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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 University of 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 Microbiology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1983 © Maureen Lynn Langsford, 1983 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 o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree 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 agree 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 copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) Abstract The c e l l u l a s e s of C. fimi have been p a r t i a l l y characterized. The e x t r a c e l l u l a r CMCase had optimal a c t i v i t y at pH 7.0 and 52°C. However, the enzyme was not stable at high temperatures. Since the enzyme was inacti v a t e d more slowly at lower temperatures, subsequent CMCase a c t i v i t y was determined at 37°C. CMCase was inducible by c e l l u l o s e and appeared to be regulated by c a t a b o l i t e repression. When cultures were grown on glucose or c e l l o b i o s e in the presence of inducing l e v e l s of c e l l u l o s e , no c e l l u l a s e was detected. CMC induced lower l e v e l s of CMCase and fewer proteins than d i d a v i c e l . Analysis by non-denaturing PAGE revealed that 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 avicel-induced c e l l u l a s e s . CMCase in culture supernatants continued to increase during growth for 9 days on CMC or a v i c e l . In older cultures, the active components had f a s t e r m o b i l i t i e s on polyacrylamide gels. The c e l l u l a s e s of C. fimi are probably derived from only 3 - 4 gene products. The apparent m u l t i p l i c i t y of components may be the r e s u l t of enzymatic modification, stoichiometric differences in complex formation, or v a r i a t i o n in enzyme a f f i n i t y for substrate. i i i Table of Contents Page Abstract i i L i s t of Tables i v L i s t of Figures v Acknowledgement v i Introduction 1 Materials and Methods 3 1. Culture conditions . 3 2. Preparation of culture supernatants 3 3. Enzyme and protein assays 3 4. Electrophoretic analysis of culture supernatants . . . . 4 Results 5 1. Properties of CMCase in culture supernatants 5 2. The e f f e c t s of d i f f e r e n t carbon sources on c e l l u l a s e production 16 3. A v i c e l and CMC induction of c e l l u l a s e 16 4. E f f e c t s of culture age on avicel-induced culture supernatants 24 Discussion 33 References Cited 38 L i s t of Tables Page Table 1. Enzyme a c t i v i t i e s in C. fimi culture supernatants . . . . 17 V L i s t of Figures Page Figure 1. The pH optimum of C. fimi CMCase 6 Figure 2. E f f e c t s of pH and buffer on the DNS reaction 8 Figure 3. Temperature optimum of C. fimi c e l l u l a s e 10 Figure 4. E f f e c t of temperature on CMCase s t a b i l i t y 12 Figure 5. Kinetics of heat i n a c t i v a t i o n of CMCase 14 Figure 6. Non-denaturing PAGE showing the e f f e c t s of carbon source on e x t r a c e l l u l a r protein patterns 18 Figure 7. Denaturing PAGE showing the e f f e c t s of carbon source on e x t r a c e l l u l a r protein patterns 20 Figure 8. CMCase a c t i v i t i e s in supernatants from C. fimi cultures grown on a v i c e l and CMC 22 Figure 9. -Avicel and CMC induction of CMCase over 9 days 25 Figure 10. S p e c i f i c a c t i v i t i e s of CMCase during growth on a v i c e l and CMC 27 Figure 11. Protein composition of culture supernatants at d i f f e r e n t stages of growth .29 Figure 12. A c t i v i t y p r o f i l e s of avicel-induced culture supernatants a f t e r 3, 6, and 9 days of growth 31 Acknowledgement I would l i k e to thank Drs. Kilburn, M i l l e r and Warren for t h e i r guidance and support throughout t h i s work. I am e s p e c i a l l y g r a t e f u l to W. Wakarchuk for his endless patience and encouragement. 