Comparison of four purified extracellular 1,4-β-d -Glucan cellobiohydrolase enzymes from Trichoderma Viride

Biochimica et Biophysica Acta, 492 (1977) 225-231

© Elsevier/North-HollandBiomedicalPress BBA 37663 COMPARISON OF FOUR PURIFIED EXTRACELLULAR 1,4-fl-D-GLUCAN CELLOBIOHYDROLASE ENZYMES FROM T R I C H O D E R M A VIRIDE

ERNEST K. GUM, Jr. and ROSS D. BROWN, Jr.* Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061 (U.S.A.)

(Received December 27th, 1976)

SUMMARY Four electrophoretically distinct 1,4-fl-D-glucan cellobiohydrolase enzymes (exo-cellobiohydrolase, EC 3.2.1.91) from Trichoderma viride have been purified to homogeneity. Three enzymes (A, B, and C) were from a commercial T. viride preparation whereas the other (D) was from T. viride QM 9123 grown on cellulose in submerged culture. The enzymes were similar with respect to ultraviolet light absorption, amino acid and amino sugar composition, heat stability, molecular weight, specific activity, and carboxyterminal residues, indicating very nearly identical polypeptide portions. The enzymes also exhibited immunological cross-reactivity. The enzymes differed most in the content and composition of covalently bound neutral carbohydrate.

INTRODUCTION In 1970, small quantities of three glycoprotein exoglucanase enzymes were purified from a commercial preparation of Trichoderrna viride cellulase and, based on their uniformly synergistic participation in the degradation of crystalline cellulose, were suggested to be different forms of the same enzyme [1]. Since then several cellulase enzymes, now classified as 1,4-fl-D-glucan cellobiohydrolases (exo-cellobiohydrolase, EC 3.2.1.91), have been isolated from other Trichoderma extracellular preparations [2-8]. We have purified suitable quantities of several electrophoretically distinct cellobiohydrolase enzymes from T. viride and have compared these enzymes in order to determine whether they are true isoenzymes with different roles in cellulose degradation or different glycoprotein forms of the same polypeptide, with identical roles in cellulose degradation. The latter, which we are now convinced is the case, enables a more direct comparison of the results from different laboratories investigating cellobiohydrolase structure or function. Our results also permit comparison of cellobiohydrolases from a commercial enzyme preparation with that prepared from

" To whom correspondence should be addressed.

226 a culture supernatant produced by a readily available T. viride strain grown under laboratory conditions. MATERIALS AND METHODS Meicelase P, an enzyme preparation of T. viride grown on bran, was purchased from Meiji Seika Kaisha, Ltd., Tokyo, Japan. In order to produce enzyme in submerged culture, a 20-1 culture of T. viride QM 9123 (ATCC 2449) was grown on the medium of Mandels and Weber [9] and maintained at pH 5.4 by the alternate addition of 1.0 M NaOH or 1.0 M HC1 in a Model C M F 1283 Microferm Fermentor equipped with a Model PH-22 pH controller (New Brunswick Scientific Co., New Brunswick, N.J.). The extracellular enzyme system was harvested by filtering through glass wool. The filtrates were concentrated on an Amicon PM 30 membrane (Amicon Corp., Lexington, Mass.) and brought to pH 5 in 0.05 M sodium acetate buffer containing 3 mM NAN3. Protein was estimated in crude preparations by the equation: 1.45 (A280 nm) - - 0.74 (A260 nm) = mg protein/ml solution [10] and in purified cellobiohydrolase preparations by the experimentally determined value E ~ = 14.2 [8]. Large scale cellulose column affinity chromatography, DEAE-Sephadex batch separation and analytical gel electrophoresis techniques have been reported previously [8]. The "Tris" preparative disc gel electrophoretic system described by Maurer [11] was modified by adjusting the separation gel to pH 8.1 and 10.5~ acrylamide. The separation gel volume was 12 ml. The stacking gel was 4 ml of 3.5 ~ acrylamide (pH 6.9) and the sample contained 16-52 mg protein in 8 ml of 0.75 M sucrose (pH 6.9). The electrophoresis apparatus consisted of a Canalco PD 1 basic unit, PD 2/320 upper column and Model 300 power supply. The separation was performed at 10 °C with a current of 6 mA for the first 3.5 h and then 12 mA for the duration. Ultraviolet spectra of the purified enzymes were measured on a Beckman Acta 11I. Total neutral carbohydrate was estimated by the phenol-HzSO4 method [12], using mannose as a standard. Neutral monosaccharides were identified and quantified by gas-liquid chromatography of their alditol acetates [13] on a 3 m x 2 mm column of 1 ~ OV 225 on Chromosorb G HP in a Varian Aerograph Model 2740 instrument. A Beckman Model 121 Automatic Amino Acid Analyzer was used for the amino acid analyses [14] and amino sugar analyses [15]. For amino acid analysis proteins were hydrolyzed with 6 M HC1 at 110 °C for 24 h. Amino sugars were analyzed in hydrolysates which were prepared with 4 M HC1 at 110 °C for 6 h, and then dried and treated with Dowex I acetate to remove interfering peptides. Activities of the purified cellobiohydrolase enzymes were measured at 40 °C with a suspension of phosphoric acid-swollen cellulose [16] and the reducing equivalents quantified by the method of Nelson [17] and Somogyi [18]. A Waters Associates ALC 202/401 high pressure liquid chromatograph equipped with the Model 6000 Solvent Delivery System and a 30-cm Microbondapak ® carbohydrate column was used to separate and quantify the soluble products of enzyme action. The solvent was acetonitrile and water (75:25, w/w) and the flow rate 1.1 ml/min. Thin-layer gel filtration chromatography on 40 x 20 cm plates with a 0.6 mm layer of Sephadex G-100 Superfine was done using 6 M guanidine-HCl as the developing solvent. Development was performed at 7 °C for 4 h at an angle of 20 ° using a Pharmacia (Piscataway, N.J.) TLG-apparatus. Replicas of the developed

