Addition of substrate-binding domains increases substrate-binding ...

FEMS Microbiology Letters 198 (2001) 57^63

www.fems-microbiology.org

Addition of substrate-binding domains increases substrate-binding capacity and speci¢c activity of a chitinase from Trichoderma harzianum M. Carmen Limo¨n a

a;b

, Emilio Margolles-Clark b , Tah|¨a Ben|¨tez a , Merja Penttila«

b;

*

Departamento de Gene¨tica, Facultad de Biolog|¨a, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain b VTT Biotechnology, P.O. Box 1500, FIN-02044 Espoo, Finland Received 9 January 2001; received in revised form 28 February 2001; accepted 28 February 2001

Abstract Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Chitinase ; Cellulose-binding domain ; Chitin-binding domain; Enzyme engineering; Trichoderma harzianum

1. Introduction Chitin, the second most abundant biopolymer in nature is composed of 1,4-L-linked N-acetyl-D-glucosamine (GlcNAc) and it is degraded by chitinases produced by a wide array of organisms. Chit42 is an endo-chitinase from the fungus Trichoderma harzianum CECT 2413 [1,2] that belongs to the chitinase class V [3] and does not contain a chitin-binding domain (ChBD). ChBDs are found mainly at the N-termini of plant chitinases [4,5], but in bacterial or fungal chitinases they can be located either at the Cterminal or N-terminal end [3,6,7]. Only a few of the fungal chitinases have been shown to contain a ChBD. The functionality of the ChBD of the CTS1 chitinase from Saccharomyces cerevisiae in substrate binding has been demonstrated. Based on amino acid sequence comparison with the yeast ChBD, similar domains were claimed to be present in Rhizopus niveus and Rhizopus oligosporus chitinases. However, no other fungal ChBD have been de* Corresponding author. Tel. : +358 (9) 456 4504; Fax: +358 (9) 455 2103; E-mail : merja.penttila@vtt.¢ Abbreviations : ChBD, chitin-binding domain; CBD, cellulose-binding domain

scribed. ChBDs from plant chitinases have six or eight conserved cysteine residues, with the two C-terminal ones often lacking [4]. Similarly, cellulose-binding domains (CBDs) of fungal cellulases contain conserved Cys residues but this Cys spacing is not found in the bacterial ChBDs [8,9]. On the other hand, the three aromatic amino acids tryptophan, phenylalanine and/or tyrosine, are found among the residues conserved in the bacterial and plant ChBDs, and are also present in CBDs. These aromatic residues have been implicated in the binding of chitinases to chitin [10] and cellulases to cellulose [11^15]. Another common feature of ChBDs and CBDs is that they are connected to the catalytic domains of the enzyme by a linker region that is Gly/Pro-rich in plant chitinases [9,5], and Thr/Ser/Pro-rich in fungal cellulases [16^20]. There are several reports describing the a¤nity of some CBDs towards chitin [21^24], and some chitinases have certain af¢nity for cellulosic materials [6]. This is explained by the structural similarities of chitin and cellulose [25]. The substrate-binding domains increase the enzyme concentration at the substrate surface and help to target the enzyme. The binding e¤ciency of the cellulases is much enhanced by the presence of the CBD and the enhanced binding clearly seems to correlate with better activity towards insoluble cellulose [26,18,27^31]. Analogous to cel-