1 Introduction The conversion of c e l l u l o s i c wastes to glucose has been a r a p i d l y growing f i e l d of study in the past 20 years. Cellulose i s one of the most abundant renewable resources, and e f f i c i e n t methods are desirable for the conversion of f o r e s t r y and a g r i c u l t u r a l wastes to single c e l l protein, l i q u i d f u e l s , organic acids and other i n d u s t r i a l l y useful compounds ( F l i c k i n g e r , 1980; Detroy and St. J u l i a n , 1983). Enzymatic degradation i s an e f f i c i e n t way to break down c e l l u l o s e , i f the enzymes are available in mass qua n t i t i e s . There are three types of enzymes which form the c e l l u l a s e complex: endoglucanase which randomly cleaves within the chain, exoglucanase which cleaves glucose or c e l l o b i o s e units from the non-reducing ends, and 6-glucosidase which hydrolyses ce l l o b i o s e (King and Vessal, 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 vary in t h e i r e f f i c i e n c y (either) because of the c a t a l y t i c properties of the enzymes themselves or because of the proportions of the d i f f e r e n t enzymes within a given system. Molecular cloning would allow the construction of str a i n s producing optimal proportions of the enzymes within 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 reported the cloning of c e l l u l a s e s from the bacterium Cellulomonas fimi (Whittle et a l . , 1982; Gilkes et a l . , manuscript submitted). The successful reconstruction by molecular genetics of the en t i r e C. fimi c e l l u l a s e system depends on an understanding of the s t r u c t u r a l genes involved. Although the genetics of C. fimi i s e s s e n t i a l l y unknown, 2 chara c t e r i z a t i o n of the enzymes in the system should indicate the c r i t i c a l number of genes. Here, the basic constituents, properties and regulation of C. find'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 reported. 3 Materials and Methods 1. Culture conditions. Cellulomonas fimi ATCC 484 was grown in a basal medium (Stewart and Leatherwood, 1976) consisting of 1 g NaNO^, 1 g K^HPO^, 0.5 g KC1, 0.5 g Difco yeast extract, 0.5 g MgSO^H^O and 1 g carbon source (either a v i c e l , CMC or glucose) in 1 1 d i s t i l l e d H 20, pH 7.0. Cultures were grown on a rotary shaker at approximately 200 RPM at 30°C. S o l i d media contained 11 g Difco bacto-agar and 1 g glucose. Inocula were made from a single colony picked into basal medium containing glucose, grown overnight to l a t e log phase, then d i l u t e d 100 f o l d into 1 l i t r e of medium. 2. Preparation of culture supernatants. C e l l s and insoluble c e l l u l o s e were removed by centrifugation for 20 minutes at 10,000 X g and 4°C. The supernatants were decanted and made 0.02% in Na^N and 0.3 mM in phenylmethylsulfonylfluoride (PMSF). Supernatants were f i l t e r e d twice with suction through 2 Whatman gf/c f i l t e r s , then concentrated 50-100 f o l d by u l t r a f i l t r a t i o n through an Amicon PM10 membrane in an Amicon pressure c e l l . 3. Enzyme and protein assays. C e l l u l o l y t i c 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 assaying for the production of reducing groups from carboxymethylcellulose (CMC). The reaction mixtures contained 0.5 ml 4% CMC (w/v) in 50 mM P°^i pH 6.8, and 0.25 ml culture supernatant or whole culture (a suspension of whole c e l l s , c e l l u l o s e and culture medium) d i l u t e d in 50 mM PO , pH 6.8. The 4 reactions were incubated at 37°C for 30 minutes, terminated by the addition of 0.8 ml DNS reagent ( M i l l e r et a l . , 1960) and 50 ug Sigma Standard Glucose, then steamed for 15 minutes. When the reactions had cooled to room temperature, the absorbance at 550 nm was determined with a Unicam SP800 spectrophotometer. The number of reducing groups produced was estimated from a glucose standard curve and expressed as ug glucose equivalents produced min ^  ml ^ enzyme. Protein was determined by the method of Lowry et a l . (1951) using Sigma lysozyme as standard. When measuring t o t a l culture protein, samples were boi l e d f i r s t for 5 min in 1% SDS. The corresponding control reaction also contained 1% SDS. 4. Electrophoretic analysis of culture supernatants. Concentrated culture supernatants were electrophoresed in 1.5 mm thick polyacrylamide slab gels, either 6% gels under non-denaturing conditions (Jovin et a l . , 1964) or 10% gels containing sodium dodecyl sulphate (SDS) (Laemmli, 1970). Gels were stained for protein with Coomassie Blue (Biorad) or with s i l v e r by the method of Tsai and Frasch (1982). Protein p r o f i l e s were obtained by scanning Coomassie stained gels on a Helena Quick Scan. C e l l u l a s e a c t i v i t y in a non-denaturing gel was detected as follows: a lane was excised from the gel and cut into 2 mm s l i c e s ; each s l i c e was incubated with 0.5 ml 50 mM phosphate, pH 6.8, for 48 hours at 4°C; the eluates were assayed for CMCase a c t i v i t y as described above. 