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1,4-β-d -Glucan cellobiohydrolase from Sclerotium rolfsii
[32]
S. rolfsii
CELLOBIOHYDROLASE
307
Substrate Specificity. All three cellulases partially degraded native cellulose. Cellulase I, but not cellulases II and III, readily hydrolyzed the mixed fl-l,3//3-1,6-polysaccharides such as carboxymethylpachyman, yeast glucan, and laminarin. Both cellulase I and the fl-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes. Lichenan (/3-1,4;/3-I ,3) was degraded by all three cellulases. Cellulase II could not degrade CM cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-/3-1,4-glucan cellobiosylhydrolase. Degradation of cellulose (filter paper) can be catalyzed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation.~l Mode of Action. The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins. ~ The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of CM-cellulose, confirmed that cellulase III was an endocellulase. The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length. 11 M. G. Shepherd, C. C. Tong, and A. L. Cole, Biochem. J. 193, 67 (1981).
[32] 1 , 4 - f l - D - G l u c a n C e l l o b i o h y d r o l a s e f r o m S c l e r o t i u m rolfsii B y JAI C . SADANA a n d RAJKUMAR V . PATIL
The fungus Sclerotium rolfsii, when grown on cellulose as the sole carbon source, produces an oxidative enzyme, cellobiose dehydrogenase, 1and three different types of hydrolytic enzymes, 2-5 (I) a 1,4-fl-oglucan cellobiohydrolase (EC 3.2.1.91, cellulose 1,4-/3-cellobiosidase) 2 (cellobiohydrolase), (2) three endo-fl-glucanases, 3 and (3) four/3-o-glucosidases. 4,5 All these enzymes have been obtained in a homogeneous state. The hydrolytic enzymes form the cellulase complex of S. rolfsii. All of the J. C. Sadana and R. V. Patil, J. Gen. Microbiol. 131, 1917 (1985). 2 R. V. Patil and J. C. Sadana, Can. J. Biochem. Cell Biol. 62, 920 (1984). 3 j. C. Sadana, A. H. Lachke, and R. V. Patil, Carbohydr. Res. 133, 297 (1984). 4 j. G. Shewale and J. C. Sadana, Arch. Biochem. Biophys. 207, 185 (1981). 5 j. C. Sadana, J. G. Shewale, and R. V. Patil, Carbohydr. Res. 118, 205 (1983).
METHODS 1N ENZYMOLOGY, VOL. 160
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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CELLULOSE
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hydrolytic enzymes are required for the efficient hydrolysis of crystalline cellulose. The cellobiose dehydrogenase plays only a minor role in the overall degradation of crystalline cellulose to glucose when S. rolfsii is grown on cellulose.1 1,4-fl-D-Glucan cellobiohydrolase shows high activity toward phosphoric acid-swollen cellulose but no viscosity-lowering activity toward carboxymethylcellulose. It functions in the hydrolysis of crystalline cellulose. 2 The enzyme acts in an endwise fashion by removing successive cellobiosyl units (93-96%) from the nonreducing end of the fl-l,4-glucan chains; glucose (4-7%) is also detected. In addition, it shows an initial endo-type mode of action on cotton sliver (dewaxed deltapine cotton) producing short fibers.6 A similar type of enzyme which shows an endotype mode of action on crystalline cellulose, besides its endwise action of removing cellobiosyl units from the nonreducing chain ends of cellulose, has been reported in Trichoderma reesei. 7
Assay Method Principle. The enzyme is assayed by determining the soluble sugars released in the supernatant fluid from phosphoric acid-swollen cellulose by the Somogyi-Nelson 8 or p-hydroxybenzoic acid hydrazide9 method or by determining p-nitrophenol from p-nitrophenyl-fl-D-cellobioside. The range of the Somogyi-Nelson method is 5-100 tzg, and that ofp-hydroxybenzoic acid hydrazide method 1-50/zg of reducing sugars as glucose. The method of estimation of cellobiohydrolase activity, using phosphoric acid-swollen cellulose as substrate, has certain limitations and is applicable to preparations which are free of endo-fl-glucanase and fl-Dglucosidase activities. The S. rolfsii cellobiohydrolase is much more active against phosphoric acid-swollen cellulose than is fl-glucosidase, but is much less active than endo-fl-glucanase. The interfering effect of fl-glucosidase is overcome by the addition of 2 m M D-glucono-1,5-a-lactone. The Sclerotium cellobiohydrolase is not inhibited by 10 m M D-glucono-I,5-8lactone. In the presence of endo-fl-glucanase, the assay will give erroneous values of cellobiohydrolase activity. A general assay technique for the determination of cellobiohydrolase activity does not exist as no specific substrate is available. However, in cases where the sample solution does not contain either fl-glucosidase or 6 j. C. Sadana and R. V. Patil, Can. J. Biochem. Cell Biol. 63, 1250 (1985). 7 H. Chanzy, B. Henrissat, and R. Vuong, FEBS Lett. 172, 193 (1984). 8 M. Somogyi, J. Biol. Chem. 195, 19 (1952). 9 p. L. Hurst, J. Nielsen, P. A. Sullivan, and M. G. Shepherd, Biochem. J. 165, 33 (1977).