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 2 4 - 0

FEMSLE 9884 18-4-01

58

M.C. Limo¨n et al. / FEMS Microbiology Letters 198 (2001) 57^63

lulases, removal of the ChBDs of the plant and bacterial chitinases decrease their activities on insoluble but not on soluble substrates [5,32,7]. In the present work, we report the production and biochemical characterization of hybrid chitinases that are composed of the catalytic domain from the fungal chitinase, T. harzianum Chit42, and the substrate-binding domains from a plant chitinase and a fungal cellulase. The CBD of Trichoderma reesei cellobiohydrolase II (CBHII) was chosen for this study because it has previously been shown to have a¤nity for chitin [23]. 2. Materials and methods 2.1. Bacterial strains and growth conditions Escherichia coli strain DH5 K (F3 , end A1, hsdR17(rKÿ , mKÿ ), supE44, thi-1, l, recA1, gyrA96, relA1, D (argF-lacZYA)U169, f80dlacZDM15) was used as the cloning host, 3 and E. coli strain BL21 (F3 , ompT, hsdS (r3 B , mB ), gal) was used for protein production. All strains were grown in LB medium supplemented with 100 mg l31 ampicillin. The chit42 gene [2], which encodes the 42-kDa chitinase, originates from T. harzianum Rifai CECT 2413 [33]. 2.2. Construction of chit42^CBD and chit42^ChBD gene fusions All DNA manipulations were performed using standard protocols. Plasmid pGEX-4T-2 was from Pharmacia Biotech. Vector pGEX-4T-2-Chit42 contains the mature chit42 gene translationally fused to the glutathione-Stransferase gene (GST) (Fig. 1). The EcoRI and SmaI sites in pGEX-4T-2-Chit42, indicated in Fig. 1, were used for insertion of the polymerase chain reaction (PCR) products (described below) (Fig. 1A). The linker and CBD of cbh2 from T. reesei were ampli¢ed from plasmid pTTC9-CBHII [34] with PCR using oligonucleotides 3c (5P-CCGGAATTCCTTGCTCAAGCGTCTGGGGCC-3P) and 4c (5P-CCCCCGGGTCCCGATCCGACTGGAGGTAC-3P). This construction was named pGEX4T-2-Chit42-CBD (Fig. 1B). In order to fuse a plant ChBD to Chit42 we used the ChBD of chiA from Nicotiana tabacum L. cv. Havana 425 and as a linker region between them the linker from CBHII. This ChBD was chosen because it belongs to a chitinase with antifungal activity [5]. The ChBD was ampli¢ed from plasmid pSCH10vP+B [35,36] using oligonucleotides 5c (5P-CCGGAATTCTAGCACAATGTGGTTCCCAGGCCGGA-3P) and 6c (5P-CCACTAGTACCAGGGCACTGCCTCTGCCA-3P) as sense and antisense primers. The resulting ChBD di¡ers from the original sequence [37] in two amino acids. These are not present in most plant ChBDs. The changes were Trp instead of Cys42 and Arg instead of Ser44. The CBHII linker was ampli¢ed from plasmid pTTC9-CBHII [34], with 7c (5P-

CCACTAGTCCCGGCGCTCAAGCTCAAGC-3P) and 4c (5P-CCCCCGGGTCCCGATCCGACTGGAGGTAC3P) primers. The newly constructed plasmid was called pGEX4T-2-Chit42-ChBD. The PCR reaction mixtures (50 Wl) contained: 200 ng of the templates, plasmids pTTC9-CBHII [34] and pSCH10vP+B [35], 1U DynaZyme1 bu¡er (Finnzymes Oy), 250 WM of dNTPs, 100 pmol of the primers, and 2.0 units of DynaZyme (Finnzymes Oy). The ampli¢cation (35 cycles) consisted of denaturation at 94³C for 1 min, annealing at 50³C for 1 min and polymerization at 72³C for 2 min. An additional cycle having a 5-min polymerization step was added. The ampli¢ed fragments were digested and ligated to plasmid pGEX4T-2-Chit42 and transformed into strain DH5K. The nucleotide sequences of the ampli¢ed regions were veri¢ed by manual DNA sequencing. 2.3. Puri¢cation of Chit42, Chit42^CBD and Chit42^ChBD E. coli strain BL21 was transformed with the constructions described, and grown overnight at 37³C in LB medium containing 100 Wg ampicillin ml31 . Cultures were diluted 1:100 and grown at 37³C to OD600 = 1.5^2.0. Expression of the chitinases was induced with 1 mM isopropyl L-D-thiogalactopyranoside and the culture was shifted to 30³C for 4 h. Fusion proteins were puri¢ed from E. coli lysates by a¤nity chromatography with glutathioneSepharose 4B (Pharmacia Biotech) according to the manufacturer's instructions. The puri¢ed fusion proteins were eluted with reduced glutathione and treated with thrombin for 12 h at 25³C in the presence of 0.5 mM EDTA, 0.3 WM aprotinin and 1 mM phenylmethylsulfonyl £uoride to reduce proteolysis. After the cleavage, the GST was eliminated through glutathione-Sepharose after removal of reduced glutathione. Bu¡ers were exchanged using BioGel0 P-6DG (Bio-Rad) chromatography columns. 2.4. Determination of protein concentration The concentration of puri¢ed proteins were estimated by UV optical density at 280 nm. Molar extinction coef¢cients (O280 ) were calculated on the basis of the tryptophan, tyrosine and cysteine content of each amino acid sequence of the proteins [38]. O280 (M31 cm31 ) = (No. Trp)U(5500)+(No. Tyr)U(1490)+(No. Cys)U(125). The molar extinction coe¤cients used were: CBHII: 97 300 M31 cm31 , Chit42: 86 875 M31 cm31 , Chit42^CBD : 103 095 M31 cm31 , Chit42^ChBD : 93 375 M31 cm31 . 2.5. Binding assays Di¡erent amounts of protein, ranging from 500 to 5000 nM, were incubated with 1 mg (dry weight) of ground crab-shell chitin (Sigma), in 1 ml of 30 mM phosphate

