Role of cellulose-binding domain of exocellulase I from White rot basidiomycete Irpex lacteus

Jotrm~ALoF BIOSCmNCBANDBmr,r~omEE~J~O Vol. 91, No. 4, 359--362. 2001

Role of Cellulose-Binding Domain of Exocellulase I from White Rot Basidiomycete lrpex lacteus NAOKO HAMADA, 1 RITSUKO KODAIRA, I MASAHIRO NOGAWA, I KAZUNORI SHINJI, 2 RIE ITO, 2 Y O S H I H I K O AMANO, 2 MAKOTO SHIMOSAKA, l TAKAHISA KANDA, 2 ArCD MITSUO OKAZAKP ,3.

Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu Univergity, 3-15-1 Tokida, Ueda 386-8567,1 Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu University, 4-17-1 Wakasato, Nagano 380-8553,2 and Gene Research Center, Shinshu University, 3-15-1 Tokida, Ueda 386-8567,3 Japan Received 28 July 2000/Accepted 8 January 2001 The core fragment (designated P-42), devoid of the cellulose-binding domain (CBD) in the C-terminus and prepared from Irpex lacteus exocellulase I (Ex-1), was isolated by limited proteolysis using papain. Both the hydrolytic activity and binding ability of the isolated P-42 toward insoluble cellulose were lower than those of the native Ex-1, whereas Ex-1 and P-42 showed similar hydrolytic activities toward soluble substrates. These results indicate that the CBD of I. lacteus Ex-1 is the important domain which could enhance hydrolytic activity and binding ability of the enzyme toward insoluble cellulose. In addition, the isolated P-42 was different from the native Ex-1 in terms of enzymatic properties such as pH and temperature stabilities. These differences in stability, with regard to pH and temperature, between P-42 and the native Ex-1 are probably due to the O-linked sugar chains existing in the linker region. [Key words:

cellulose-binding domain, lrpex lacteus, O-linked sugar chains, limited proteolysis]

Efficient enzymatic degradation of insoluble polysaccharides often requires a tight interaction between enzymes and their substrates. In the case of cellulose degradation, many cellulases are known to bind to crystalline and/or amorphous celluloses via cellulose-binding domains (CBDs) which are distinct from catalytic domains. In most cases, CBDs are separated from catalytic domains by linker sequences which are highly enriched in proline and hydroxyamino acids. Klysov reported that not only the quantity of cellulases but also the ability to be adsorbed tightly onto crystalline cellulose is essential for effective hydrolysis (1). By tight binding to cellulose, the enzyme induces expansion of the cellulose surface followed by the release of small particles. The ability of enzymes to bind to cellulose is likely to be one of the important factors in the degradation of cellulose, since removal of CBDs from some cellulases severely reduced their hydrolytic activities against insoluble cellulose (2, 3). The CBD from Cellulomonas fimi CenA expressed in Escherichia coli disrupted the structure of cellulose fibers and fragments, even though it did not exhibit any catalytic activity and produced no detectable amount of reducing sugars (4). Conversely, artificial addition of a heterologous CBD (Thermomonospora fusca E2 CBD) to a catalytic domain of Pruvotella ruminicola carboxymethyl cellulase (CMCase) increased its specific activities against insoluble cellulose allomorphs (5). These results indicate that CBDs have a possible role in the disruption and dispersion of crystalline cellulose. On the other hand, Nidetzky et al. reported the limited function of CBDs, which simply enhanced the cellulosehydrolyzing activity of the catalytic domain of the enzyme by increasing the adsorption partition coefficient toward crystalline cellulose (6). The estimated functions

