r non-denaturing  denaturing ( F i g . 7). As shown by Stewart  and Leatherwood (1976), no c e l l u l a s e was  i n c u l t u r e s grown on g l u c o s e or c e l l o b i o s e even i n the presence inducing l e v e l s of c e l l u l o s e . of  ( F i g . 6)  the uninduced  detected  of  C o r r e s p o n d i n g l y , the p r o t e i n g e l p a t t e r n s  or r e p r e s s e d supernatants were much s i m p l e r than  induced s u p e r n a t a n t s .  T h i s suggested t h a t the CMCase a c t i v i t y was  the derived  from m u l t i p l e components.  3.  A v i c e l and CMC The  i n d u c t i o n of c e l l u l a s e .  amount of c e l l u l a s e a c t i v i t y  supernatants was supernatants  found  o n l y 70% of t h a t found  (Table 1).  i n CMC-induced c u l t u r e  in avicel-induced culture  T h i s c o u l d be due  simply to a decrease  i n the  l e v e l o f a g i v e n enzyme or a l t e r n a t i v e l y , t o the i n d u c t i o n o f d i f f e r e n t c e l l u l a s e s w i t h lower a c t i v i t i e s .  The  SDS  g e l pattern ( F i g . 7 lanes 3 &  7) i n d i c a t e d t h a t the p r o t e i n m o b i l i t i e s were s i m i l a r although CMC-induced supernatant p a t t e r n appeared profile  simpler.  However, the  from a n a t i v e g e l i n d i c a t e d t h a t the a c t i v e components  different mobilities  ( F i g . 8).  the activity had  17  T a b l e 1.  Enzyme a c t i v i t i e s  i n C. f i m i  Growth s u b s t r a t e  c u l t u r e supernatants  CMCase  activity  ( u n i t s ml "S  0.1% A v i c e l  130  0.1% A v i c e l + 1% g l u c o s e  0  0.1% CMC  90  0.1% CMC + 1% g l u c o s e  0  1% g l u c o s e  0  1% c e l l o b i o s e  0  C. f i m i was grown i n b a s a l medium w i t h v a r i o u s carbon a c t i v i t y was measured i n the unconcentrated MATERIALS AND METHODS.  sources.  supernatants  CMCase  as d e s c r i b e d i n  18  F i g u r e 6.  Non-denaturing PAGE showing the e f f e c t s o f carbon  e x t r a c e l l u l a r protein patterns.  Supernatants  were prepared  grown f o r 3 days on 0.1% a v i c e l o r CMC w i t h or without c e l l o b i o s e and a n a l y z e d by non-denaturing METHODS).  l a n e 4:  1% g l u c o s e o r  Lane 1:  1% c e l l o b i o s e and 0.1% a v i c e l ; l a n e 3:  1% glucose and 0.1% a v i c e l ; lane 5:  g l u c o s e and 0.1% CMC; l a n e 7:  0.1% CMC.  from c u l t u r e s  PAGE (see MATERIALS AND  The g e l was s t a i n e d w i t h Coomassie Blue.  c e l l o b i o s e ; l a n e 2:  source on  1% g l u c o s e ;  1% 0.1% a v i c e l ;  lane 6:  1%  19  I 2  3 4 5 6  7  20  F i g u r e 7.  Denaturing  PAGE showing the e f f e c t s of carbon source  e x t r a c e l l u l a r protein patterns.  The  samples d e s c r i b e d i n the legend  f i g u r e 6 were e l e c t r o p h o r e s e d under d e n a t u r i n g silver  stained.  a v i c e l ; lane 3:  Lane 1: 0.1%  l a n e 5:  1% g l u c o s e ;  lane 8:  molecular  fl g a l a c t o s i d a s e bovine  conditions.  1% c e l l o b i o s e ; lane 2:  a v i c e l ; lane 4: lane 6:  17. g l u c o s e  1% glucose  weight standards  on  and 0.1%  The  gel  1% c e l l o b i o s e and and 0.1% CMC;  l a n e 7:  0.1%  B (97,400 d a l t o n s ) ,  serum albumin (66,000 d a l t o n s ) , ovalbumin (45,000 d a l t o n s ) ,  c a r b o n i c anhydrase (29,000 d a l t o n s ) .  