[32]
S. rolfsii CELLOBIOHYDROLASE
309
endo-fl-glucanase, or the endo-fl-glucanase does not hydrolyze cellobiose, p-nitrophenyl fl-o-cellobioside can be used as a specific substrate for the determination of cellobiohydrolase activity. The cellobiohydrolase from S. rolfsii hydrolyzes p-nitrophenyl fl-o-cellobioside to p-nitrophenol and cellobiose but it has no action on cellobiose or p-nitrophenyl/3-0glucopyranoside, z The three endo-/3-glucanases, A, B, and C from S. rolfsii, which have been obtained in a homogeneous state, do not hydrolyze cellobiose, p-nitrophenyl/3-o-glucopyranoside or p-nitrophenyl/3-0cellobioside. 3
Reagents Phosphoric acid-swollen cellulose suspension in 50 m M citrate buffer, pH 4.5, 20 mg/ml Citric acid-trisodium citrate buffer, 50 mM, pH 4.5 Sodium azide, l0 m M in 50 m M citrate buffer, pH 4.5 o-Glucono-1,5-~-lactone, 20 and also 60 m M in 50 m M citrate buffer, pH 4.5 p-Nitrophenyl /3-o-cellobioside, 2 m M in 50 m M citrate buffer, pH 4.5 Sodium carbonate, 2% Preparation o f Phosphoric Acid-Swollen Cellulose. Avicel P.H. 101 (Honeywill and Stein Ltd., U.K.) (10 g) is suspended in o-phosphoric acid (88%, w/v) and left for 1 hr with occasional stirring at 4°. The mixture is poured into ice-cold water (4 liters) and left for 3 rain. The swollen cellulose is washed several times with cold water by decantation. After washing with 1% (w/v) NaHCO3 solution, the thick suspension of swollen Avicel is dialyzed against water in a cold room. After 1 min treatment in a Waring blender, 50 m M citrate buffer, pH 4.8, is added to the suspension so that 1 ml of suspension contains 20 mg of cellulose. Cellulose content is estimated by the anthrone-sulfuric acid method.~° Phosphoric acid-swollen cellulose prepared in this way is in the form of an even suspension. Phosphoric acid-swollen cellulose is stored at 4-5 °. Procedure
With Phosphoric Acid-Swollen Cellulose as Substrate. A 1-ml suspension of phosphoric acid-swollen cellulose is mixed with 0.3 ml of 10 m M sodium azide, 0.1 ml of D-glucono-l,5-8-1actone (if the sample solution contains/3-glucosidase), and 1.5 ml of 50 m M citrate buffer, pH 4.5, in a Pyrex test tube. The enzyme solution (0.1 ml) containing 10 to 15 /xg to D. M. Updegraff,Anal. Biochern. 32, 420 (1969).
310
CELLULOSE
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enzyme protein (0.6-1 mU) is added to the reaction mixture to give a final volume of 3 ml, mixed thoroughly, and incubated at 50° for 4 hr. A control is run with boiled enzyme. The reaction is stopped by immersing the tubes in a boiling water bath for 5 min and is centrifuged after cooling. The released soluble sugars are determined in a 1-ml aliquot of the supernatant fluid by the Somogyi-Nelson 8 or hydroxybenzoic acid hydrazide 9 method. The crude filtrate from S. rolfsii shows high endo-fl-glucanase and flglucosidase activities. The interference due to/3-glucosidase is overcome by adding D-glucono-1,5-8-1actone at a final concentration of 3 m M which inhibits fl-glucosidase. The interference due to endo-fl-glucanase cannot be overcome in the above assay procedure. With p-Nitrophenyl-fl-o-Cellobioside as Substrate. In this assay the activity is measured as the amount of p-nitrophenol released from pnitrophenyl-fl-o-cellobioside. One mole ofp-nitrophenol is released from 1 mol of p-nitrophenyl-/3-o-cellobioside. A suitably diluted enzyme (0.1 ml) containing 50-100/xg enzyme protein is added to a small Pyrex test tube containing 0.8 ml 2 m M p-nitrophenyl-fl-o-cellobioside in 50 m M citrate buffer, pH 4.5, and 0. I ml of 20 m M D-glucono-1,5-8-1actone which has been previously preincubated at 37°. After incubation at 37° for 30 min, the reaction is stopped by adding 1 ml 2% sodium carbonate. The absorbancy of p-nitrophenol released is determined spectrophotometrically at 410 mm in a 1-cm light path cuvette after 10-15 min. The amount ofp-nitrophenol released is calculated from the absorbancy index of 18.5 cmZ/tzmol for p-nitrophenol at 410 nm. 11 Linearity is obtained when a value of 0.02-0.05/xmol of p-nitrophenol is released per minute. Definition of Unit of Activity. One unit of cellobiohydrolase activity is defined as the amount of enzyme that releases 180 tzg reducing sugars (1 /zmol as o-glucose equivalent and corresponds to the production of one reducing end group) per minute at pH 4.5 and 50°, or 1/zmol ofp-nitrophenol from p-nitrophenyl-/3-o-cellobioside per minute at 37°, pH 4.5. Purification
Preparation of Crude Filtrate. Sclerotium rolfsii CPC 142,12 used as the enzyme source, is grown as given in chapter [10] of this volume for cellobiose dehydrogenase. The purification of cellobiohydrolase follows the procedure given in chapter [10] of this volume for cellobiose dehydrogenase. When the concentrated enzyme solution from ultrafiltration step (step 3) of the cellobiIID. Herr, F. Baumer, and H. Dellwig, Eur. J. Appl. Microbiol. Biotechnol. 5, 29 0978). 12 Sclerotium rolfsii CPC 142 culture (NCIM No. 1084) is available from the National Collection of Industrial Microorganisms, National Chemical Laboratory, Poona 411 008, India.