FEMSLE 9884 18-4-01

M.C. Limo¨n et al. / FEMS Microbiology Letters 198 (2001) 57^63

bu¡er pH 6.0 for 1 h, in a gel shaker at 450 rpm at 4³C (KS250 basic, Ika labortechnik Staufen). After incubation, reactions were stopped by centrifugation (13 000 rpm, 10 min, 4³C) and ¢ltered through Millex GV 0.22 Wm ¢lters (Millipore). Non-bound protein was measured in a £uorescence spectrophotometer (LS-B; Perkin-Elmer) with excitation at 280 nm and emission at 350 nm. The bound protein was calculated as the total protein minus the free protein measured. To test the e¡ect of the ionic strength, 965 nM (V50 Wg) of each protein was incubated at pH 6.0 with di¡erent NaCl concentrations (0^1 M). The e¡ect of the pH was measured under the same conditions as described above but using the following bu¡ers: Gly/HCl bu¡er, pH 3.0; sodium acetate bu¡er, pH 5.0; NaH2 PO4 bu¡er, pH 6.0, and Gly/NaOH bu¡er, pH 10. The CBHII protein was a gift of Dr. Linder (VTT Biotechnology). 2.6. Substrate preparation The S. cerevisiae GRF167, Botrytis cinerea CECT 2100 and T. harzianum CECT 2413 [33] cell walls were prepared according to Fleet and Pha¡ [39], lyophilized and kept at room temperature. Ground chitin, prepared as described in Linder et al. [23], was a gift of Dr. Linder (VTT Biotechnology). Colloidal chitin was prepared using crab-shell chitin (Sigma) according to Jeuniaux [40]. The concentration of ground chitin and colloidal chitin stock solutions were determined measuring the dry weight of lyophilized aliquots. Glycol chitin was prepared from chitosan as described by Molano et al. [41]. 2.7. Substrate speci¢city of chitinases Chitinase activity against high molecular mass substrates was assayed according to Boller et al. [42]. The substrate concentration in the reaction mixtures were 1.5 mg ml31 for fungal cell walls (dry weight) and 1.3 mg ml31 for glycol chitin, colloidal chitin and ground chitin. Fungal cell walls were sonicated (250 Soni¢er, Branson Ultrasonics) prior to incubation with chitinases. Incubations were done in 70 mM sodium phosphate bu¡er, pH 6.0 for 20 h at 30³C in a cell mixer (CM 100, Luckham Sussex) at 30 rpm and contained 0.27 nmol of each chitinase. Brie£y, the reactions were stopped by boiling the samples for 10 min, and then the samples were centrifuged. Reaction products released by chitinases were digested to monomers with Helicase0 (Helix pomatia Juice, IBF biotechnics, France) incubating at 37³C, for 1 h. The amount of GlcNAc produced was measured according to Reissig et al. [43] using GlcNAc as a standard. An enzymatic unit was de¢ned as the amount of enzyme that released 1 Wmol GlcNAc min31 . Activity against glycol chitin was measured by a modi¢cation of the Schales' method described by [44] for detecting reduced sugars. Chitinase activity against low molecular mass substrates was measured with 4-methylumbeliferyl-N,NP,NQ-triacetyl-