was 0.1%  avicel;  which were, from top to bottom,  (116,000 d a l t o n s ) , phosphorylase  to  CMC;  21  I  2  3  4  5  6  7  8  22  F i g u r e 8.  CMCase a c t i v i t i e s  w i t h A v i c e l and CMC. on 0.1% a v i c e l  i n supernatants from C. f i m i c u l t u r e s grown  Supernatant  samples from c u l t u r e s grown f o r 3 days  (upper two p a n e l s ) o r 0.1% CMC  (lower two p a n e l s ) were  e l e c t r o p h o r e s e d i n d u p l i c a t e l a n e s i n a non-denaturing s t a i n e d w i t h Coomassie Blue and scanned. CMCase a c t i v i t y  gel.  One l a n e was  The o t h e r l a n e was assayed f o r  as d e s c r i b e d i n MATERIALS AND METHODS.  The absorbance a t  550 nm was p l o t t e d a g a i n s t g e l s l i c e number where s l i c e 1 r e p r e s e n t s the o r i g i n o f the g e l and s l i c e 40 r e p r e s e n t s the dye f r o n t .  23  24 A time course o f CMC and a v i c e l i n d u c t i o n o f c e l l u l a s e was by a s s a y i n g c u l t u r e s a t d a i l y cells.  determined  i n t e r v a l s f o r CMCase, p r o t e i n and v i a b l e  The c e l l numbers and t o t a l p r o t e i n p l a t e a u e d a f t e r the f i r s t  days o f growth ( F i g . 9 ) .  three  CMCase c o n t i n u e d t o i n c r e a s e through-  out the 9 day growth p e r i o d ; t h i s was p a r t i c u l a r l y n o t i c e a b l e i n the avicel-grown c u l t u r e  ( F i g s . 9 and 10).  C. f i m i was a b l e t o grow w e l l on  the m i c r o - c r y s t a l l i n e c e l l u l o s e and reached a c e l l d e n s i t y g r e a t e r than 2 9 X 10  -1 c e l l s ml  .  The b a c t e r i a grew l e s s e f f i c i e n t l y on the  c a r b o x y m e t h y l - s u b s t i t u t e d c e l l u l o s e , perhaps u t i l i z e CM-glucose o r C M - c e l l o b i o s e 4.  because  they were unable t o  produced.  E f f e c t s o f c u l t u r e age on a v i c e l - i n d u c e d c u l t u r e s u p e r n a t a n t s . To determine what changes were t a k i n g p l a c e i n supernatants as CMCase  i n c r e a s e d w i t h c u l t u r e age, supernatants were prepared from c u l t u r e s grown on a v i c e l f o r 3, 6 and 9 days.  A n a l y s i s by non-denaturing  PAGE ( F i g . 11,  lanes 1-3) and d e n a t u r i n g PAGE ( F i g . 11, l a n e s 4-6) showed t h a t the e x t r a c e l l u l a r p r o t e i n s became more mobile as the c u l t u r e s aged. activities  The major  i n a 3 day c u l t u r e supernatant m i g r a t e d more s l o w l y than the  major a c t i v i t i e s  i n 6 o r 9 day supernatants ( F i g . 12).  g e l these changes corresponded  In the d e n a t u r i n g  t o the l o s s o f h i g h e r m o l e c u l a r weight  components (110, 65 and 56 k i l o d a l t o n s ) p r e s e n t a f t e r 3 days o f growth and the appearance  o f lower m o l e c u l a r weight  k i l o d a l t o n s ) by 9 days o f c u l t u r e growth.  components (45, 40 and 25  25  F i g u r e 9. One l i t r e  A v i c e l and CMC  i n d u c t i o n o f CMCase over 9 days.  c u l t u r e s w i t h 0.1% a v i c e l  B, D and F) were grown f o r 9 days. removed from each c u l t u r e . counts culture  (panels A & B ) .  (panels A, C & E) o r 0.1% CMC  A t 24 hour i n t e r v a l s 25 ml were  T o t a l v i a b l e c e l l s were determined  (panels E & F ) . -1  c e l l s were expressed as colony forming u n i t s ml CMCase was expressed as u n i t s ml 1  from  plate  P r o t e i n and CMCase were measured i n the t o t a l  (panels C & D), and i n the supernatant  expressed as mg ml  (panels  (•—•  +  1  A—A).  ( o — o + A—A);  Viable  -9 X 10 protein  (o—o); was  27  F i g u r e 10. Specific  Specific  a c t i v i t y o f CMCase d u r i n g growth on a v i c e l and CMC  a c t i v i t y was c a l c u l a t e d as CMCase u n i t s mg  from the d a t a shown i n F i g u r e 9.  Panel A:  supernatant  ( o — o ) and i n c u l t u r e  (o—•);  supernatant  ( A — A ) and i n c u l t u r e  (A—A).  -1  p r o t e i n x 10  -3  a v i c e l - i n d u c e d CMCase i n p a n e l B:  CMC-induced CMCase  Specific Activity -  o  to b  «  o  29  F i g u r e 11.  P r o t e i n composition i n c u l t u r e supernatants a t d i f f e r e n t  stages of growth. avicel.  At 3, 6 and 9 days 250 ml were removed, and supernatants were  prepared. and  A 1 l i t r e c u l t u r e o f C. f i m i was grown w i t h 0.1%  Samples were e l e c t r o p h o r e s e d i n a non-denaturing  i n a d e n a t u r i n g g e l ( l a n e s 4-6).  Blue.  The g e l s were s t a i n e d w i t h Coomassie  P r o t e i n p a t t e r n s o f a 9 day supernatant  supernatant  g e l ( l a n e s 1-3)  ( l a n e s 2 and 5 ) , 3 day supernatant  ( l a n e s 1 & 6 ) , 6 day (lanes 3 & 4 ) .  30  31  F i g u r e 12.  Activity  p r o f i l e s o f a v i c e l - i n d u c e d c u l t u r e supernatants  3, 6 and 9 days o f growth.  A c t i v i t y p r o f i l e s were o b t a i n e d  i n MATERIALS AND METHODS f o r the samples d e s c r i b e d 11.  CMCase a c t i v i t y p r o f i l e s  day  9 (panel E) supernatants;  after  as d e s c r i b e d  i n the legend  to figure  from day 3 ( p a n e l A ) , day 6 ( p a n e l C) and native g e l protein p r o f i l e s  ( p a n e l B), day 6 ( p a n e l D) and day 9 ( p a n e l F)  from day 3  supernatants.  32  Slice  Number  33 Discussion  The  o p t i m a l c o n d i t i o n s f o r the CMCases present i n c u l t u r e  supernatants o f Cellulomonas 52-55°C.  Although  the enzymes were more a c t i v e at h i g h e r  they were a l s o more l a b i l e ; remained  f i m i ATCC484 were found t o be pH 7.0 and  l e s s than 50% o f the o r i g i n a l  a f t e r 45 minutes at 55°C.  d u r i n g 15 minutes at 37°C.  activity  The enzyme a c t i v i t y was c o n s t a n t  These r e s u l t s compare w e l l w i t h the  p u b l i s h e d c o n d i t i o n s determined 1978;  temperatures,  f o r o t h e r cellulomonads  (Choi e_t a l . ,  Kim and Wimpenny, 1981; Stoppok e t a l . , 1982; S t o r v i c k and King,  1960).  In c o n t r a s t , c e l l u l a s e from the t h e r m o p h i l i c b a c t e r i u m  Clostridium  o thermocellum 1982). this  had o p t i m a l a c t i v i t y  at 70 C and pH 6.1 (Johnson  The f u n g a l enzymes g e n e r a l l y were more a c t i v e a t pH 4.5 - 5.0;  i s i n keeping w i t h the a c i d environments  they were found The  et a l . ,  o f r o t t i n g wood on which  (Rho e t a l . , 1982; P a r r y e t a l . , 1983).  c e l l u l a s e s o f C. f i m i were induced by c e l l u l o s e and r e g u l a t e d by  catabolite repression.  In the presence o f high l e v e l s o f g l u c o s e o r  c e l l o b i o s e and i n d u c i n g l e v e l s o f c e l l u l o s e , no CMCase a c t i v i t y d e t e c t e d i n the c u l t u r e s u p e r n a t a n t s .  