S. rolfsii CELLOBIOHYDROLASE
[32]
311
TABLE I PURIFICATION OF CELLOBIOHYDROLASE FROM
S. rolfsii"
Activity toward (total units) Fraction Culture filtrate Ammonium sulfate, 0-3.4 M Sephadex G-75 chromatography fraction A Ultrafiltration of fraction A (Amicon PM10) DEAE-Sephadex A-50 chromatography Preparative polyacrylamide gel electrophoresis
Protein (mg)
H3PO4-swollen cellulose
17,202 13,937
27,523 26,480
520,109 45,284 462,620 42,785
10,774
22,324
t70,210
8,470
21,260
122,300 21,430
329
206
4.8
57
52
3.4
0.018
0
CMC
PNPG
28,370
From Ref. 2. One unit of activity towards H3PO4-swollen cellulose or CMC (carboxymethylcellulose) is defined as the amount of enzyme that releases 180 /xg reducing sugars (1 /zmol as D-glucose equivalent) per minute at pH 4.5 and 50°, using a 4-hr incubation for H3PO4-swollen cellulose and a 30-min incubation for CMC.6 fl-D-Glucosidase is determined as described in chapter [10] of this volume. PNPG, p-Nitrophenyl/3-D-glucopyranoside.
ose d e h y d r o g e n a s e p r o c e d u r e is c h r o m a t o g r a p h e d on a D E A E - S e p h a d e x A-50 column, endo-fl-glucanase and/3-glucosidase activities are not adsorbed on the column, whereas a major amount of protein containing cellobiohydrolase is adsorbed. This is eluted by 0.1 M citrate buffer, p H 4.5 (fraction n u m b e r s 50-70, see Fig. 1 in chapter [10] of this volume for cellobiose dehydrogenase). This fraction is concentrated by lyophilization and dialyzed against 5 m M T r i s - g l y c i n e buffer, p H 8.5. Further purification of cellobiohydrolase is obtained by preparative polyacrylamide gel electrophoresis as described for cellobiose dehydrogenase in this volume [10]. The results of a typical purification of cellobiohydrolase from S. rolfsii are presented in Table I. Properties of the Purified E n z y m e
Purity. The purified e n z y m e shows one protein band on polyacrylamide gel electrophoresis at p H 2.9 and 8.9, with or without SDS treat-
312
CELLULOSE
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ment, and in analytical isoelectric focusing in 7.5% polyacrylamide gel over the pH range 3.5-10.0. Stability. The enzyme is stable when stored in 50 m M citrate buffer, pH 4.5, at - 15°. The enzyme is most stable at pH 4.5-5.0. The enzyme is stable to repeated freezing and thawing. Physical Properties. The relative molecular weight, Mr, estimated by gel filtration on BioGel P-150,13 electrophoresis by slope method, TMand by its migration in SDS-polyacrylamide gel 15,16is 41,500, 41,700, and 42,000, respectively. The p I of the pure S. rolfsii enzyme is 4.32. Chemical Properties. The enzyme is composed of a single polypeptide chain as the carboxyamidomethylated derivative of the reduced form of" cellobiohydrolase gives one protein band with a relative molecular weight (Mr 42,000) corresponding to the native protein. The enzyme is a glycoprotein containing 7.0% total carbohydrate; it contains 3.6 residues of glucosamine per molecule of enzyme but no galactosamine. The enzyme is high in acidic and low in basic amino acids and contains no cystine or half-cystine. 2 Enzymatic Properties. With phosphoric acid-swollen cellulose, the pH and temperature optima are 4.5 and 50° (4 hr assay), and with Avicel (24 hr assay) they are 4,2 and 37°. The rate of phosphoric acid-swollen cellulose hydrolysis is linear up to 15/-~g enzyme protein (4 hr assay), and with 10 ~g enzyme, the hydrolysis is linear up to 8 hr under standard assay conditions. The purified enzyme (10/zg) produces 40.8/xg reducing sugars as glucose equivalent from phosphoric acid-swollen cellulose in 4 hr, 50°, pH 4.5. With phosphoric acid-swollen cellulose, the activation energy, calculated from Arrhenius plot, is 8.55 cal/mol (1 cal = 4.1868 J). The cellobiohydrolase shows no S-factor activity, ~7 though it potentiates Sfactor activity of endo-fl-glucanases. 3 Kinetics. The [S]0.5 and Vmaxvalues for phosphoric acid-swollen cellulose at pH 4.5, 50 °, calculated from Lineweaver-Burk plot, are 1.66 mg/ ml and 42/.~g reducing sugars/mg protein per minute. The [S]0.s for cellotriose is 2.2 m M (glucose liberated is measured by glucose oxidase-peroxidase method ~8) and for ceUotriose it is 1.81 m M (cellobiose liberated is measured by p-hydroxybenzoic acid hydrazide method9). The Vmaxfor cellotriose is 46/zg glucose/mg protein per minute, and for cellotetraose 208 ~g reducing sugar/mg protein per minute. 13 p. Andrews, Biochem. J. 91, 222 (1964). 14 j. L. Hedrick and A. J. Smith, Arch. Biochem. Biophys. 126, 155 (1968). 15 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). 16 A. L. Shapiro, E. Vinuela, and J. V. Maizel, Biochem. Biophys. Res. Commun. 28, 815 (1967). 17 C. B. Marsh, G. V. Merola, and M. E. Simpson, Text. Res. 23, 831 (1953). ts H. U. Bergmeyer, K. Gawein, and M. Grassl, Methods Enzymatic Anal. 1, 457 (1974).