of CBDs vary depending on their origins. Since the mid-1980's, cellulase genes from various origins have been cloned and sequenced; however, relatively little is known about the molecular structures of the cellulases from white rot basidiomycetes. The white rot basidiomycete, lrpex lacteus is known to possess a high capacity for cellulose degradation, and two types of cellulases, exocellulase I (Ex-1) and endocellulase I (En-1), have been purified and their properties have been characterized (7, 8). In addition, three cellulase genes, cell (9), cel2 (10) and cel3 (I 1), have been isolated and characterized. We have previously determined that cel2 encodes Ex-1 (10). In this study, we isolated the core fragment (designated P-42), devoid of the CBD, from Ex-1 by limited proteolysis using papain, and compared its hydrolytic activity and binding ability toward insoluble cellulose with those of the native Ex-1 to evaluate the role of the CBD. In addition, the effect of sugar chains on pH and temperature stabilities of Ex-1 is discussed. MATERIALS AND M E T H O D S Materials The cellulosic substrates, namely, carboxymethyl cellulose (CMC), Avicel, bacterial cellulose and cotton, were obtained as in the previous paper (10). 4MeUmbLac (4-methylumbelliferyl-~-D-lactoside) was purchased from Seikagaku Kogyo, K.K. Enzyme assay The enzyme activity on 4-MeUmbLac was measured as follows. The reaction mixture consisted of 0.4 ml of 5 mM 4-MeUmbLac, 0.4 ml of enzyme solution, and 0.8ml of 50raM sodium acetate buffer (pH 5.0). The mixture was incubated at 30°C for 60 min, and then 2.0ml of 1% (w/v) sodium carbonate solution was added to the reaction mixture. The 4-methylumbelliferon liberated was measured at 347 nm. The enzyme activities on CMC, Avicel, bacterial cellulose, and cotton were measured by the method of Somogi-Nelson (12, 13)

* Corresponding author, e-mail: [email protected] phone: +81-(0)268-21-5340 fax: +81-(0)268-21-5331 359

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HAMADA ET AL.

under the same reaction conditions as mentioned above except for the substrate concentration (1%). Proteolytic digestion and isolation of core proteins Papain (30 U/mg, Roche Diagnostics K.K., Tokyo) digestion of native Ex-1 was performed at 30°C in a reaction buffer (300pl) composed of 50mM phosphate buffer (pH 7.0), 5 mM ~-cysteine, and 2 mM EDTA with addition of 22 pg of papain. The aliquots (30/A) were taken at specified time intervals and used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resultant core protein was purified by gel filtration using Bio Gel P-100 (140 × 2 cm2). Analytical methods For amino acid composition analysis, 1 mg each of the purified enzymes (Ex-1 and P-42) was hydrolyzed for 24 h at 110°C in an evacuated and sealed tube containing 4 N methanesulfonic acid with 0.2% 3-(2-aminoethyi) indole (Wako Pure Chemical Industries Ltd., Osaka). After neutralization with 4 N NaOH, the hydrolysates were analyzed using a Shimadzu L-10A amino acid analyzer. C-Terminal amino acid sequence analysis of P-42 was carried out after carboxypeptidase Y digestion. Methods for determination of protein and sugar content were described in the previous paper (10). Endo-/~-N-acetylgulcosaminidase (endoH) treatment for deglycosylation of the native Ex-1 (20mg) was performed at 37°C for 24h in 10raM Tris-HCl buffer (pH7.2) with addition of endoH (0.1 U, Seikagaku Kogyo, K.K., Tokyo). The deglycosylated enzyme was purified by column chromatography using a combination of concanavalin A Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) and Toyopearl HW-50S (Tosoh, Tokyo). Cellulose-binding assay One mg of purified Ex-1 or P-42 and 10 mg of insoluble cellulose (Avicel, bacterial cellulose, and cotton) were added to 50 mM ammonium acetate buffer (pH 5.0) and the mixture was incubated at 4°C for 1 h to allow binding of the protein to the cellulose. Samples were mixed every 10 rain. The samples were then centrifuged at 12,000 x g at 4°C for 5 rain and the amount of unabsorbed protein in the supernatant was measured. In addition, the reducing sugar content in the supernatant was also determined to confirm that hydrolysis of the polysaccharide did not occur under the conditions used in this study. RESULTS AND DISCUSSION

Limited proteolysis of Ex-1 by papain As detected by SDS-PAGE (Fig. 1), Ex-1 (53 kDa) was readily converted into a 42-kDa fragment (P-42) by papaln treatment. Under the conditions given, a 12G-rain digestion resulted in complete proteolytic cleavage and yielded a 42-kDa core protein that was not further degraded even with longer incubation. Thus, Ex-1 (25 mg in 50raM phosphate buffer, pH7.0) was digested for 14h with 10/~g papain to prepare a large amount of 42-kDa fragments for further analysis. The fragments resulting from the digestion were purified by gel filtration, and the purity of the core protein was confirmed for homogeneity by SDS-PAGE. Characterization of the sites of papain cleavage in ExI by amino acid sequence determination Both Ex-I and P-42 peptides were recovered from the SDS-PAGE gels by electroblotting and the N-terminal amino acids were sequenced using an automated protein sequencer. However, no sequence could be determined for either