These r e s u l t s agree with those  o t h e r s t u d i e s (Stewart and Leatherwood, 1976; Beguin §_t &1., et a l . , 1982; Choi e t a l . , 1978).  was  Beguin  e t a l . (1977) found  from  1977; Stoppok that  c e l l o b i o s e r e p r e s s e d the s y n t h e s i s o f c e l l u l a s e s even a f t e r the a d d i t i o n of 1 mM cAMP.  Priest  (1977) noted t h a t the e x t r a c e l l u l a r enzymes o f many  b a c t e r i a are r e g u l a t e d by c a t a b o l i t e r e p r e s s i o n mechanisms which do not r e q u i r e cAMP, cGMP o r the h i g h l y p h o s p h o r y l a t e d n u c l e o t i d e s .  34 C. carbon growth. or due  f i m i grew l e s s e f f i c i e n t l y and source  as compared to a v i c e l .  T h i s may  0.1%  CMC  have been because the carbon  source had  p e r c e n t C M - c e l l u l o s e may this  been d e p l e t e d ; have been  A l t e r n a t i v e l y , the s m a l l e r breakdown p r o d u c t s , CM-glucose  C M - c e l l o b i o s e , were not m e t a b o l i z a b l e .  idea.  overcome t h i s  CMC  limitation.  Some p u b l i s h e d  results  as on 2.5%  avicel.  Ilbc  In c o n t r a s t , C.  uda  grew b e t t e r and degraded more s u b s t r a t e when c u l t u r e d w i t h 2% a v i c e l w i t h 2% CMC  (Stoppok e t a l . , 1982).  b e t t e r growth of CS1-1  Choi  e t a l . (1978) a l s o  w i t h 1% a v i c e l than w i t h 1% CMC.  reached  However, i n the  a maximum l e v e l a f t e r 2 - 5  i n c r e a s e as r e p o r t e d f i m i was  environment.  days of growth, then  The  i t either  c e l l u l a s e a c t i v i t y d i d not c o n t i n u e  to  here.  very e f f i c i e n t  CMCase a c t i v i t y  at t r a n s p o r t i n g c e l l u l a s e s  i n c u l t u r e supernatants  to the  appeared to  f o r a l l the a c t i v i t y d e t e c t e d i n whole c u l t u r e ( F i g s . 9 & 10). c e l l - b o u n d c e l l u l a s e must c o n t r i b u t e v e r y l i t t l e However the CMCase assay of whole c u l t u r e may a c t i v i t y and may  almost  In a l l t h r e e of these p u b l i s h e d r e p o r t s the CMCase  remained c o n s t a n t or d e c l i n e d .  C.  than  observed  CMC-grown c u l t u r e the amount of CMCase produced per v i a b l e c e l l was 30 f o l d h i g h e r .  and  Growing the organism on a h i g h e r  Beguin §_t a l . (1977) r e p o r t e d t h a t C e l l u l o m o n a s  grew twice as f a s t on 2.5%  as  appeared to be l i m i t i n g f o r  to the s u b s t i t u t i o n s l a r g e unusable fragments may  generated.  support  produced l e s s c e l l u l a s e w i t h CMC  miss c y t o p l a s m i c  a n a l y s i s " o f CMCase l o c a t i o n  activity.  to the t o t a l  account  Therefore, activity.  d e t e c t o n l y membrane-bound T h e r e f o r e , a thorough  s h o u l d be made w i t h f r a c t i o n a t e d  cells.  As  avicel-grown  c o r r e l a t e d with polyacrylamide  c u l t u r e s aged, the i n c r e a s e  activity  an i n c r e a s e i n the m o b i l i t y o f a c t i v e components on gels.  I t seemed u n l i k e l y t h a t a s i n g l e organism would make  as many as 10 unique gene products The  in cellulase  to hydrolyze  apparent m u l t i p l i c i t y o f the C. f i m i  changing a f f i n i t y o f c e l l u l a s e s possbile modifications  a simple  glucose  polymer.  