[32]
S. rolfsii CELLOBIOHYDROLASE
313
Specificity. The enzyme is specific for/3-D-(1---~4) linkage. Action of Cellobiohydrolase on Insoluble and Soluble Substrates. The cellobiohydrolase hydrolyzes Cellulose-123 (Carl Schliecher and Schull Co., W. Germany), Avicel, a-cellulose, phosphoric acid-swollen cellulose, cellooligosaccharide G37 (i.e., degree of polymerization 37), and cotton. The principal product from each substrate is cellobiose (93-96%); glucose (4-7%) is also detected. The enzyme hydrolyzes carboxymethylcellulose to a small extent which is not due to its contamination with endo-/3-glucanase; cellobiose is the only product detected. The cellobiohydrolase has no action on cellobiose or p-nitrophenyl-/3-o-glucopyranoside; however, it hydrolyzes p-nitrophenyl fl-o-cellobioside to pnitrophenol and cellobiose. The enzyme hydrolyzes cellotriose and higher molecular weight cellodextrins; the rate of hydrolysis increases with increasing polymerization of the cellodextrin. Cellotriose is hydrolyzed to cellobiose and glucose, cellotetraose to cellobiose, cellopentaose to cellobiose and cellotriose initially and finally to cellobiose and glucose, and cellohexaose to cellobiose and cellotetraose initially and finally to only cellobiose. Inhibitors. D-Glucono-l,5-8-1actone at l0 m M is not inhibitory. NBromosuccinimide, a tryptophan-specific reagent, at 0.1 m M inhibits the enzyme activity completely. Endo-type Mode of Action of Cellobiohydrolase. The enzyme forms insoluble short fibers from native dewaxed cotton initially. 6 The formation of insoluble short fibers from cotton by S. rolfsii cellobiohydrolase suggests an endo-type mode of action, besides its endwise action of removing cellobiosyl units from the nonreducing end of cellulose chains. Synergism between Cellobiohydrolase and Endo-[3-Glucanase. The pure cellobiohydrolase and endo-/3-glucanases from S. rolfsii, when acting in concert, show active synergistic effects in reconstitution experiments in the solubilization of Avicel and cotton. With phosphoric acidswollen cellulose, the synergistic effect is much smaller. 19 Function
Initiation of Enzymatic Degradation of Crystalline Cellulose. The greater solubilization of Avicel by cellobiohydrolase and of phosphoric acid-swollen cellulose by endo-/3-glucanases when acting alone, and the beneficial effect of pretreatment of Avicel with cellobiohydrolase, and of phosphoric acid-swollen cellulose by endo-/3-glucanases, prior to the addition of the alternative type of enzyme, ~9 suggest that endo-fl-glucanase initiates the attack on amorphous cellulose (creating more ends for the J9 j. C. Sadana and R. V. Patil, Carbohydr. Res. 140, 111 (1985).
314
CELLULOSE
[33]
cellobiohydrolase to act), and cellobiohydrolase initiates the attack on crystalline cellulose (thereby making the substrate more accessible for hydrolysis~9). The observation that cellobiohydrolase also displays an initial endo-type mode of action besides its endwise action of removing cellobiosyl units from the nonreducing end of the cellulose chains lends support to this concept. 6
[33] C e l l u l a s e s o f Therrnomonospora fusca
By DAVID B. WILSON Introduction
Thermomonosporafusca YX is a filamentous actinomycete that has a doubling time of about 4 hr when grown at 55 ° in defined medium with cellulose (Solka-Floc) as a carbon source. The supernatant from a culture of cellulose-grown T. fusca contains a high level of cellulase activity, xylanase activity, and an active protease which modifies the different cellulases without altering their cellulase activity.~ Even though the protease activity can be inhibited by phenylmethylsulfonyl fluoride (PMSF), treated supernatant still contains proteolytically derived isozymes and is extensively degraded when it is heated in SDS. We have isolated a mutant strain of T. fusca, ER-12 that produces no detectable extracellular protease activity and the supernatant from this strain has been used to purify the different cellulase activities produced by T. fusca. Polyacrylamide gel electrophoresis of ER-1 supernatant in the presence of SDS separates 16 protein bands of varying intensity, with none of the bands containing more than 10% of the total protein present in the culture supernatant. We have isolated five different/3-1,4-endoglucanases and one xylanase from the culture supernatant of ER-1 cells grown on cellulose. At the present time we have not detected any enzyme that is a true exocellulase. However, one enzyme, E3, appears to resemble the fungal exocellulases in that it has relatively little activity on CMC and it shows synergism when it is assayed on filter paper with two of the other four enzymes. i R. E. Calza, D. C. Irwin, and D. B. Wilson, Biochemistry 24, 7797 (1985). 2 E. Lin, unpublished observations (1985).