J. BIoscI. Bto~so.,

kDa 97.4 66.2

~+ ~

31.0

"--

21.5

1 2

3

4 5

6

7

8

FIG. 1. SDS-PAGE analysis of Ex-I digested with papain. Lane 1: Intact Ex-1; lanes 2-8: papain digests after 5, 10, 20, 30, 40, 60, and 120 rnin, respectively.

peptide because of possible N-terminal blocking. To determine the C-terminal end of P-42, it was digested with carboxypeptidase Y and the amino acids released were quantified and identified. The sequence was determined to be Phe-Thr-Gly-COOH and a corresponding sequence could be found between amino acid positions 450-452 of the Ex-1 deduced from the primary structure (10) (Fig. 2). Thus, the papain cleavage site is located at approximately 70 residues from the Ex-1 C-terminus and is accounted for the release of a 10-kDa peptide, which could not be detected by SDS-PAGE under the above conditions used (Fig. 1). The 10-kDa peptide fragment was not obtained by gel filtration chromatography that was used to purify the P42 enzyme. We presumed that the peptide could be further degraded by papain. Considering the putative signal peptide sequence (amino acid positions 1-18) and the C-terminal end of P-42, the deduced structure of P-42 corresponded to the amino acid sequence between amino acid positions 19 and 452 in the primary structure of Ex-1 (10). The molecular mass calculated based on the deduced amino acid sequence of P-42 was consistent with that determined by SDS-PAGE. Moreover, the amino acid compositions of Ex-1 and P-42 were determined and compared with that based on the deduced primary structure which is shown in Table 22~ Olu

|

231 Glu

447 Ash

Ex-1 I8

i

449

P -42 18

487

GDINTT~T

FIG. 2. Schematic representation of the deduced Ex-I and P-42 proteins. Ex-1 is composed of the N-terminal signal peptide (horizontal lines), catalytic domain (white), tinker region (light grey), and C-terminal CBD (black). The suggested catalytic residues are indicated by solid circles. The putative cleavage site by limited proteolysis using papain is indicated by an arrow. The amino acids with double underlines are C-terminal amino acids of P-42 determined by the carboxypeptidase method. N-Glycosylation site is indicated by an asterisk.

VOL 91, 2001

CBD OF EX-1 FROM L LACTEUS

TABLE 2. Comparison of adsorption and catalytic properties of Ex-1 and P-42 using various cellulosic substrates

TABLE 1. Amino acid composition of Ex-I and P-42 Amino acid Asx Thr Set Glx Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp Total

Ex-1 (mol %)

P-42 (mol %)

Expected residues (19-452p (tool %)

15.0 10.9 9.8 7.2 4.3 11.3 8.4 1.8 5.9 1.4 2.7 5.6 3.3 4. I 1.3 4.2 2.9 ND

14.9 11.2 9.1 7.5 3.4 11.0 7.9 4.0 5.8 1.9 3.3 5.2 3.6 4.0 1.0 2.9 3.2 ND

15.02 11.27 9.16 7.51 3.29 11.03 7.98 4.23 5.87 1.87 3.29 5.16 3.52 3.99 0.94 2.81 3.06 (1.87)

100.1

99.9

100.00

361

Adsorption (pmol/g)a

Substrate BCc Avicel c Cottonc CMC c 4-MeUmbLaca

Specificactivity (mU/mg)b

Ex-I

P-42

Ex-1

P-42

1.4 1.3 1.1 ---

0.6 0.5 0.4 ---

14.5 20.9 14.2 26.0 24.3

2.1 1.2 1.1 31.1 24.4

a Values indicate the quantity of adsorption per g of each cellulose. b Specific activity is defined as activity m U / m g of enzyme protein. c One unit is defined as the amount of CMC, Avicel, or bacterial cellulose (BC) saccharification activity which produces a reducing power equivalent to 1.0 pmol of//-D-glucose per min. d One unit is defined as the amount of enzyme activity which releases 1.0 pmol of MeUmb.