complex c o u l d be a r e s u l t o f  f o r substrate or of modification.  Some  i n c l u d e enzymatic p r o c e s s i n g o f the enzymes,  changes i n the s t o i c h i o m e t r y o f the c e l l u l a s e complex, o r microheterogeneity  of glycoproteins.  E r i k s s o n and P e t t e r s s o n S p o r o t r i c h u m pulverulentum from t h a t fungus.  (1982) have r e p o r t e d the enhancement o f  e n d o c e l l u l a s e a c t i v i t y by p u r i f i e d  They p o s t u l a t e d t h a t the p r o t e a s e s  a c t i v a t e a zymogen, d e s t r o y  proteases  functioned to  an i n h i b i t o r o r a i d i n r e l e a s e o f the  c e l l u l a s e s from the f u n g a l c e l l w a l l .  I f C. f i m i has an e x t r a c e l l u l a r  protease  cleavage  l i k e the B a c i l l u s  must be s p e c i f i c .  subtilisin,  The SDS g e l s  of "precursor"  cellulases  ( F i g . 11) show a r e p r o d u c i b l e p a t t e r n o f  d i s c r e e t bands, not a smear o f s m a l l e r fragments, which i s c o n s i s t e n t w i t h this  idea.  The advantage o f having  a c t i v e enzymes with  access  a protease  to regions  may be t o generate  smaller  of c e l l u l o s e that l a r g e r proteins or  complexes would not have. Saddler  and Kahn (1981) c h a r a c t e r i z e d 2 endoglucanases from  Acetivibrio cellulolyticus. and  10,400 d a l t o n s .  c u l t u r e age w h i l e  The enzymes had m o l e c u l a r  weights o f 33,000  The p r o p o r t i o n o f the l a r g e r enzyme decreased  the s m a l l e r enzyme i n c r e a s e d .  a p r o t e o l y t i c cleavage  product  or a subunit  with  The s m a l l e r enzyme may be  o f the l a r g e r enzyme.  The  36 r e l a t e d n e s s o f the two enzymes was not determined.  Enger and S l e e p e r  (1965) found t h a t 3 o f 5 e l e c t r o p h o r e t i c a l l y d i s t i n c t Streptomyces The al.  a n t i b i o t i c u s were r e l a t e d  endogluconases i n  immunologically.  s t o i c h i o m e t r y o f the complex a l s o may be changing.  Yoshikawa e t  (1974) grew Pseudomonas f l u o r e s c e n s v a r c e l l u l o s a on a v i c e l f o r  s e v e r a l days.  A t v a r i o u s times o f c u l t u r e growth, s u p e r n a t a n t s were  passed over a g e l f i l t r a t i o n  column.  They found t h a t as the c u l t u r e s  aged, the a c t i v e f r a c t i o n s were e l u t i n g o f f the column l a t e r i n c l u d e d volume.  When the a c t i v e f r a c t i o n s  i n the  were a n a l y z e d  e l e c t r o p h o r e t i c a l l y , they were found t o c o n s i s t o f o n l y 2 components: (fast-moving) o r B (slow-moving).  A  Both o f these components were  g l y c o s y l a t e d , but A c o n t a i n e d more g a l a c t o s e , w h i l e B had more g l u c o s e i n the c a r b o h y d r a t e moiety  (Yamane e t a l . , 1970).  Early i n culture  growth,  the a c t i v e column f r a c t i o n s c o n t a i n e d o n l y B; but as t h e c u l t u r e aged, t h e peaks c o n t a i n e d more A and l e s s B. B by enzymatic  They concluded t h a t A was d e r i v e d from  modification.  R e c e n t l y the o b s e r v a t i o n t h a t c e l l u l a s e s b i n d t o c e l l u l o s e has g a i n e d significance.  T h i s phenomenon has been observed  i n f u n g a l and b a c t e r i a l  systems (Berghem e t a l . , 1976; Manning and Wood, 1983; Choi §_t a l . , 1978; Beguin  and E i s e n , 1978).  