METHODS 1N ENZYMOLOGY, VOL. 160
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
S. rolfsii
CELLOBIOHYDROLASE
307
Substrate Specificity. All three cellulases partially degraded native cellulose. Cellulase I, but not cellulases II and III, readily hydrolyzed the mixed fl-l,3//3-1,6-polysaccharides such as carboxymethylpachyman, yeast glucan, and laminarin. Both cellulase I and the fl-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes. Lichenan (/3-1,4;/3-I ,3) was degraded by all three cellulases. Cellulase II could not degrade CM cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-/3-1,4-glucan cellobiosylhydrolase. Degradation of cellulose (filter paper) can be catalyzed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation.~l Mode of Action. The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins. ~ The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of CM-cellulose, confirmed that cellulase III was an endocellulase. The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length. 11 M. G. Shepherd, C. C. Tong, and A. L. Cole, Biochem. J. 193, 67 (1981).
[32] 1 , 4 - f l - D - G l u c a n C e l l o b i o h y d r o l a s e f r o m S c l e r o t i u m rolfsii B y JAI C . SADANA a n d RAJKUMAR V . PATIL
The fungus Sclerotium rolfsii, when grown on cellulose as the sole carbon source, produces an oxidative enzyme, cellobiose dehydrogenase, 1and three different types of hydrolytic enzymes, 2-5 (I) a 1,4-fl-oglucan cellobiohydrolase (EC 3.2.1.91, cellulose 1,4-/3-cellobiosidase) 2 (cellobiohydrolase), (2) three endo-fl-glucanases, 3 and (3) four/3-o-glucosidases. 4,5 All these enzymes have been obtained in a homogeneous state. The hydrolytic enzymes form the cellulase complex of S. rolfsii. All of the J. C. Sadana and R. V. Patil, J. Gen. Microbiol. 131, 1917 (1985). 2 R. V. Patil and J. C. Sadana, Can. J. Biochem. Cell Biol. 62, 920 (1984). 3 j. C. Sadana, A. H. Lachke, and R. V. Patil, Carbohydr. Res. 133, 297 (1984). 4 j. G. Shewale and J. C. Sadana, Arch. Biochem. Biophys. 207, 185 (1981). 5 j. C. Sadana, J. G. Shewale, and R. V. Patil, Carbohydr. Res. 118, 205 (1983).
METHODS 1N ENZYMOLOGY, VOL. 160
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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CELLULOSE
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hydrolytic enzymes are required for the efficient hydrolysis of crystalline cellulose. The cellobiose dehydrogenase plays only a minor role in the overall degradation of crystalline cellulose to glucose when S. rolfsii is grown on cellulose.1 1,4-fl-D-Glucan cellobiohydrolase shows high activity toward phosphoric acid-swollen cellulose but no viscosity-lowering activity toward carboxymethylcellulose. It functions in the hydrolysis of crystalline cellulose. 2 The enzyme acts in an endwise fashion by removing successive cellobiosyl units (93-96%) from the nonreducing end of the fl-l,4-glucan chains; glucose (4-7%) is also detected. In addition, it shows an initial endo-type mode of action on cotton sliver (dewaxed deltapine cotton) producing short fibers.6 A similar type of enzyme which shows an endotype mode of action on crystalline cellulose, besides its endwise action of removing cellobiosyl units from the nonreducing chain ends of cellulose, has been reported in Trichoderma reesei. 7
Assay Method Principle. The enzyme is assayed by determining the soluble sugars released in the supernatant fluid from phosphoric acid-swollen cellulose by the Somogyi-Nelson 8 or p-hydroxybenzoic acid hydrazide9 method or by determining p-nitrophenol from p-nitrophenyl-fl-D-cellobioside. The range of the Somogyi-Nelson method is 5-100 tzg, and that ofp-hydroxybenzoic acid hydrazide method 1-50/zg of reducing sugars as glucose. The method of estimation of cellobiohydrolase activity, using phosphoric acid-swollen cellulose as substrate, has certain limitations and is applicable to preparations which are free of endo-fl-glucanase and fl-Dglucosidase activities. The S. rolfsii cellobiohydrolase is much more active against phosphoric acid-swollen cellulose than is fl-glucosidase, but is much less active than endo-fl-glucanase. The interfering effect of fl-glucosidase is overcome by the addition of 2 m M D-glucono-1,5-a-lactone. The Sclerotium cellobiohydrolase is not inhibited by 10 m M D-glucono-I,5-8lactone. In the presence of endo-fl-glucanase, the assay will give erroneous values of cellobiohydrolase activity. A general assay technique for the determination of cellobiohydrolase activity does not exist as no specific substrate is available. However, in cases where the sample solution does not contain either fl-glucosidase or 6 j. C. Sadana and R. V. Patil, Can. J. Biochem. Cell Biol. 63, 1250 (1985). 7 H. Chanzy, B. Henrissat, and R. Vuong, FEBS Lett. 172, 193 (1984). 8 M. Somogyi, J. Biol. Chem. 195, 19 (1952). 9 p. L. Hurst, J. Nielsen, P. A. Sullivan, and M. G. Shepherd, Biochem. J. 165, 33 (1977).