5 Results 1. Properties of CMCase in culture supernatants. The pH and temperature optima and temperature s t a b i l i t y were determined for the CMCase in concentrated supernatant from a culture grown for 3 days with a v i c e l . The pH optimum was 7.0 (Fig. 1). However there were some e f f e c t s of pH and buffer on the DNS reaction ( Fig. 2). The absorbance at 550 nm was enhanced by the acetate buffer compared with the phosphate buffer. The absorbance also increased with increasing pH in both buffers. Taking these e f f e c t s into consideration, the optimal pH for CMCase remained pH 7.0. The optimal temperature f or the CMCase reaction was 52-55°C (Fi g . 3). However, the enzymes were not stable at high temperatures p r i o r to incubation with substrate (Fig. 4). Aft e r heating f or 15 minutes at temperatures less than 50°C, 95% of the o r i g i n a l a c t i v i t y was retained. After heating at 58°C for 15 minutes, the enzymes were almost completely inactivated. A c t i v i t y was l o s t more slowly at lower temperatures: a f t e r 90 minutes at 37°C over 30% of the o r i g i n a l a c t i v i t y was l o s t ; at 49°C 50% of 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 l o s t (Fig. 5). The r e s u l t s indicated that hydrolysis of substrate and i n a c t i v a t i o n o of CMCase both occurred much fas t e r at higher temperatures. Above 60 C the rate of i n a c t i v a t i o n exceeded the rate of hydrolysis, and the enzyme was denatured completely before i t could hydrolyze the c e l l u l o s e . 6 Figure 1. The pH optimum of C. fimi CMCase. Concentrated supernatant was prepared from a 3 day culture grown with 0.1% a v i c e l . CMC was dissolved i n the appropriate buffer to 4% (w/v), and the f i n a l pH was determined. A buffer of the same pH was used to d i l u t e the enzyme. A constant amount of enzyme was added to each reaction and assayed for CMCase as described in MATERIALS AND METHODS. CMCase was expressed as ug glucose m i n - 1 . ( x — x ) 0.2 M acetate buffer; (o^-o) 0.05 M phosphate buffer. 8 Figure 2. E f f e c t s of pH and buffer on the DNS reaction. Standard glucose was prepared in various buffers. 100 ug glucose were added to the DNS reagent. The reactions were steamed, and the absorbance was read at 550 nm. ( x — x ) 0.2 M acetate buffer; ( o — o ) 0.05 M phosphate buffer. 9 10 Figure 3. Temperature optimum of C. fimi CMCase. Concentrated supernatant was d i l u t e d in SO mM phosphate, pH 6.8 and assayed for CMCase at various temperatures for 30 minutes as described in MATERIALS AND METHODS. CMCase was expressed as ug glucose min \ Temp °C 12 Figure 4. E f f e c t of temperature on CMCase s t a b i l i t y . Concentrated supernatant was d i l u t e d in 50 mM phosphate, pH 6.8, and incubated at various temperatures f o r 15 minutes. The enzyme then was cooled on i c e , and an aliquot 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 percent of the a c t i v i t y i n the sample incubated at 0°C. 13 —i 1 1 1 — — — t -o o o o o O CO CO ^ t\l A J I A I P V |DUi6uo % 14 Figure 5. Kinetics of heat i n a c t i v a t i o n of CMCase. Concentrated supernatant was d i l u t e d in 50 mM phosphate, pH 6.8 and incubated at 0°, 37°, 49° and 55°C. At various times, an aliquot was removed to an ice bath. A l l samples were assayed for CMCase at 37°C as described in MATERIALS AND METHODS. A c t i v i t y was expressed as a percentage of the a c t i v i t y in the sample incubated at 0°C. 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); 45 minutes (A—A); 90 minutes ( x — x ) . 15 20] 0 20 40 60 80 Temp °C 16 2. The e f f e c t s of d i f f e r e n t carbon sources on c e l l u l a s e production. C. fimi was grown on c e l l u l o s e with or without high glucose or c e l l o b i o s e to determine i f c e l l u l a s e production was regulated by c a t a b o l i t e repression. CMCase a c t i v i t y was measured in the culture supernatants (Table 1). Concentrated supernatants were also analyzed by polyacrylamide gel electrophoresis (PAGE), ei t h e r non-denaturing (Fig. 6) or denaturing (Fig. 7). As shown by Stewart and Leatherwood (1976), no c e l l u l a s e was detected in cultures grown on glucose or c e l l o b i o s e even in the presence of inducing l e v e l s of c e l l u l o s e . Correspondingly, the protein gel patterns of the uninduced or repressed supernatants were much simpler than the induced supernatants. This suggested that the CMCase a c t i v i t y was derived from multiple components. 