Beguin  e t a l . (1977) have observed  t h a t the  amount o f enzyme bound t o a v i c e l d e c r e a s e d w i t h c u l t u r e age, c o r r e s p o n d i n g to an i n c r e a s e i n s o l u b l e enzyme.  C e l l u l a s e s may b i n d more r e a d i l y t o the  amorphous r e g i o n s , and as these become h y d r o l y z e d , t h e enzymes may become f r e e i n the s u p e r n a t a n t . enzymatic  Once f r e e they may be more s u s c e p t i b l e t o  m o d i f i c a t i o n , whether i t be p r o t e o l y s i s o r changes i n  glycosylation.  A p o s s i b l e outcome o f the m o d i f i c a t i o n s may  a f f i n i t y of the c e l l u l a s e s f o r s u b s t r a t e . the components one  f o r the o t h e r may  be an  altered  A l t e r n a t i v e l y the a f f i n i t y  result  of  in stoichiometric  rearrangements. The  changes i n m o b i l i t y o f a c t i v e components may  a c t u a l l y represent  s y n t h e s i s of d i f f e r e n t c e l l u l a s e s . E n r i q u e z (1981) observed growth p a t t e r n e a r l y Cellulomonas  Ilbc.  i n the f e r m e n t a t i o n o f sugar cane bagasse by The  first  l o g phase corresponded  amorphous and h e m i c e l l u l o s e r e g i o n s .  A l a g o f 10-20  a second  t o a decrease  l o g phase which corresponded  substrate.  a diauxic  He p o s t u l a t e d t h a t s y n t h e s i s o f the  d u r i n g the l a g phase.  to d e g r a d a t i o n o f hours was  in c r y s t a l l i n i t y  Perhaps a s i m i l a r p a t t e r n occurs w i t h C.  Any  fimi.  Once the  amorphous r e g i o n s have been h y d r o l y s e d , enzymes more a c t i v e on be  crystalline  induced.  or a l l o f these mechanisms may  occur  i n C. f i m i .  R e c e n t l y , some  of the c e l l u l a s e s from C. f i m i have been shown to b i n d a v i c e l and c o n t a i n c a r b o h y d r a t e groups ( L a n g s f o r d e_t a l . , manuscript publication).  of  component o c c u r r e d  A v i c e l c o n t a i n s both amorphous and c r y s t a l l i n e r e g i o n s .  r e g i o n s may  f o l l o w e d by  An e x t r a c e l l u l a r p r o t e a s e has  to  accepted f o r  a l s o been d e t e c t e d .  U l t i m a t e l y the enzymes must be p u r i f i e d t o determine  i f the many CMCase  activities  from a more l i m i t e d  are unique  gene p r o d u c t s or are generated  number o f p r e c u r s e r m o l e c u l e s .  Antibody  c r o s s - r e a c t i v i t y and  peptide  mapping s h o u l d a l l o w the d e t e c t i o n of s i m i l a r i t i e s  between the  components.  in identifying  Then these techniques c o u l d be u s e f u l  p r o d u c t s from the c l o n e d genes.  different the  38 References  1.  Beguin,  P. and E i s e n , H.  characterization  2.  P u r i f i c a t i o n and p a r t i a l  of three e x t r a c e l l u l a r c e l l u l a s e s  sp. European J o u r n a l of B i o c h e m i s t r y  87:525-531.  Beguin,  (1977).  P., E i s e n , H. and Roupas, A.  cellulose-bound c e l l u l a s e s General M i c r o b i o l o g y  3.  (1978).  Cited  i n a Cellulomonas  from  Cellulomonas  Free and species.  Journal of  101:191-196.  Berghem, L.E.R., P e t t e r s s o n , L.G. and A x i o - F r e d e r i k s s o n , (1976).  