[32]
S. rolfsii CELLOBIOHYDROLASE
309
endo-fl-glucanase, or the endo-fl-glucanase does not hydrolyze cellobiose, p-nitrophenyl fl-o-cellobioside can be used as a specific substrate for the determination of cellobiohydrolase activity. The cellobiohydrolase from S. rolfsii hydrolyzes p-nitrophenyl fl-o-cellobioside to p-nitrophenol and cellobiose but it has no action on cellobiose or p-nitrophenyl/3-0glucopyranoside, z The three endo-/3-glucanases, A, B, and C from S. rolfsii, which have been obtained in a homogeneous state, do not hydrolyze cellobiose, p-nitrophenyl/3-o-glucopyranoside or p-nitrophenyl/3-0cellobioside. 3
Reagents Phosphoric acid-swollen cellulose suspension in 50 m M citrate buffer, pH 4.5, 20 mg/ml Citric acid-trisodium citrate buffer, 50 mM, pH 4.5 Sodium azide, l0 m M in 50 m M citrate buffer, pH 4.5 o-Glucono-1,5-~-lactone, 20 and also 60 m M in 50 m M citrate buffer, pH 4.5 p-Nitrophenyl /3-o-cellobioside, 2 m M in 50 m M citrate buffer, pH 4.5 Sodium carbonate, 2% Preparation o f Phosphoric Acid-Swollen Cellulose. Avicel P.H. 101 (Honeywill and Stein Ltd., U.K.) (10 g) is suspended in o-phosphoric acid (88%, w/v) and left for 1 hr with occasional stirring at 4°. The mixture is poured into ice-cold water (4 liters) and left for 3 rain. The swollen cellulose is washed several times with cold water by decantation. After washing with 1% (w/v) NaHCO3 solution, the thick suspension of swollen Avicel is dialyzed against water in a cold room. After 1 min treatment in a Waring blender, 50 m M citrate buffer, pH 4.8, is added to the suspension so that 1 ml of suspension contains 20 mg of cellulose. Cellulose content is estimated by the anthrone-sulfuric acid method.~° Phosphoric acid-swollen cellulose prepared in this way is in the form of an even suspension. Phosphoric acid-swollen cellulose is stored at 4-5 °. Procedure
With Phosphoric Acid-Swollen Cellulose as Substrate. A 1-ml suspension of phosphoric acid-swollen cellulose is mixed with 0.3 ml of 10 m M sodium azide, 0.1 ml of D-glucono-l,5-8-1actone (if the sample solution contains/3-glucosidase), and 1.5 ml of 50 m M citrate buffer, pH 4.5, in a Pyrex test tube. The enzyme solution (0.1 ml) containing 10 to 15 /xg to D. M. Updegraff,Anal. Biochern. 32, 420 (1969).
310
CELLULOSE
[32]
enzyme protein (0.6-1 mU) is added to the reaction mixture to give a final volume of 3 ml, mixed thoroughly, and incubated at 50° for 4 hr. A control is run with boiled enzyme. The reaction is stopped by immersing the tubes in a boiling water bath for 5 min and is centrifuged after cooling. The released soluble sugars are determined in a 1-ml aliquot of the supernatant fluid by the Somogyi-Nelson 8 or hydroxybenzoic acid hydrazide 9 method. The crude filtrate from S. rolfsii shows high endo-fl-glucanase and flglucosidase activities. The interference due to/3-glucosidase is overcome by adding D-glucono-1,5-8-1actone at a final concentration of 3 m M which inhibits fl-glucosidase. The interference due to endo-fl-glucanase cannot be overcome in the above assay procedure. With p-Nitrophenyl-fl-o-Cellobioside as Substrate. In this assay the activity is measured as the amount of p-nitrophenol released from pnitrophenyl-fl-o-cellobioside. One mole ofp-nitrophenol is released from 1 mol of p-nitrophenyl-/3-o-cellobioside. A suitably diluted enzyme (0.1 ml) containing 50-100/xg enzyme protein is added to a small Pyrex test tube containing 0.8 ml 2 m M p-nitrophenyl-fl-o-cellobioside in 50 m M citrate buffer, pH 4.5, and 0. I ml of 20 m M D-glucono-1,5-8-1actone which has been previously preincubated at 37°. After incubation at 37° for 30 min, the reaction is stopped by adding 1 ml 2% sodium carbonate. The absorbancy of p-nitrophenol released is determined spectrophotometrically at 410 mm in a 1-cm light path cuvette after 10-15 min. The amount ofp-nitrophenol released is calculated from the absorbancy index of 18.5 cmZ/tzmol for p-nitrophenol at 410 nm. 11 Linearity is obtained when a value of 0.02-0.05/xmol of p-nitrophenol is released per minute. Definition of Unit of Activity. One unit of cellobiohydrolase activity is defined as the amount of enzyme that releases 180 tzg reducing sugars (1 /zmol as o-glucose equivalent and corresponds to the production of one reducing end group) per minute at pH 4.5 and 50°, or 1/zmol ofp-nitrophenol from p-nitrophenyl-/3-o-cellobioside per minute at 37°, pH 4.5. Purification
Preparation of Crude Filtrate. Sclerotium rolfsii CPC 142,12 used as the enzyme source, is grown as given in chapter [10] of this volume for cellobiose dehydrogenase. The purification of cellobiohydrolase follows the procedure given in chapter [10] of this volume for cellobiose dehydrogenase. When the concentrated enzyme solution from ultrafiltration step (step 3) of the cellobiIID. Herr, F. Baumer, and H. Dellwig, Eur. J. Appl. Microbiol. Biotechnol. 5, 29 0978). 12 Sclerotium rolfsii CPC 142 culture (NCIM No. 1084) is available from the National Collection of Industrial Microorganisms, National Chemical Laboratory, Poona 411 008, India.