3. A v i c e l and CMC induction of c e l l u l a s e . The amount of c e l l u l a s e a c t i v i t y found in CMC-induced culture supernatants was only 70% of that found in avicel-induced culture supernatants (Table 1). This could be due simply to a decrease in the l e v e l of a given enzyme or a l t e r n a t i v e l y , to the induction of d i f f e r e n t c e l l u l a s e s with lower a c t i v i t i e s . The SDS gel pattern (Fig. 7 lanes 3 & 7) indicated that the protein m o b i l i t i e s were s i m i l a r although the CMC-induced supernatant pattern appeared simpler. However, the a c t i v i t y p r o f i l e from a native gel indicated that the active components had d i f f e r e n t m o b i l i t i e s ( Fig. 8). 17 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 Growth substrate CMCase a c t i v i t y (units ml "S 0.1% A v i c e l 130 0.1% A v i c e l + 1% glucose 0 0.1% CMC 90 0.1% CMC + 1% glucose 0 1% glucose 0 1% ce l l o b i o s e 0 C. fimi was grown in basal medium with various carbon sources. CMCase a c t i v i t y was measured in the unconcentrated supernatants as described in MATERIALS AND METHODS. 18 Figure 6. Non-denaturing PAGE showing the e f f e c t s of carbon source on e x t r a c e l l u l a r protein patterns. Supernatants were prepared from cultures grown f or 3 days on 0.1% a v i c e l or CMC with or without 1% glucose or ce l l o b i o s e and analyzed by non-denaturing PAGE (see MATERIALS AND METHODS). The gel was stained with Coomassie Blue. Lane 1: 1% ce l l o b i o s e ; lane 2: 1% cellobiose and 0.1% a v i c e l ; lane 3: 0.1% a v i c e l ; lane 4: 1% glucose and 0.1% a v i c e l ; lane 5: 1% glucose; lane 6: 1% glucose and 0.1% CMC; lane 7: 0.1% CMC. 19 I 2 3 4 5 6 7 20 Figure 7. Denaturing PAGE showing the e f f e c t s of carbon source on e x t r a c e l l u l a r protein patterns. The samples described in the legend to figure 6 were electrophoresed under denaturing conditions. The gel was s i l v e r stained. Lane 1: 1% c e l l o b i o s e ; lane 2: 1% c e l l o b i o s e and 0.1% a v i c e l ; lane 3: 0.1% a v i c e l ; lane 4: 17. glucose and 0.1% a v i c e l ; lane 5: 1% glucose; lane 6: 1% glucose and 0.1% CMC; lane 7: 0.1% CMC; lane 8: molecular weight standards which were, from top to bottom, fl galactosidase (116,000 daltons), phosphorylase B (97,400 daltons), bovine serum albumin (66,000 daltons), ovalbumin (45,000 daltons), carbonic anhydrase (29,000 daltons). 21 I 2 3 4 5 6 7 8 22 Figure 8. CMCase a c t i v i t i e s in supernatants from C. fimi cultures grown with A v i c e l and CMC. Supernatant samples from cultures grown for 3 days on 0.1% a v i c e l (upper two panels) or 0.1% CMC (lower two panels) were electrophoresed in duplicate lanes in a non-denaturing g e l . One lane was stained with Coomassie Blue and scanned. The other lane was assayed f o r CMCase a c t i v i t y as described in MATERIALS AND METHODS. The absorbance at 550 nm was plotted against gel s l i c e number where s l i c e 1 represents the o r i g i n of the gel and s l i c e 40 represents the dye front. 23 24 A time course of CMC and a v i c e l induction of c e l l u l a s e was determined by assaying cultures at d a i l y i n t e r v a l s for CMCase, protein and viable c e l l s . The c e l l numbers and t o t a l protein plateaued a f t e r the f i r s t three days of growth ( F i g . 9). CMCase continued to increase through-out the 9 day growth period; t h i s was p a r t i c u l a r l y noticeable in the avicel-grown culture (Figs. 9 and 10). C. fimi was able to grow well 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 density greater than 2 9 -1 X 10 c e l l s ml . The bacteria grew less e f f i c i e n t l y on the carboxymethyl-substituted c e l l u l o s e , perhaps because they were unable to u t i l i z e CM-glucose or CM-cellobiose produced. 4. Ef f e c t s of culture age on avicel-induced culture supernatants. To determine what changes were taking place in supernatants as CMCase increased with culture age, supernatants were prepared from cultures grown on a v i c e l f o r 3, 6 and 9 days. Analysis by non-denaturing PAGE ( F i g . 11, lanes 1-3) and denaturing PAGE (Fig. 11, lanes 4-6) showed that the e x t r a c e l l u l a r proteins became more mobile as the cultures aged. The major a c t i v i t i e s in a 3 day culture supernatant migrated more slowly than the major a c t i v i t i e s i n 6 or 9 day supernatants (Fig. 12). In the denaturing gel these changes corresponded to the loss of higher molecular weight components (110, 65 and 56 kilodaltons) present a f t e r 3 days of growth and the appearance of lower molecular weight components (45, 40 and 25 kilodaltons) by 9 days of culture growth. 