227 plates made with Whatman 3MM paper were washed four times with acetone/ ethanol (50:50, v/v) and stained with Canalco Coomassie Blue RDSL concentrate/ methanol (1:1, v/v). Paper was destained by washing with water followed by methanol/ water/acetic acid (45:45:10, v/v). Bovine serum albumin, ovalbumin, chymotrypsinogen and myoglobin were used to calibrate the system. Immunodiffusion was carried out on a lantern slide cover glass (8.3 × 10.2 cm) coated with 20 ml of 1 ~ ion-flee agar in 5 mM NAN3. Antiserum had been prepared against the large scale cellobiohydrolase C preparations [19]. The thermal inactivation of each cellobiohydrolase enzyme was determined using phosphoric acid-swollen cellulose as a substrate after preincubation of a solution containing approx. 30 #g enzyme/ml at 55, 60, or 65 °C for 30 min. Diisopropyl phosphofluoridate-treated carboxypeptidase A was used to identify the amino acid residues near the C-terminals of the cellobiohydrolases [20]. Released amino acids were identified and quantified in the supernatant of the acidprecipitated incubation mixture using the Automated Amino Acid Analyzer. RESULTS Cellobiohydrolase C was separated from cellobiohydrolases A and B present in the Meicelase by adsorption to cellulose at pH 5.0 with a protein:cellulose ratio of 1:10. After a batch separation on DEAE-Sephadex, the cellobiohydrolase C was subjected to preparative gel electrophoresis. The center fractions of the single peak were pooled and dialyzed on an Amicon PM 10 membrane. Cellobiohydrolases A and B were purified from the buffer fraction of the first cellulose column by adsorption to a second cellulose column at pH 5.0 and a protein: cellulose ratio of 1:100. The water eluate of this column contains approximately equal amounts of enzymes A and B contaminated by a slight amount of C. Preparative gel electrophoresis separated the A and B proteins; however, only the fractions during in the center of each peak were pure enough for the comparative studies. Cellobiohydrolase D was purified from the T. viride QM 9123 grown in submerged culture on purified cellulose. Since under those conditions only one cellobiohydrolase form was present in the culture supernatant after concentration on an Amicon PM 30 membrane, it was easily separated from the other proteins. The center fractions of the first major protein peak from the electrophoretic separations were pooled and dialyzed using an Amicon PM 10 membrane. The total yield of the purified cellobiohydrolases were 8.9, 5.7, 12.7, and 15.3 mg of A, B, C, and D, respectively. Protein-stained analytical gels of each purified preparation showed a single band after electrophoresis in the presence or absence of sodium dodecyl sulfate. At a basic pH, the electrophoretic mobility of A is the largest, D the smallest and B is greater than C. Each enzyme preparation could not be contaminated with greater than 0.2 ~ fl-glucosidase or 0.3 ~ endoglucanase proteins. These purified proteins produce soluble sugars from phosphoric acid-swollen cellulose, of which cellobiose constitutes greater than 90 ~ by weight. This is demonstrated in Fig. 1 by the high pressure liquid chromatographic patterns of the deionized supernatants from enzyme-treated cellulose. The specific activities in terms of