between amino acid positions 19 and 452 was 1.23 using the Aitchison method (14). On the other hand, the statistical distance between the P-42 amino acid composition and the amino acid sequence (amino acid positions 19-452) was 0.04 by the same statistical treatment. These results indicate that the P-42 amino acid sequence is closer to the deduced amino acid sequences from amino acid positions 19 to 452 of Ex-l. Comparison of hydrolytic activity and binding ability toward various cellulosic substrates of Ex-1 with those of P-42 The specific activity and the degree of ad-

ND, Not determined (tryptophan was not determined). a From the amino acid sequence of Ex-1 [10].

1. The statistical distance between the native Ex-1 amino acid composition and the deduced amino acid sequence

B

A 100

80

60

40

2

0

I

0 2

4

6

8

10

20

pH

C

I

I

I

40 60 80 Temperature (~C)

D

100

a

60 40

4

6 pH

8

10 Temperature ( ~ )

FIG. 3. Effects of pH (A) and temperature (B) of Ex-1 and P-42 and differences in pH (C) and thermal (D) stabilities between both enzymes. (A) Buffers (50 mM concentration): [], citrate buffer; ©, sodium acetate buffer; A, sodium-potassium phosphate buffer; O, Tris-HC1 buffer. (B) The reaction was carded out using 50 mM sodium acetate buffer (pH 5.0). (C) After incubation with both enzymes in each buffer (pH 3.0-9.0) described above at 30°C for 24 h, the residual activities were measured using CMC as substrate. (D) After incubation with both enzymes in 50 mM sodium acetate buffer (pH 5.0) at various temperatures from 20°C to 80°C for 30 rain, the residual activities were measured using CMC as substrate. Symbols: ©, Ex-1; e , P-42.

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ET AL.

sorption o f Ex-1 and P-42 t o w a r d various cellulosic substrates were examined and are shown in Table 2. The specific activities o f P-42 against soluble substrates, namely, C M C and 4 - M e U m b L a c , were almost equal to those o f Ex-l; however, the specific activities o f P42 against insoluble substrates, namely, Avicel, bacterial cellulose, and cotton were m a r k e d l y lower t h a n those o f the native Ex-1. F u r t h e r m o r e , P-42 exhibited a decreased level (40%) o f a d s o r p t i o n o f insoluble celluloses as comp a r e d to Ex-1. These results indicate that CBD o f L lacteus Ex-I is resposible for the enhancement o f the hydrolyzing activity and binding ability t o w a r d insoluble cellulose. Comparison of enzymatic properties of P-42 with those of Ex-1 The p H and temperature profiles o f Ex-1 and P-42 are determined using C M C as substrate and summarized in Fig. 3. Ex-I exhibited a m a x i m u m activity at p H 5.0 and 50°C, and retained m o r e than 80% o f its m a x i m u m activity (at p H 5.0) between 25°C and 55°C and retained more than 70% o f the m a x i m u m activity within the p H range from 3.0 to 7.0. On the other hand, P-42 exhibited a m a x i m u m activity at p H 5.0 a n d 50°C, and retained m o r e than 80% o f its m a x i m u m activity (at p H 5.0) between 25°C and 40°C. P-42 had a narrower p H stability profile than Ex-I and retained more than 70% o f its m a x i m u m activity within a p H range from 5.0 to 6.0. In addition, P-42 had a lower temperature stability profile t h a n Ex-1. Therefore, the isolated P-42 was different from the native Ex-I in terms o f p H and temperature stabilities. Two possible sites for sugar chain attachment could be deduced f r o m the putative amino acid sequence o f Ex-1. One is a putative N-glycosylation site, Asn447-Thr Thr at the C-terminus o f the catalytic d o m a i n , a n d the other is a possible O-glycosylation site attached to serine or threonine residues in the linker region. To investigate the role o f the N-linked c a r b o h y d r a t e chain o f Ex-1 that leads to the differences in enzymatic properties between Ex-1 and P-42, N-linked carbohydrate-depleted enzymes were prepared f r o m Ex-1 by treatment with e n d o H . The native and N-linked carbohydrate-depleted enzymes were f o u n d to be identical in terms o f their enzymatic properties (data not shown). These results suggest that the Nlinked c a r b o h y d r a t e chain o f Ex-I is not the cause o f the differences in enzymatic properties between Ex-I and P42. The isolated P-42 might not contain O-linked sugar chains because the position o f the C-terminus o f P-42 was at G l y 452, which is consistent with the third amino acid in the linker region in Ex-I (10). Moreover, the existence o f sugar chains was not recognized by P A S staining (data not shown). Based on these results, the differences in enzymatic properties between P-42 and the native Ex-1 m a y depend on the O-linked sugar chains in the linker region. Further study is necessary to clarify the contribution o f O-linked sugar chains to the p H and temperature stabilities o f the enzyme.