25 Figure 9. A v i c e l and CMC induction of CMCase over 9 days. One l i t r e cultures with 0.1% a v i c e l (panels A, C & E) or 0.1% CMC (panels B, D and F) were grown for 9 days. At 24 hour i n t e r v a l s 25 ml were removed from each culture. Total viable c e l l s were determined from plate counts (panels A & B). Protein and CMCase were measured in the t o t a l culture (panels C & D), and in the supernatant (panels E & F). Viable -1 -9 c e l l s were expressed as colony forming units ml X 10 ( o — o ) ; CMCase was expressed as units ml 1 ( o — o + A—A); protein was expressed as mg ml 1 (•—• + A—A). 27 Figure 10. S p e c i f i c a c t i v i t y of CMCase during growth on a v i c e l and CMC -1 -3 Sp e c i f i c a c t i v i t y was calculated as CMCase units mg protein x 10 from the data shown in Figure 9. Panel A: avicel-induced CMCase in supernatant ( o — o ) and in culture (o—•); panel B: CMC-induced CMCase supernatant (A—A) and in culture ( A — A ) . Specific Activity - to « o b o 29 Figure 11. Protein composition i n culture supernatants at d i f f e r e n t stages of growth. A 1 l i t r e culture of C. fimi was grown with 0.1% a v i c e l . At 3, 6 and 9 days 250 ml were removed, and supernatants were prepared. Samples were electrophoresed in a non-denaturing gel (lanes 1-3) and in a denaturing gel (lanes 4-6). The gels were stained with Coomassie Blue. Protein patterns of a 9 day supernatant (lanes 1 & 6), 6 day supernatant (lanes 2 and 5), 3 day supernatant (lanes 3 & 4). 30 31 Figure 12. A c t i v i t y p r o f i l e s of avicel-induced culture supernatants a f t e r 3, 6 and 9 days of growth. A c t i v i t y p r o f i l e s were obtained as described in MATERIALS AND METHODS for the samples described in the legend to figu r e 11. CMCase a c t i v i t y p r o f i l e s from day 3 (panel A), day 6 (panel C) and day 9 (panel E) supernatants; native gel protein p r o f i l e s from day 3 (panel B), day 6 (panel D) and day 9 (panel F) supernatants. 32 Slice Number 33 Discussion The optimal conditions for the CMCases present in culture supernatants of Cellulomonas fimi ATCC484 were found to be pH 7.0 and 52-55°C. Although the enzymes were more active at higher temperatures, they were also more l a b i l e ; less than 50% of the o r i g i n a l a c t i v i t y remained a f t e r 45 minutes at 55°C. The enzyme a c t i v i t y was constant during 15 minutes at 37°C. These r e s u l t s compare well with the published conditions determined for other cellulomonads (Choi e_t a l . , 1978; Kim and Wimpenny, 1981; Stoppok et a l . , 1982; Storvick and King, 1960). In contrast, c e l l u l a s e from the thermophilic bacterium Clostridium o thermocellum had optimal a c t i v i t y at 70 C and pH 6.1 (Johnson et a l . , 1982). The fungal enzymes generally were more active at pH 4.5 - 5.0; t h i s i s in keeping with the acid environments of r o t t i n g wood on which they were found (Rho et a l . , 1982; Parry et a l . , 1983). The c e l l u l a s e s of C. fimi were induced by c e l l u l o s e and regulated by cata b o l i t e repression. In the presence of high l e v e l s of glucose or cel l o b i o s e and inducing l e v e l s of c e l l u l o s e , no CMCase a c t i v i t y was detected in the culture supernatants. These re s u l t s agree with those from other studies (Stewart and Leatherwood, 1976; Beguin §_t &1., 1977; Stoppok et a l . , 1982; Choi et a l . , 1978). Beguin et a l . (1977) found that c e l l o b i o s e repressed the synthesis of c e l l u l a s e s even a f t e r the addition of 1 mM cAMP. P r i e s t (1977) noted that the e x t r a c e l l u l a r enzymes of many bacteria are regulated by cat a b o l i t e repression mechanisms which do not require cAMP, cGMP or the highly phosphorylated nucleotides. 34 C. fimi grew les s e f f i c i e n t l y and produced les s c e l l u l a s e with CMC as carbon source as compared to a v i c e l . 