228 STD

A

B

C

I "= D

CB

GIc

I

I

c,B

c~

1-'4 n,"

<3

0

4

8

)

4

8 12 0

4

ELUTION

8 I

)

TIME

4

8

12 0

4

8

12

(min)

Fig. I. High pressure liquid chromatographic analyses of soluble products from phosphoric acidswollen cellulose after incubation with cellobiohydro]ases. Arrows mark the points of injection;

glucose, cellobiose and cellotriose are abbreviated Glc, CB and CT; and the letter A, B, C, or D denotes the cellobiohydrolase which produced the respective array of products. 1he chromatogram denoted STD resulted from a 5-pl injection of a solution of the standard sugars glucose, cellobiose, and cellotriose, each at l ~, w/w. reducing sugar released from a suspension of swollen cellulose (8.9 mg/ml) was 1.01, 0.67, 0.56, and 0.53/~mol.mg -1. min -1 for enzymes A, B, C, and D, respectively. Ultraviolet spectra of the cellobiohydrolases showed the same absorbance characteristics: minima at 249 nm, maxima at 278 nm, and a shoulder at 290 nm. The molecular weights of the cellobiohydrolases were compared by thin-layer gel filtration chromatography. Each preparation gave a single spot. The molecular weights of all the cellobiohydrolases were estimated to be 53 000 4- 1 0 ~ when in the presence of 6 M guanidine. HC1. The amino acid compositions of 24-h hydrolysates, shown in Table I, for the cellobiohydrolases are not significantly different. Each cellobiohydrolase contains between 0.5 and 0.8 ~ by weight glucosamine (presumably as the N-acetylated form) and no galactosamine. The neutral sugar composition of each cellobiohydrolase enzyme is presented in Table II. Cellobiohydrolase A contains the least neutral carbohydrate, whereas B and D contain about equal amounts. However, the ratio of glucose to mannose is 2.6 for cellobiohydrolase B and only 1.9 for D. The C enzyme contains the largest amount of neutral sugar and is the only form which contains galactose. An Ouchterlony double-diffusion pattern of the cellobiohydrolases is shown in