59

chitotriose (4-MU-(GlNAc)3 ; Sigma) as a substrate. Twenty-¢ve pmol of each enzyme was incubated with 20 Wl of 250 mM 4-MU-(GlNAc)3 in 0.1 M sodium citrate bu¡er, pH 3.0, at 30³C for 30 min. The reaction was then diluted in 2.9 ml of 0.5 M Gly bu¡er, pH 10.4. The amount of 4-methylumbeliferone released was measured spectro£uorometrically with excitation at 360 nm and emission at 450 nm. 3. Results and discussion 3.1. Production and puri¢cation of hybrid proteins The two hybrid chitinases were constructed by fusion of a tobacco ChBD or a T. reesei CBHII CBD to chitinase Chit42 of T. harzianum (Fig. 1). In both proteins, a Ser/ Pro-rich linker from T. reesei CBHII was used to separate the catalytic domain from the binding domain. The hybrid chitinases were produced as GST fusions in E. coli, puri¢ed, cleaved with thrombin and further puri¢ed by removal of the GST moiety. The wild-type Chit42 was produced similarly. 3.2. Chitin binding Wild-type Chit42 and CBHII adhered slightly to ground chitin as shown in Fig. 2, the binding capacity of the latter being superior. This data is comparable to that reported previously [23]. Both hybrid chitinases, Chit42^CBD and Chit42^ChBD, bound similarly to chitin at the initial enzyme concentration of 500 nM, but with increasing enzyme concentrations the di¡erences between them became apparent. Chit42^CBD adhered to chitin more e¤ciently than Chit42^ChBD (Fig. 2). The increase in binding capacity of Chit42^CBD at higher concentration was much higher than expected. The explanation could be that when a fusion of separate domains having di¡erent a¤nities is assayed at a low protein concentration, there is no competition for the sites between the domains because there are easily accessible sites on the substrate [19]. At higher protein concentrations, however, the number of binding sites becomes limiting and the molecules with more a¤nity ¢nd more sites than the catalytic domain alone. Another explanation could be a co-operative e¡ect of the catalytic and the binding domains as has been described by Linder et al. [23] for a double CBD consisting of CBDs from the two cellobiohydrolases, CBHI and CBHII. The CBD of CBHI had a very low a¤nity for chitin while the CBD of CBHII had a high a¤nity, and the double CBD showed a co-operative e¡ect. Both hybrid chitinases showed higher binding capacities to chitin than Chit42 at all pH values tested (Fig. 3). The hybrid chitinases also retained good binding properties at lower pH values unlike Chit42.

FEMSLE 9884 18-4-01

60

M.C. Limo¨n et al. / FEMS Microbiology Letters 198 (2001) 57^63

Fig. 1. Scheme of gene constructions. Chitinase Chit42 was fused to the GST gene and expressed as a mature protein without its signal peptide and proregion. Vectors pGEX-4T-2-Chit42 (A), pGEX-4T-2-Chit42-CBD (B) and pGEX-4T-2-Chit42-ChBD (C) were constructed amplifying the CBD, ChBD, and linker regions by PCR with primers containing strategic restriction enzyme cleavage sites.

Stabilizing salts such as NaCl and MgSO4 have been reported to increase the binding of both bacterial and fungal cellulases to crystalline cellulose [45,46]. The increase in the adsorption could be caused by the masking e¡ect of salt ions on the ionic interactions between the protein molecules, allowing a more dense packing [13].

Increases in salt concentration resulted in a decrease in the amount of Chit42 bound to chitin (Fig. 4). The levels of bound enzyme were higher for hybrid chitinases than for the native Chit42 but the binding of the fusion molecules did not decrease signi¢cantly with increasing salt concentration.