The mechanism of enzymatic  cellulose  p u r i f i c a t i o n and p r o p e r t i e s o f two d i f f e r e n t from Trichoderma v i r i d e .  U.B.  degradation: 1,4-B-glucanohydrolases  European J o u r n a l o f B i o c h e m i s t r y  61:621-630.  4.  C h o i , W.Y.,  Haggett, K.D.  c o t t o n wool degrading ability Science  5.  Detroy,  and Dunn, N.W.  (1978).  s t r a i n of Cellulomonas:  to degrade c o t t o n wool.  Australian  Isolation  mutants w i t h  of a altered  Journal of B i o l o g i c a l  34:553-564.  R.W.  and S t . J u l i a n , G.  f e r m e n t a t i o n chemicals Microbiology  and f u e l s .  10:203-228.  (1983).  Biomass c o n v e r s i o n :  CRC C r i t i c a l  Reviews i n  39 6.  Enger, M.D.  and S l e e p e r , B.P.  (1965).  from Streptomyces a n t i b i o t i c u s .  7.  E n r i q u e z , A. bagasse.  8.  (1981).  Multiple cellulase  Journal of Bacteriology  Growth o f c e l l u l o l y t i c  Biotechnology  and B i o e n g i n e e r i n g  of Cellulomonas  89:23-27.  b a c t e r i a on sugarcane  23:1423-1429.  E n r i q u e z , A., Montalvo, R., and C a n a l e s , M. bagasse c r y s t a l l i n i t y  system  (1981).  V a r i a t i o n of  and c e l l u l a s e a c t i v i t y d u r i n g the f e r m e n t a t i o n  bacteria.  Biotechnology  and B i o e n g i n e e r i n g  23:1431-1436.  9.  E r i k s s o n , K.-E., and P e t t e r s s o n , B.  (1982).  P u r i f i c a t i o n and  p a r t i a l c h a r a c t e r i z a t i o n o f two a c i d i c p r o t e a s e s fungus Sporotrichum  pulverulentum.  from the w h i t e - r o t  European J o u r n a l o f B i o c h e m i s t r y  124:635-642.  10.  F l i c k i n g e r , M.C. of c e l l u l o s i c come?  11.  (1980).  carbohydrates  Biotechnology  G i l k e s , N.R.,  into l i q u i d  & Bioengineering  K i l b u r n , D.G.,  Wakarchuk, W.W., (manuscript  Current b i o l o g i c a l research i n conversion fuels:  how f a r have we  22 ( s u p p l . l ) : 2 7 - 4 8 .  L a n g s f o r d , M.L.,  M i l l e r , R.C., J r . ,  Warren, R.A.J., W h i t t l e , D.J. and Wong, W.K.R.  accepted  for publication).  c h a r a c t e r i z a t i o n of E s c h e r i c h i a c o l i genes from Cellulomonas  fimi.  I s o l a t i o n and  clones expressing  cellulase  40 12.  Johnson, A.L.  E.A.,  (1982).  Sakajoh, M.,  H a l l i w e l l , G.,  Madia, A.,  S a c c h a r i f i c a t i o n o f complex c e l l u l o s i c  the c e l l u l a s e system from C l o s t r i d i u m thermocellum. Environmental M i c r o b i o l o g y  13.  and Demain, s u b s t r a t e s by Applied  &  43:1125-1132.  J o v i n , T., Chrambach, A. and Naughton, M.A.  (1964).  An  apparatus  f o r temperature-regulated polyacrylamide g e l e l e c t r o p h o r e s i s . A n a l y t i c a l Biochemistry  14.  Kim,  B.H.  9:351-369.  and Wimpenny, J.W.T.  (1981).  a c t i v i t y o f Cellulomonas f l a v i g e n a .  Growth and  cellulolytic  Canadian J o u r n a l o f M i c r o b i o l o g y  27:1260-1266.  15.  King, K.W. complex.  and V e s s a l , M.I. In Gould, R.F.  (ed.)  Advances i n Chemistry s e r i e s  16.  Laemmli, U.K. assembly  17.  (1970).  (1969).  Enzymes o f the c e l l u l a s e  Cellulases  M i l l e r , R.C., publication).  Applications,  95:17-25.  Cleavage o f s t r u c t u r a l p r o t e i n s d u r i n g  o f the head o f b a c t e r i o p h a g e T4.  