Exocellulase of Irpex lacteus (Polyporus tulipiferae)

EXOCELLULASEOF I. lacteus

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403

endo-1,4-/3-glucanase components in solubilizing cotton fiber. Cellobiohydrolase I and II differ, however, in their carbohydrate contents (I, 9%; II, 19%) their heat stabilities (destroyed during 40 min at 60°: II, 97%, I, 65%), and their pH optima (I, 2.5; II, 4.5). Further, cellobiohydrolase II does not react with anticellobiohydrolase I antiserum, and cellobiohydrolase I, but not II, has appreciable activity on cellotriose. D-glucose (200 m M ) stimulates the action of cellobiohydrolase I on H3PO4-swollen cellulose, but inhibits cellobiohydrolase II. The converse is true for ceHobiose.

[46] E x o c e l l u l a s e o f Irpex lacteus (Polyporus tulipiterae)

By TAKAHISA KANDA and KAZUTOSI NISlZAWA The cellulase system of Irpex lacteus (Polyporus tulipiferae) includes various kinds of endocellulases of different randomness and at least one kind of exocellulase. The exocellulase may be a 1,4-fl-o-glucan cellobiohydrolase (EC 3.2.1.91, cellulose 1,4-/~-cellobiosidase) based on its substrate specificity. The fungus seems to attack native cellulose to obtain reducing sugars in vivo, using the synergistic action of the different kinds of cellulases. Assay Method

Principle. The assays are based on the following measurements: (1) the increase in reducing power formed during reaction, and (2) the decrease in the average degree of polymerization (DP) of cellulose.l,2 Both methods for measuring these changes should be performed in parallel to detect the exocellulase activity. The first method is conveniently carried out during the purification of exocellulase which is higher in saccharification activity, and in addition is simpler than the second procedure. In this section, therefore, we describe the reducing sugar method. Reagents Sodium acetate buffer, 0.05 M, pH 5.0 1% Avicel (microcrystalline cellulose) suspension in water. The suspension may be used for at least 1 month if stored in a refrigerator A. Donetzhuber, Sven. Papperstidn. 63, 447 (1960). 2 T. Kanda, K. Wakabayashi, and K. Nisizawa, J. Biochem. (Tokyo) 87, 1635 (1980).

METHODS IN 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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Copper reagent3,4: (a) dissolve 25 g of N a 2 C O 3 (anhydrous), 25 g of Rochelle salt, 20 g of NaHCO3, and 200 g of NazSO4 (anhydrous) in 800 ml of distilled water in this order, and dilute up to 1 liter. (b) Dissolve 150 g of CuSO4" 5H20 in distilled water and make up to 1 liter. Before use, reagents (a) and (b) are mixed in a ratio of 25 : 1 (v/v) Nelson reagent3,4: dissolve 25 g of (NH4)6Mo7024" 4H20 in 500 ml of distilled water, add 42 g of concentrated H2SO4, 3 g of NazHAsO4" 7H20, and dilute up to 1 liter Procedure. 5 The reaction mixture consists of 1 ml of 1% Avicel suspension, 2 ml of 0.05 M sodium acetate buffer, pH 5.0, and 1 ml of enzyme solution. The mixture is incubated at 30 ° with shaking for I hr in most cases and centrifuged. To 0.5 ml of the supernatant, are added 1 ml of copper reagent and 0.5 ml of distilled water. The mixture is shaken and heated for 10 min in a boiling water bath. After cooling for 5 min, 1 ml of Nelson reagent and 2 ml of distilled water are added and mixed. The reducing power of the mixture is estimated colorimetrically by the absorbance at 660 nm. Definition o f Unit and Specific Activity. One unit of the Avicel saccharification activity is defined as the amount of enzyme which produces reducing power equivalent to 1 /~mol of glucose per minute under the assay conditions described above. Specific activity is expressed as enzyme units per milligram protein. Protein is determined by the method of Lowry et al. 6 using bovine serum albumin as standard.