S. rolfsii CELLOBIOHYDROLASE
[32]
311
TABLE I PURIFICATION OF CELLOBIOHYDROLASE FROM
S. rolfsii"
Activity toward (total units) Fraction Culture filtrate Ammonium sulfate, 0-3.4 M Sephadex G-75 chromatography fraction A Ultrafiltration of fraction A (Amicon PM10) DEAE-Sephadex A-50 chromatography Preparative polyacrylamide gel electrophoresis
Protein (mg)
H3PO4-swollen cellulose
17,202 13,937
27,523 26,480
520,109 45,284 462,620 42,785
10,774
22,324
t70,210
8,470
21,260
122,300 21,430
329
206
4.8
57
52
3.4
0.018
0
CMC
PNPG
28,370
From Ref. 2. One unit of activity towards H3PO4-swollen cellulose or CMC (carboxymethylcellulose) is defined as the amount of enzyme that releases 180 /xg reducing sugars (1 /zmol as D-glucose equivalent) per minute at pH 4.5 and 50°, using a 4-hr incubation for H3PO4-swollen cellulose and a 30-min incubation for CMC.6 fl-D-Glucosidase is determined as described in chapter [10] of this volume. PNPG, p-Nitrophenyl/3-D-glucopyranoside.
ose d e h y d r o g e n a s e p r o c e d u r e is c h r o m a t o g r a p h e d on a D E A E - S e p h a d e x A-50 column, endo-fl-glucanase and/3-glucosidase activities are not adsorbed on the column, whereas a major amount of protein containing cellobiohydrolase is adsorbed. This is eluted by 0.1 M citrate buffer, p H 4.5 (fraction n u m b e r s 50-70, see Fig. 1 in chapter [10] of this volume for cellobiose dehydrogenase). This fraction is concentrated by lyophilization and dialyzed against 5 m M T r i s - g l y c i n e buffer, p H 8.5. Further purification of cellobiohydrolase is obtained by preparative polyacrylamide gel electrophoresis as described for cellobiose dehydrogenase in this volume [10]. The results of a typical purification of cellobiohydrolase from S. rolfsii are presented in Table I. Properties of the Purified E n z y m e
Purity. The purified e n z y m e shows one protein band on polyacrylamide gel electrophoresis at p H 2.9 and 8.9, with or without SDS treat-
312
CELLULOSE
[32]
ment, and in analytical isoelectric focusing in 7.5% polyacrylamide gel over the pH range 3.5-10.0. Stability. The enzyme is stable when stored in 50 m M citrate buffer, pH 4.5, at - 15°. The enzyme is most stable at pH 4.5-5.0. The enzyme is stable to repeated freezing and thawing. Physical Properties. The relative molecular weight, Mr, estimated by gel filtration on BioGel P-150,13 electrophoresis by slope method, TMand by its migration in SDS-polyacrylamide gel 15,16is 41,500, 41,700, and 42,000, respectively. The p I of the pure S. rolfsii enzyme is 4.32. Chemical Properties. The enzyme is composed of a single polypeptide chain as the carboxyamidomethylated derivative of the reduced form of" cellobiohydrolase gives one protein band with a relative molecular weight (Mr 42,000) corresponding to the native protein. The enzyme is a glycoprotein containing 7.0% total carbohydrate; it contains 3.6 residues of glucosamine per molecule of enzyme but no galactosamine. The enzyme is high in acidic and low in basic amino acids and contains no cystine or half-cystine. 2 Enzymatic Properties. With phosphoric acid-swollen cellulose, the pH and temperature optima are 4.5 and 50° (4 hr assay), and with Avicel (24 hr assay) they are 4,2 and 37°. The rate of phosphoric acid-swollen cellulose hydrolysis is linear up to 15/-~g enzyme protein (4 hr assay), and with 10 ~g enzyme, the hydrolysis is linear up to 8 hr under standard assay conditions. The purified enzyme (10/zg) produces 40.8/xg reducing sugars as glucose equivalent from phosphoric acid-swollen cellulose in 4 hr, 50°, pH 4.5. With phosphoric acid-swollen cellulose, the activation energy, calculated from Arrhenius plot, is 8.55 cal/mol (1 cal = 4.1868 J). The cellobiohydrolase shows no S-factor activity, ~7 though it potentiates Sfactor activity of endo-fl-glucanases. 3 Kinetics. The [S]0.5 and Vmaxvalues for phosphoric acid-swollen cellulose at pH 4.5, 50 °, calculated from Lineweaver-Burk plot, are 1.66 mg/ ml and 42/.~g reducing sugars/mg protein per minute. The [S]0.s for cellotriose is 2.2 m M (glucose liberated is measured by glucose oxidase-peroxidase method ~8) and for ceUotriose it is 1.81 m M (cellobiose liberated is measured by p-hydroxybenzoic acid hydrazide method9). The Vmaxfor cellotriose is 46/zg glucose/mg protein per minute, and for cellotetraose 208 ~g reducing sugar/mg protein per minute. 13 p. Andrews, Biochem. J. 91, 222 (1964). 14 j. L. Hedrick and A. J. Smith, Arch. Biochem. Biophys. 126, 155 (1968). 15 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). 16 A. L. Shapiro, E. Vinuela, and J. V. Maizel, Biochem. Biophys. Res. Commun. 28, 815 (1967). 17 C. B. Marsh, G. V. Merola, and M. E. Simpson, Text. Res. 23, 831 (1953). ts H. U. Bergmeyer, K. Gawein, and M. Grassl, Methods Enzymatic Anal. 1, 457 (1974).