0.1% CMC appeared to be l i m i t i n g f o r growth. This may have been because the carbon source had been depleted; or due to the substitutions large unusable fragments may have been generated. A l t e r n a t i v e l y , the smaller breakdown products, CM-glucose and CM-cellobiose, were not metabolizable. Growing the organism on a higher percent CM-cellulose may overcome t h i s l i m i t a t i o n . Some published r e s u l t s support t h i s idea. Beguin §_t a l . (1977) reported that Cellulomonas Ilbc grew twice as f a s t on 2.5% CMC as on 2.5% a v i c e l . In contrast, C. uda grew better and degraded more substrate when cultured with 2% a v i c e l than with 2% CMC (Stoppok et a l . , 1982). Choi et a l . (1978) also observed better growth of CS1-1 with 1% a v i c e l than with 1% CMC. However, in the CMC-grown culture the amount of CMCase produced per v i a b l e c e l l was almost 30 f o l d higher. In a l l three of these published reports the CMCase reached a maximum l e v e l a f t e r 2 - 5 days of growth, then i t e i t h e r remained constant or declined. The c e l l u l a s e a c t i v i t y d i d not continue to increase as reported here. C. fimi was very e f f i c i e n t at transporting c e l l u l a s e s to the environment. CMCase a c t i v i t y in culture supernatants appeared to account f o r a l l the a c t i v i t y detected in whole culture (Figs. 9 & 10). Therefore, cell-bound c e l l u l a s e must contribute very l i t t l e to the t o t a l a c t i v i t y . However the CMCase assay of whole culture may detect only membrane-bound a c t i v i t y and may miss cytoplasmic a c t i v i t y . Therefore, a thorough analysis"of CMCase l o c a t i o n should be made with f r a c t i o n a t e d c e l l s . As avicel-grown cultures aged, the increase in c e l l u l a s e a c t i v i t y c o r r e l a t e d with an increase i n the m o b i l i t y of active components on polyacrylamide gels. I t seemed u n l i k e l y that a single organism would make as many as 10 unique gene products to hydrolyze a simple glucose polymer. The apparent m u l t i p l i c i t y of the C. fi m i complex could be a r e s u l t of changing a f f i n i t y of c e l l u l a s e s for substrate or of modification. Some possbile modifications include enzymatic processing of the enzymes, changes in the stoichiometry of the c e l l u l a s e complex, or microheterogeneity of glycoproteins. Eriksson and Pettersson (1982) have reported the enhancement of Sporotrichum pulverulentum endocellulase a c t i v i t y by p u r i f i e d proteases from that fungus. They postulated that the proteases functioned to activate a zymogen, destroy an i n h i b i t o r or aid in release of the c e l l u l a s e s from the fungal c e l l w a l l . I f C. fimi has an e x t r a c e l l u l a r protease l i k e the B a c i l l u s s u b t i l i s i n , cleavage of "precursor" c e l l u l a s e s must be s p e c i f i c . The SDS gels ( F i g . 11) show a reproducible pattern of dis c r e e t bands, not a smear of smaller fragments, which is consistent with t h i s idea. The advantage of having a protease may be to generate smaller active enzymes with access to regions of c e l l u l o s e that larger proteins or complexes would not have. Saddler and Kahn (1981) characterized 2 endoglucanases from A c e t i v i b r i o c e l l u l o l y t i c u s . The enzymes had molecular weights of 33,000 and 10,400 daltons. The proportion of the la r g e r enzyme decreased with culture age while the smaller enzyme increased. The smaller enzyme may be a p r o t e o l y t i c cleavage product or a subunit of the larger enzyme. The 36 relatedness of the two enzymes was not determined. Enger and Sleeper (1965) found that 3 of 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 endogluconases in Streptomyces a n t i b i o t i c u s were r e l a t e d immunologically. The stoichiometry of the complex also may be changing. Yoshikawa et a l . (1974) grew Pseudomonas fluorescens var c e l l u l o s a on a v i c e l f o r several days. At various times of culture growth, supernatants were passed over a gel f i l t r a t i o n column. They found that as the cultures aged, the active 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 in the included volume. When the active f r a c t i o n s were analyzed e l e c t r o p h o r e t i c a l l y , they were found to consist of only 2 components: A (fast-moving) or B (slow-moving). Both of these components were glycosylated, but A contained more galactose, while B had more glucose i n the carbohydrate moiety (Yamane et a l . , 1970). Early i n cult u r e growth, the active column f r a c t i o n s contained only B; but as the culture aged, the peaks contained more A and less B. They concluded that A was derived from B by enzymatic modification. Recently the observation that c e l l