Purification Procedure 5 All operations are performed at 4-6 °. Step 1. Ammonium Sulfate Fractionation. Driselase powder (50 g), a commercial enzyme preparation from Irpex lacteus by Kyowa Hakko Co., Japan, is extracted with about 300 ml of water. The suspension is centrifuged at 11,900 g for 15 min. The brown supernatant is brought to 20% saturation by addition of solid ammonium sulfate, and the precipitate which ordinarily has no cellulase activity is removed by centrifugation at 11,900 g for 30 min. The supernatant is brought to 80% saturation by 3 M. Somogyi, J. Biol. Chem. 195, 19 (1952). 4 N. Nelson, J. Biol. Chem. 153, 375 (1944). 5 T. Kanda, S. Nakakubo, K. Wakabayashi, and K. Nisizawa, J. Biochem. (Tokyo) 84, 1217 (1978). 6 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

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EXOCELLULASEOF I. lacteus

405

further addition of solid ammonium sulfate. The resulting precipitate which contains a high cellulase activity is collected by centrifugation and dissolved in about 200 ml of water. The aqueous cellulase solution is dialyzed against running tap water at 5° for 24 hr. The dialyzed solution is concentrated by ultrafiltration with Diaflo UM 10 membrane (Amicon) and lyophilized (17.5 g). Step 2. First DEAE-Sephadex A-50 Chromatography. The crude lyophilized preparation obtained above is dissolved in 150 ml of 0.02 M ammonium acetate buffer, pH 5.0, and applied to a DEAE-Sephadex A-50 column (5.0 x 50 cm) preequilibrated with the same buffer. Elution is carried out stepwise with 0.02 and 0.1 M of the same buffer, and maintained at a flow rate of 1 ml/min. Each 20-ml fraction of the eluates is collected. Four peaks of protein are obtained, and the third fraction being eluted with 0.1 M acetate buffer is most active toward Avicel and comprises the greatest amount of protein among the four. This peak is pooled and concentrated by ultrafiltration and lyophilized (6.2 g). Step 3. First BioGel P-IO0 Gel Filtration. The lyophilized preparation is dissolved in about 30 ml of 0.1 M sodium acetate buffer, pH 5.0, and applied to a BioGel P-100 column (2.7 x 130 cm) preequilibrated with the same buffer, and eluted with the same buffer. The column is operated at a flow rate of 5 ml/hr, and 2.5-ml fractions of eluate are collected. Two peaks of Avicel saccharification activity are obtained, of which the second peak is higher in cellulase activity and protein content. This fraction is concentrated and lyophilized as above (1.5 g). Step 4. CM-Sephadex C-50 Chromatography. The lyophilized preparation is dissolved in about 10 ml of 0.01 M sodium acetate buffer, pH 5.0, and applied to CM-Sephadex C-50 column (3.0 x 30 cm) preequilibrated with the same buffer. The elution is carried out stepwise with 0.01 and 0.1 M sodium acetate buffers. The column is operated at a flow rate of 20 ml/ hr and 10-ml fractions of the eluate are collected. The Avicel saccharification activity is separated further into three peaks, and the first peak fraction shows relatively high activity and amount of protein. This cellulase fraction is pooled, concentrated, and lyophilized (228 mg). Step 5. Second DEAE-Sephadex A-50 Chromatography. The lyophilized preparation is dissolved in about 5 ml of 0.02 M ammonium acetate buffer, pH 5.0 and again subjected to DEAE-Sephadex A-50 column chromatography under conditions similar to those of the first DEAE-Sephadex A-50 chromatography except for column size (3.0 x 30 cm), flow rate (26 ml/hr), and fraction volume (10 ml). Two cellulase peaks are obtained; the second peak being eluted with 0.1 M ammonium acetate buffer shows a typical Avicel saccharification activity as compared with CMC saccharification activity (carboxymethyl cellulose which is ordinarily used as sub-

406

CELLULOSE

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TABLE I PURIFICATION OF EXOCELLULASE FROM

Fraction 1. Crude extract 2. Ammonium sulfate fractionation 3. First DEAE-Sephadex A-50 4. First BioGel P-100 5. CM-Sephadex C-50 6. Second DEAE-Sephadex A-50 7. Second BioGel P-100