[32]
S. rolfsii CELLOBIOHYDROLASE
313
Specificity. The enzyme is specific for/3-D-(1---~4) linkage. Action of Cellobiohydrolase on Insoluble and Soluble Substrates. The cellobiohydrolase hydrolyzes Cellulose-123 (Carl Schliecher and Schull Co., W. Germany), Avicel, a-cellulose, phosphoric acid-swollen cellulose, cellooligosaccharide G37 (i.e., degree of polymerization 37), and cotton. The principal product from each substrate is cellobiose (93-96%); glucose (4-7%) is also detected. The enzyme hydrolyzes carboxymethylcellulose to a small extent which is not due to its contamination with endo-/3-glucanase; cellobiose is the only product detected. The cellobiohydrolase has no action on cellobiose or p-nitrophenyl-/3-o-glucopyranoside; however, it hydrolyzes p-nitrophenyl fl-o-cellobioside to pnitrophenol and cellobiose. The enzyme hydrolyzes cellotriose and higher molecular weight cellodextrins; the rate of hydrolysis increases with increasing polymerization of the cellodextrin. Cellotriose is hydrolyzed to cellobiose and glucose, cellotetraose to cellobiose, cellopentaose to cellobiose and cellotriose initially and finally to cellobiose and glucose, and cellohexaose to cellobiose and cellotetraose initially and finally to only cellobiose. Inhibitors. D-Glucono-l,5-8-1actone at l0 m M is not inhibitory. NBromosuccinimide, a tryptophan-specific reagent, at 0.1 m M inhibits the enzyme activity completely. Endo-type Mode of Action of Cellobiohydrolase. The enzyme forms insoluble short fibers from native dewaxed cotton initially. 6 The formation of insoluble short fibers from cotton by S. rolfsii cellobiohydrolase suggests an endo-type mode of action, besides its endwise action of removing cellobiosyl units from the nonreducing end of cellulose chains. Synergism between Cellobiohydrolase and Endo-[3-Glucanase. The pure cellobiohydrolase and endo-/3-glucanases from S. rolfsii, when acting in concert, show active synergistic effects in reconstitution experiments in the solubilization of Avicel and cotton. With phosphoric acidswollen cellulose, the synergistic effect is much smaller. 19 Function
Initiation of Enzymatic Degradation of Crystalline Cellulose. The greater solubilization of Avicel by cellobiohydrolase and of phosphoric acid-swollen cellulose by endo-/3-glucanases when acting alone, and the beneficial effect of pretreatment of Avicel with cellobiohydrolase, and of phosphoric acid-swollen cellulose by endo-/3-glucanases, prior to the addition of the alternative type of enzyme, ~9 suggest that endo-fl-glucanase initiates the attack on amorphous cellulose (creating more ends for the J9 j. C. Sadana and R. V. Patil, Carbohydr. Res. 140, 111 (1985).
314
CELLULOSE
[33]
cellobiohydrolase to act), and cellobiohydrolase initiates the attack on crystalline cellulose (thereby making the substrate more accessible for hydrolysis~9). The observation that cellobiohydrolase also displays an initial endo-type mode of action besides its endwise action of removing cellobiosyl units from the nonreducing end of the cellulose chains lends support to this concept. 6
[33] C e l l u l a s e s o f Therrnomonospora fusca
By DAVID B. WILSON Introduction
Thermomonosporafusca YX is a filamentous actinomycete that has a doubling time of about 4 hr when grown at 55 ° in defined medium with cellulose (Solka-Floc) as a carbon source. The supernatant from a culture of cellulose-grown T. fusca contains a high level of cellulase activity, xylanase activity, and an active protease which modifies the different cellulases without altering their cellulase activity.~ Even though the protease activity can be inhibited by phenylmethylsulfonyl fluoride (PMSF), treated supernatant still contains proteolytically derived isozymes and is extensively degraded when it is heated in SDS. We have isolated a mutant strain of T. fusca, ER-12 that produces no detectable extracellular protease activity and the supernatant from this strain has been used to purify the different cellulase activities produced by T. fusca. Polyacrylamide gel electrophoresis of ER-1 supernatant in the presence of SDS separates 16 protein bands of varying intensity, with none of the bands containing more than 10% of the total protein present in the culture supernatant. We have isolated five different/3-1,4-endoglucanases and one xylanase from the culture supernatant of ER-1 cells grown on cellulose. At the present time we have not detected any enzyme that is a true exocellulase. However, one enzyme, E3, appears to resemble the fungal exocellulases in that it has relatively little activity on CMC and it shows synergism when it is assayed on filter paper with two of the other four enzymes. i R. E. Calza, D. C. Irwin, and D. B. Wilson, Biochemistry 24, 7797 (1985). 2 E. Lin, unpublished observations (1985).
METHODS 1N ENZYMOLOGY, VOL. 160
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.