u l a s e s bind to c e l l u l o s e has gained s i g n i f i c a n c e . This phenomenon has been observed in fungal and b a c t e r i a l systems (Berghem et a l . , 1976; Manning and Wood, 1983; Choi §_t a l . , 1978; Beguin and Eisen, 1978). Beguin et a l . (1977) have observed that the amount of enzyme bound to a v i c e l decreased with culture age, corresponding to an increase in soluble enzyme. Cel l u l a s e s may bind more r e a d i l y to the amorphous regions, and as these become hydrolyzed, the enzymes may become free in the supernatant. Once free they may be more susceptible to enzymatic modification, whether i t be pro t e o l y s i s or changes in g l y c o s y l a t i o n . A possible outcome of the modifications may be an a l t e r e d a f f i n i t y of the c e l l u l a s e s f o r substrate. A l t e r n a t i v e l y the a f f i n i t y of the components one f o r the other may r e s u l t in stoichiometric rearrangements. The changes in mobility of active components may a c t u a l l y represent synthesis of d i f f e r e n t c e l l u l a s e s . Enriquez (1981) observed a diauxic growth pattern early in the fermentation of sugar cane bagasse by Cellulomonas I l b c . The f i r s t log phase corresponded to degradation of amorphous and hemicellulose regions. A lag of 10-20 hours was followed by a second log phase which corresponded to a decrease in c r y s t a l l i n i t y of substrate. He postulated that synthesis of the component occurred during the lag phase. Perhaps a s i m i l a r pattern occurs with C. f i m i . A v i c e l contains both amorphous and c r y s t a l l i n e regions. Once the amorphous regions have been hydrolysed, enzymes more active on c r y s t a l l i n e regions may be induced. Any or a l l of these mechanisms may occur in C. f i m i . Recently, some of the c e l l u l a s e s from C. fimi have been shown to bind a v i c e l and to contain carbohydrate groups (Langsford e_t a l . , manuscript accepted for p u b l i c a t i o n ) . An e x t r a c e l l u l a r protease has also been detected. Ultimately the enzymes must be p u r i f i e d to determine i f the many CMCase a c t i v i t i e s are unique gene products or are generated from a more l i m i t e d number of precurser molecules. Antibody c r o s s - r e a c t i v i t y and peptide mapping should allow the detection of s i m i l a r i t i e s between the d i f f e r e n t components. Then these techniques could be useful in i d e n t i f y i n g the products from the cloned genes. 38 References Cited 1. Beguin, P. and Eisen, H. (1978). 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 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 from Cellulomonas sp. European Journal of Biochemistry 87:525-531. 2. Beguin, P., Eisen, H. and Roupas, A. (1977). Free and cellulose-bound c e l l u l a s e s in a Cellulomonas species. Journal of General Microbiology 101:191-196. 3. Berghem, L.E.R., Pettersson, L.G. and Axio-Frederiksson, U.B. (1976). The mechanism of enzymatic c e l l u l o s e degradation: p u r i f i c a t i o n and properties of two d i f f e r e n t 1,4-B-glucanohydrolases from Trichoderma v i r i d e . European Journal of Biochemistry 61:621-630. 4. Choi, W.Y., Haggett, K.D. and Dunn, N.W. (1978). I s o l a t i o n of a cotton wool degrading s t r a i n of Cellulomonas: mutants with a l t e r e d a b i l i t y to degrade cotton wool. Au s t r a l i a n Journal of B i o l o g i c a l Science 34:553-564. 5. Detroy, R.W. and St. J u l i a n , G. (1983). Biomass conversion: fermentation chemicals and f u e l s . CRC C r i t i c a l Reviews in Microbiology 10:203-228. 39 6. Enger, M.D. and Sleeper, B.P. (1965). M u l t i p l e c e l l u l a s e system from Streptomyces a n t i b i o t i c u s . Journal of Bacteriology 89:23-27. 7. Enriquez, A. (1981). Growth of c e l l u l o l y t i c b a c t e r i a on sugarcane bagasse. Biotechnology and Bioengineering 23:1423-1429. 8. Enriquez, A., Montalvo, R., and Canales, M. (1981). V a r i a t i o n of bagasse c r y s t a l l i n i t y and c e l l u l a s e a c t i v i t y during the fermentation of Cellulomonas b a c t e r i a . Biotechnology and Bioengineering 23:1431-1436. 9. Eriksson, K.-E., and Pettersson, 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 of two a c i d i c proteases from the white-rot fungus Sporotrichum pulverulentum. European Journal of Biochemistry 124:635-642. 