J. BiOSCl. B1o~No.,

ACKNOWLEDGMENTS We thank Dr. R. Naganawa of Hokkaido University for his teehnicai assistance in the determination of the C-terminal amino acid sequence. We are grateful to Dr. M. Miura of Shinshu University for valuable and helpful discussion about statistical treatment. This work was supported by a Grant-in-Aid for COE Research (10CE2003) from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES 1. Klysov, A.A.: Trends in biochemistry and enzymology of cellulose degradation. Biochemistry, 29, 10577-10585 (1990). 2. Tomme, P., Tilbeurgh, H.V., Pettersson, G., Damme, J.V., Vandekerckhove, J., Knowles, J., Teeri, T., and Claeyssens, M.: Studies of the cellulolytic system of Trichoderma reesei QM9414. Eur. J. Biochem., 170, 575-581 (1988). 3. Gilkes, N.R., Warren, R . A . J . , Miller, R.C., Jr., and Kiihurn, D. G.: Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J. Biol. Chem., 263, 1040110407 (1988). 4. Din, N., Gilkes, N.R., Tekant, B., Miller, R. C., Jr., Warren, R . A . J . , and Kilhurn, D.G.: Non-hydrolytic disruption of cellulose fibers by the binding domain of a bacterial cellulases. Bio/Technology, 9, 1096-1099 (1991). 5. Maglione, G., Matsudhita, O., Russell, J.B., and Wilson, D. B.: Properties of a genetically reconstructed Prevotella ruminiola endoglucanase. Appl. Environ. Microbiol., 58, 35933597 (1992). 6. Nidetzky, B., Steiner, W., and Claeysseus, M.: Cellulose hydrolysis by the cellulases from Trichiderma reesei: adsorptions of two ceUobiohydrolases, two endoglucanases and their core proteins on filter paper and their relation to hydrolysis. Biochem. J., 303, 817-823 (1994). 7. Kanda, T., Nakakubo, S., Wakabayashi, K., and Nisizawa, K.: Purification and properties of an exo-cellulase of avicelase type from a wood-rotting fungus, lrpex lacteus (Polyporus tulip~ferae). J. Biochem., 84, 1217-1226 (1978). 8. Kanda, T., Wakahayashi, K., and Nisizawa, K.: Purification and properties of a lower-molecular-weight endo-cellulase from Irpex lacteus (Polyporus tulipiferae). J. Biochem., 87, 16251634 (1980). 9. Hamada, N., Okumura, R., Fuse, N., Kodaira, R., Shimosaka, M., Kanda, T., and Okazaki, M.: Isolation and transcriptionai analysis of a cellulase gene (cell) from the basidiomycete Irpex lacteus. J. Biosci. Bioeng., 87, 97-102 (1999). 10. Hamada, N., Ishikawa, K., Fuse, N., Kodalra, R., Shimosaka, M., Kanda, T., and Okazaki, M.: Purification, characterization and gene analysis of exo-ceUulase II (Ex-2) from the basidiomycete Irpex lacteus. J. Biosci. Bioeng., 87, 442--451 (1999). 11. Hamada, N., Fuse, N., Shimosnka, M., Kodaira, R., Amano, Y., Kanda, T., and Okazaki, M.: Cloning and characterization of a new exo-cellulase gene, cel3, in Irpex lacteus. FEMS Microbiol. Lett., 172, 231-237 (1999). 12. Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 195, 19-23 (1952). 13. Nelson, N.: A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153, 375-380 (1944). 14. Aitchison, J.: The statistical analysis of compositional data, p. 1-416. Chabman and Hall, London (1986).