229 TABLE 1 AMINO ACID COMPOSITION OF CELLOBIOHYDROLASES tool percent

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine

A

B

C

D

3.1 0.9 1.6 13.6 9.6 10.4 8.4 6.0 13.3 7.1 3.2 5.0 1.7 2.2 5.9 4.8 3.3

2.7 0.9 1.9 13.6 10.6 10.2 8.2 5.7 13.0 7.1 4.1 4.7 1.6 2.2 5.6 4.9 3.1

2.5 1.0 1.7 12.5 10.7 10.4 8.2 6.4 13.2 6.8 4.1 4.9 1.6 2.3 5.5 5.0 3.0

2.7 1.1 2.1 11.2 11.4 10.0 9.1 6.6 13.5 6.2 3.8 4.8 1.8 2.3 5.3 4.8 3.1

TABLE 11 NEUTRAL CARBOHYDRATE COMPOSITION OF CELLOBIOHYDROLASES Weight percent A

B

C

D

Mannose Glucose Galactose

1.1 0.3 0

4.2 1.6 0

7.6 1.9 0.9

4.4 2.3 0

Total

1.4

5.8

10.4

6.7

Fig. 2. A l l enzymes reacted with the a n t i b o d i e s p r o d u c e d to the purified cellobioh y d r o l a s e C. Spurs o f n o n - i d e n t i t y are o b s e r v e d between c e l l o b i o h y d r o l a s e s B a n d C as well as between c e l l o b i o h y d r o l a s e A a n d the c e l l o b i o h y d r o l a s e C present in the crude p r e p a r a t i o n . There is a b a r e l y visible spur indicating some n o n - i d e n t i t y between c e l l o b i o h y d r o l a s e s C a n d D. T h e r e a p p e a r to be no significant differences between the h e a t stabilities o f the v a r i o u s enzymes in 0.05 M s o d i u m acetate, p H 5.0, at either 55, 60, o r 65 °C. A t 55 °C there was negligible loss o f activity o f a n y o f the f o u r enzymes. The activity r e m a i n i n g after 30 min at 60 °C was 64, 68, 65, a n d 65 ~o for A, B, C, a n d D, respectively, whereas there was less t h a n 10 ~ activity r e m a i n i n g when each o f the enzymes was p r e i n c u b a t e d at 65 °C. O n l y c e l l o b i o h y d r o l a s e s C a n d D were i n c u b a t e d with c a r b o x y p e p t i d a s e A. Both C a n d D have h y d r o p h o b i c C - t e r m i n a l ends with a T y r - L e u - C O 2 H sequence.

230

Fig. 2. Ouchterlony double diffusion pattern of the cellobiohydrolases. Wells contained the following: S, 35 ttl antiserum to cellobiohydrolase C; A, 51/~g cellobiohydrolase A ; B, 38/~g cellobiohydrolase B; C, 41/~g cellobiohydrolase C; D, 50/~g cellobiohydrolase D; P, 33 #g of the commercial T. viride enzyme preparation containing cellobiohydrolases A, B, and C. All antigens were in 25/~1. Diffusion was allowed to proceed at room temperature for 24 h before photographing plate. DISCUSSION The evidence indicates that all four cellobiohydrolase enzymes purified in this study are differentially glycosylated forms of the same polypeptide. Comparison of ultraviolet spectra, amino acid and amino sugar compositions, heat stabilities, molecular weights, specific activities, and C-terminal residues indicates identical (or very nearly identical) polypeptide regions. The immunological cross-reactivity of the enzymes supports this conclusion. Peptide mapping, which would normally confirm identical polypeptide sequences, would have been difficult to interpret due to the differences in the neutral carbohydrate content and composition of the enzymes and the attachment of the carbohydrate at several sites per molecule [8]. Several techniques utilizing l-dimethylaminonaphthalene-5-sulfonyl chloride [21], which gave the expected results with myoglobin, failed to identify the N-terminal residue of each cellobiohydrolase enzyme. These negative results again point to the similarity of the four cellobiohydrolase enzymes but fail to confirm the report of Berghem et al. [6] of alanine as the N-terminal residue of a T. viride cellobiohydrolase. In addition to differences in the content and composition of the neutral carbohydrate, each cellobiohydrolase has a different electrophoretic mobility. Although the difference in glycosylation could account for the non-identity observed by immunodiffusion, it does not account for the difference in electrophoretic mobility. With the exception of cellobiohydrolase D, the mobility is inversely related to the amount of carbohydrate. However, a slight difference in primary structure, such as partial deamidation of C, would explain the observed difference in mobility of C and D.

231 In a recent report, N a k a y a m a et al. [22] concluded that partial proteolysis o f an endo-cellulase (cellulase, EC 3.2.1.4) from T. viride m a y in part be responsible for the multiplicity o f endo-cellulases in vivo. Limited proteolysis affected the ionic properties o f the cellulase without significantly changing the molecular weight. Since neither amino acid nor carbohydrate compositions o f the modified cellulase components (whose purity is uncertain) were reported, it is impossible to ascertain what structural changes had resulted f r o m incubation with the protease. On the basis o f our study, it is now evident that the carbohydrate content is the principal factor which differentiates the cellobiohydrolase enzymes. Thus, it is consistent with the available evidence, to designate the cellobiohydrolase forms as A, B, C, and D rather than I, II, III, and IV suggested for isoenzymes [23]. The relationship o f the p r i m a r y structure o f the cellobiohydrolase to the endoglucanases and flglucosidases produced by T. viride is currently under investigation. ACKNOWLEDGEMENTS The a u t h o r s acknowledge the excellent technical assistance o f Mrs. Barbara Greenberg and Mrs. Blanche Hall. This work was supported by a grant f r o m the G u l f Oil Chemicals Co.

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