10. F l i c k i n g e r , M.C. (1980). Current b i o l o g i c a l research in conversion of c e l l u l o s i c carbohydrates into l i q u i d f u e l s : how far have we come? Biotechnology & Bioengineering 22 (suppl. l):27-48. 11. G i l k e s , N.R., Kilburn, D.G., Langsford, M.L., M i l l e r , R.C., J r . , Wakarchuk, W.W., Warren, R.A.J., Whittle, D.J. and Wong, W.K.R. (manuscript accepted f o r p u b l i c a t i o n ) . I s o l a t i o n and ch a r a c t e r i z a t i o n of Escherichia c o l i clones expressing c e l l u l a s e genes from Cellulomonas f i m i . 40 12. Johnson, E.A., Sakajoh, M., H a l l i w e l l , G., Madia, A., and Demain, A.L. (1982). S a c c h a r i f i c a t i o n of complex c e l l u l o s i c substrates by the c e l l u l a s e system from Clostridium thermocellum. Applied & Environmental Microbiology 43:1125-1132. 13. Jovin, T., Chrambach, A. and Naughton, M.A. (1964). An apparatus for temperature-regulated polyacrylamide gel elect r o p h o r e s i s . A n a l y t i c a l Biochemistry 9:351-369. 14. Kim, B.H. and Wimpenny, J.W.T. (1981). Growth and c e l l u l o l y t i c a c t i v i t y of Cellulomonas flavigena . Canadian Journal of Microbiology 27:1260-1266. 15. King, K.W. and Vessal, M.I. (1969). Enzymes of the c e l l u l a s e complex. In Gould, R.F. (ed.) C e l l u l a s e s and Their A p p l i c a t i o n s , Advances in Chemistry seri e s 95:17-25. 16. Laemmli, U.K. (1970). Cleavage of s t r u c t u r a l proteins during assembly of the head of bacteriophage T4. Nature 227:680-685. 17. Langsford, M.L., Gilkes, N.R., Wakarchuk, W.W., Kilburn, D.G., M i l l e r , R.C., J r . , and Warren, R.A.J, (manuscript accepted f o r p u b l i c a t i o n ) . The c e l l u l a s e system of Cellulomonas f i m i . 41 18. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951). Protein measurement with the F o l i n phenol reagent. Journal of B i o l o g i c a l Chemistry 193:265-275. 19. Manning, K. and Wood, D.A. (1983). Production and regulation of e x t r a c e l l u l a r endocellulase by Agaricus bisporus. Journal of General Microbiology 129:1839-1847. 20. M i l l e r , G.L., Blum, R., Glennon, W.E., and Burton, A.L. (1960). Measurement of carboxymethylcellulase a c t i v i t y . A n a l y t i c a l Biochemistry 2:127-132. 21. Parry, J.B., Stewart, J.C. and H e p t i n s t a l l , J . (1983). P u r i f i c a t i o n of the major endoglucanase from A s p e r g i l l u s fumigatus Fresenius. Biochemistry Journal 213:437-444. 22. P r i e s t , Fergus G. (1977). E x t r a c e l l u l a r enzyme synthesis in the genus B a c i l l u s . B a c t e r i o l o g i c a l Reviews 41:711-753. 23. Rho, D., Desrochers, M., Jurasek, L., Driguez, H. and Defaye, J. (1982). Induction of c e l l u l a s e in Schizophyllum commune: th i o c e l l o b i o s e as a new inducer. Journal of Bacteriology 149:47-53. 24. Saddler, J.N. and Khan, A.W. (1981). C e l l u l o l y t i c enzyme system of A c e t i v i b r i o c e l l u l o l y t i c u s . Canadian Journal of Microbiology 27:288-294. 42 25. Stewart, Bobbie, J . , and Leatherwood, J.M. (1976). Derepressed synthesis of c e l l u l a s e by Cellulomonas. Journal of Bacteriology 128:609-615. 26. Stoppok, W., Rapp, P., and Wagner, F. (1982). Formation, l o c a t i o n , and regulation of endo-1,4-B-glucanases and S-glucosidases from Cellulomonas uda. Applied and Environmental Microbiology 44:44-53. 27. Storvick, W.O. and King, K.W. (1960). The complexity and mode of action of the c e l l u l a s e system of C e l l v i b r i o g i l v u s . Journal of B i o l o g i c a l Chemistry 235:303-307. 28. T s a i , C.-M. and Frasch, C.E. (1982). A s e n s i t i v e s i l v e r s t a i n f o r detecting lipopolysaccharides in polyacrylamide gels. A n a l y t i c a l Biochemistry 119:115-119. 29. Whittle, D.J., Kilburn, D.G., Warren, R.A.J, and M i l l e r , R.C., J r . (1982). Molecular cloning of a Cellulomonas fimi c e l l u l a s e gene in Escherichia c o l i . Gene 17:139-145 . 30. Yamane, K., Suzuki, H., and Nisizawa, K. (1970). P u r i f i c a t i o n and properties of e x t r a c e l l u l a r and cell-bound c e l l u l a s e components of Pseudomonas fluorescens var. c e l l u l o s a . Journal of Biochemistry 67:19-35. A3 Yoshikawa, T., Suzuki, H. and Nisizawa, K. (1974). Biogenesis of multiple c e l l u l a s e components of Pseudomonas fluorescens var. c e l l u l o s a . I. E f f e c t s of culture and conditions on the m u l t i p l i c i t y of c e l l u l a s e . Journal of Biochemistry 75:531-540. 

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