Total volume (ml) 260 100 430 50 190 50 12

Irpex lacteus

Total Specific protein Total activity Recovery (mg) units (units/mg) (%) 14,925 2,853 1,011 245 37 14 6.8

11.95 5.14

0.0008 0.0018

(100) 43

3.24 1.27 0.95 0.61 0.39

0.0032 0.0052 0.0256 0.0438 0.0571

27 11 8 5 3

strate for endocellulase). This peak is pooled and lyophilized after concentration (87 mg). Step 6. Second BioGel P-IO0 Gel Filtration. The lyophilized preparation is dissolved in 3 ml of 0.1 M sodium acetate buffer, pH 5.0, and again applied to BioGel P-100 column under conditions similar to those of the first BioGel P-100 gel filtration except for column size (1.2 x 140 cm), flow rate (3 ml/hr), and fraction volume (3.0 ml). A protein peak containing both cellulase activities for Avicel and CMC is obtained. The peak fractions are combined and lyophilized (42 mg). The cellulase activity ratio of this enzyme preparation for Avicel to that for CMC is 9.6 when the Avicel saccharification activity is measured after incubation for 1 hr under the standard condition and CMC saccharification activity is measured after incubation for 30 min under the same conditions; this ratio shows no change upon subsequent column chromatography of the cellulase preparation. Moreover, the enzyme preparation reveals a single protein band on SDS-polyacrylamide gel electrophoresis. A summary of the purification procedure is given in Table I. Properties

p H and Temperature Optima and Stability) Exocellulase is practically stable in the pH range from 3.5 to 6.0, and the optimum pH is 5.0. The optimum activity of the enzyme is at 50°, and only 5% of its optimum activity remains after the enzyme was heated at 100° for 10 min. Molecular Weight, Amino Acid Composition, and Carbohydrate Content) The molecular weight of the exocellulase is estimated by gel filtration to be 65,000 and it contains 2.4% carbohydrate as glucose. The pattern of its amino acid content is not very different from those of other

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EXOCELLULASEOF I. lacteus

407

endocellulases from the same fungus, particularly the high content of acidic amino acids, glycine, serine, and threonine. 7,8 Comparison o f Randomness o f Exo- and Endocellulases. 7,9 The randomness of hydrolysis by the exocellulase is compared with that by endocellulases from the same fungus on the basis of the ratio of a decrease in the degree of polymerization of substrates to an increase in the reducing power produced simultaneously. The ratio for more randomness should be larger than for less randomness. The ratios for the hydrolysis of all substrates including CMC and native cellulose by the exocellulase are the smallest. This cellulase, therefore, may be regarded as a saccharification type rather than a liquefaction type. Substrate Specificity. The exocellulase seems to split off successively almost exclusively cellobiosyl residues from the nonreducing end of native and degraded celluloses as well as from CMC. However, the rconfiguration of the anomeric carbon of the ceUobiosyl residue seems not to be inversed on liberation from the substrate by the enzyme action, because an upward mutarotation is observed on addition of alkali to the reaction mixture in which cellopentaitol is used as substrate. 5 This property is entirely similar to that of endocellulase from the same fungus. 8 The exocellulase produces glucose (G~) and cellobiose (G2) from cellotriose (G3), and Gl, G2, and G3 from cellopentaose (Gs), but only G2 from cellotetraose (G4), cellohexaose (G6), CMC, and insoluble celluloses at earlier stages of hydrolysis. G3 and G4 are attacked only slowly by exocellulase as compared with G5 and G6. G2 is not hydrolyzed by the exocellulase even during a prolonged incubation, but p-nitrophenyl /3-o-cellobioside, a synthetic derivative of cellobiose, is hydrolyzed at either its holoside or aglycon bond. The hydrolysis rate of the aglycon bond is higher than that of the holoside bond. Km values for G5 and G6 are 0.190 and 0.303 mM, respectively. 5,7 In contrast to the hydrolysis mode on (1 ~ 4)-fl-o-glucan, the exocellulase hydrolyzes internal glucosidic linkages of fl-l,3;1,4-o-glucan such as barley glucan and lichenan, and it releases G~, G2, G3, and two kinds of mixed oligosaccharides, 3-O-/3-o-glucosylcellobiose and 3-O-fl-o-cellobiosylcellobiose, but does not release laminaribiose.l° In this respect, the enzyme is similar to an endo-type cellulase from Streptomyces. H:2 7 T. Kanda, S. Nakakubo, K. Wakabayashi, and K. Nisizawa, Adv. Chem. Ser. 181, 211 (1979). 8 T. Kanda, K. Wakabayashi, and K. Nisizawa, J. Biochem. (Tokyo) 87, 1625 (1980). 9 M. Takai, J. Hayashi, K. Nisizawa, and T. Kanda, J. Appl. Polym. Sci.: Apply Polym. Syrup. 37, 345 (1983). 10 T. Kanda, H. Yatomi, Y. Amano, and K. Nisizawa, manuscript in preparation. 11 F. W. Parrish, A. S. Perlin, and E. T. Reese, Can. J. Chem. 38, 2094 (1960). 12 A. S. Perlin, in "Advances in Enzymic Hydrolysis of Cellulose and Related Materials" (E. T. Reese, ed.), p. 185. Pergamon, New York, 1963.