L a n g s f o r d , M.L.,  and T h e i r  G i l k e s , N.R.,  Nature  Wakarchuk, W.W.,  227:680-685.  Kilburn,  D.G.,  J r . , and Warren, R.A.J, (manuscript a c c e p t e d f o r The c e l l u l a s e  system o f Cellulomonas  fimi.  41 18.  Lowry, O.H., Rosebrough, N.J., F a r r , A.L., and R a n d a l l , R.J. (1951).  P r o t e i n measurement w i t h t h e F o l i n phenol  of B i o l o g i c a l Chemistry  19.  20.  (1983).  P r o d u c t i o n and r e g u l a t i o n o f  extracellular  e n d o c e l l u l a s e by A g a r i c u s b i s p o r u s .  Microbiology  129:1839-1847.  Journal  P r i e s t , Fergus G. genus B a c i l l u s .  J.  (1983).  Extracellular  Bacteriological  27:288-294.  Fresenius.  Reviews  enzyme s y n t h e s i s i n the 41:711-753.  M., J u r a s e k , L., D r i g u e z , H. and Defaye, J .  as a new i n d u c e r .  S a d d l e r , J.N. and Khan, A.W. Acetivibrio  Purification  213:437-444.  Induction of c e l l u l a s e  thiocellobiose  24.  J.C. and H e p t i n s t a l l ,  (1977).  Rho, D., D e s r o c h e r s , (1982).  (1960).  Analytical  the major endoglucanase from A s p e r g i l l u s fumigatus  Biochemistry  23.  A.L.  2:127-132.  P a r r y , J.B., Stewart, of  22.  J o u r n a l of General  M i l l e r , G.L., Blum, R., Glennon, W.E., and Burton, Measurement o f c a r b o x y m e t h y l c e l l u l a s e a c t i v i t y .  21.  Journal  193:265-275.  Manning, K. and Wood, D.A.  Biochemistry  reagent.  cellulolyticus.  i n Schizophyllum  commune:  Journal of Bacteriology  (1981).  149:47-53.  C e l l u l o l y t i c enzyme system o f  Canadian J o u r n a l o f M i c r o b i o l o g y  42 25.  Stewart, Bobbie, J . , and Leatherwood, J.M. s y n t h e s i s o f c e l l u l a s e by C e l l u l o m o n a s .  (1976).  Derepressed  J o u r n a l of B a c t e r i o l o g y  128:609-615.  26.  Stoppok, W.,  Rapp, P., and Wagner, F.  (1982).  Formation,  location,  and r e g u l a t i o n o f endo-1,4-B-glucanases and S - g l u c o s i d a s e s from Cellulomonas uda.  27.  S t o r v i c k , W.O.  A p p l i e d and E n v i r o n m e n t a l M i c r o b i o l o g y  and King, K.W.  a c t i o n of the c e l l u l a s e B i o l o g i c a l Chemistry  28.  T s a i , C.-M.  29.  and F r a s c h , C.E.  (1982).  A sensitive silver  i n polyacrylamide gels.  Analytical  Warren, R.A.J, and M i l l e r ,  R.C., J r .  M o l e c u l a r c l o n i n g o f a C e l l u l o m o n a s f i m i c e l l u l a s e gene i n Gene 17:139-145 .  Yamane, K., S u z u k i , H., and N i s i z a w a , K. properties of e x t r a c e l l u l a r Pseudomonas 67:19-35.  stain for  119:115-119.  Escherichia c o l i .  30.  Journal of  235:303-307.  W h i t t l e , D.J., K i l b u r n , D.G., (1982).  The c o m p l e x i t y and mode o f  system o f C e l l v i b r i o g i l v u s .  detecting lipopolysaccharides Biochemistry  (1960).  44:44-53.  (1970).  P u r i f i c a t i o n and  and c e l l - b o u n d c e l l u l a s e components o f  fluorescens var. c e l l u l o s a .  Journal of Biochemistry  A3 Yoshikawa, T., S u z u k i , H. and N i s i z a w a ,  K.  m u l t i p l e c e l l u l a s e components o f Pseudomonas cellulosa.  I.  of c e l l u l a s e .  (1974).  Biogenesis of  fluorescens var.  E f f e c t s o f c u l t u r e and c o n d i t i o n s on the m u l t i p l i c i t y Journal of Biochemistry  75:531-540.  

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