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CELLULOSE

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This enzyme seems to require a cellobiosyl residue adjacent to a (1 ~ 3)-/3-D-linked glucosyl residue, and it splits the (1 ~ 4)-fl-D-glucosidic linkage contiguous to a (1 ~ 4)-/3-D-linked glucosyl residue. This fact may be explained by the assumption that the stereochemical structure of the successive (1 ~ 4)-/3- and (1 ~ 3)-/3-glucosyl linkages is permissible in the action of this cellulase which requires two contiguous (1 ~ 4)-/3-1inked sequences.l° Furthermore, the exocellulase is able to attack (1 ~ 3)-fl-Dlinkage positioned between (1 ~ 4)-/3-D-linkages of oligosaccharides such as 3-O-/3-cellobiosylcellobiose to produce cellobiose almost exclusively. 10 Other Properties. A synergistic action of exo- and endocellulase in the hydrolysis of cotton, viscose rayon and alkali cellulose has been observed. Due to this effect the hydrolysis of cotton proceeds 44% farther than the sum of the hydrolysis extents by each single cellulase of a different type. Under similar conditions, the hydrolysis effects on viscose rayon and alkali cellulose are 28 and 16%, respectively. 2 The exocellulase transfers the cellobiosyl residue at the nonreducing end of donor substrates such a s G 3 , G s , and p-nitrophenyl /3-D-cellobioside to appropriate acceptors. It can, however, transfer the glucosyl residue at the nonreducing end of these substrates to a small extent. The transcellobiosylation activity of exocellulase is, however, far higher than that of endocellulase from the same fungus. 13 13 T. Kanda, I. Noda, K. Wakabayashi, and K. Nisizawa, J. Biochem. (Tokyo) 93, 787 (1983).

[47] f l - G l u c o s i d a s e f r o m R u m i n o c o c c u s albus By K U N I O O H M I Y A a n d SHOICHI S H I M I Z U p-Nitrophenyl-/3-D-glucoside + H20 ~ glucose + p-nitrophenol Cellooligomers + nH20 ~ n glucose + cellooligomers (glucose)m (glucose),. _n

A fl-glucosidase is purified to homogeneity from the cells of Ruminococcus albus. The enzyme catalyzes not only the hydrolysis of p-nitrophenyl-/3-D-glucoside (PNPG), but also the degradation of such cellooligomers as cellobiose, cellotriose, cellotetraose, and cellopentaose to glucose by cleaving/3-glucoside linkages from nonreducing terminal ends. Therefore, the enzyme is 1,4-fl-D-glucan glucohydrolase (EC 3.2.1.74, glucan 1,4-fl-glucosidase). METHODS IN ENZYMOLOGY, VOL. 160

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.