FEMSLE 9884 18-4-01

M.C. Limo¨n et al. / FEMS Microbiology Letters 198 (2001) 57^63

Fig. 2. Adsorption of the native and hybrid proteins to ground chitin. Di¡erent amounts of protein, ranging from 500 to 5000 nM, were incubated with 1 mg of chitin (dry weight) in 30 mM sodium phosphate bu¡er pH 6.0 for 1 h, in a gel shaker at 450 rpm at 4³C. Free protein was spectro£uorometrically measured. Bound protein = total protein3 free protein.

3.3. Chitinase activity The modi¢ed chitinases showed identical speci¢c chitinase activities on the low molecular mass substrate 4-mU(GlcNAc)3 to the wild-type Chit42 (Table 1). The activity was also similar on the soluble substrate glycol chitin. With amorphous colloidal chitin, there was a slight di¡erence between the di¡erent chitinase activities : the Chit42^ CBD chitinase activity was a little higher and the Chit42^ ChBD little lower than the wild-type Chit42 activity. Signi¢cant di¡erences were observed between the chitinases when using insoluble ground chitin, which has a crystalline structure. Both chitinases with a binding domain degraded ground chitin better than Chit42: Chit42^CBD had 52% and Chit42^CBD 36% higher activity than Chit42. No activity was detected on S. cerevisiae cell walls, which contain very little chitin. Signi¢cant di¡erences were, however, found when fungal cell walls were used as a sub-

61

Fig. 3. Adsorption of the native and the hybrid proteins at di¡erent pH values. 965 nM (50Wg) of each protein were incubated with bu¡ers of di¡erent pHs : glycine/HCl bu¡er, pH 3.0; sodium acetate bu¡er, pH 5.0; sodium phosphate bu¡er, pH 6.0, and glycine/NaOH bu¡er, pH 10.

strate: Chit42^CBD had the highest activity on Botrytis cell walls whereas Chit42^ChBD had the highest activity on Trichoderma cell walls (Table 1). The increase found for Chit42^ChBD activity on the T. harzianum cell walls was eight times higher than for Chit42. In conclusion, the presence of a binding domain did not a¡ect the activity of the chitinases on small or soluble substrates. Activity levels on high molecular mass substrates were clearly enhanced by the presence of a CBD or ChBD. There were minor changes in the activities of the modi¢ed enzymes on the soluble and amorphous substrates, and major changes on the insoluble substrates. Di¡erences between Chit42^CBD and Chit42^ChBD activity on B. cinerea and T. harzianum cell walls could be explained by di¡erences in the cell wall composition and/or in the cross-linking of their structural components [47]. We have shown that protein engineering can produce chitinases with better binding properties, leading to higher enzymatic activity. Production of the proteins in Tricho-

Table 1 Substrate speci¢cities of native and hybrid Chit42 Substrate

S. cerevisiae cell walls B. cinerea cell walls T. harzianum cell walls Ground chitin Colloidal chitinb Glycol chitin 4-MU-(GlcNAc)c3

Speci¢c chitinase activity (U mmol31 enzyme)a Chit42

Chit42^CBD

Chit42^ChBD

0 166.6 þ 14.6 178.3 þ 34.6 515.2 þ 39.4 4014 þ 91.2 4600.2 þ 311.4 7.8 þ 0.45

0 463.2 þ 8.4 135.7 þ 29.0 783.3 þ 57.3 4123 þ 93.1 4733.6 þ 88.9 9.25 þ 0.02

0 261.1 þ 14.0 1434.6 þ 82.3 703.3 þ 18.7 3238 þ 86.5 4600.2 þ 222.8 8.09 þ 0.19

a

1 U = 1 Wmol GlcNAc liberated min31 . 1 U = 1 Wmol reduced sugars min31 . c 1 U = 1 Wmol 4-MU liberated min31 . b

FEMSLE 9884 18-4-01

62

M.C. Limo¨n et al. / FEMS Microbiology Letters 198 (2001) 57^63

Fig. 4. Binding of native and hybrid chitinases at di¡erent NaCl concentration. 965 nM (50Wg) of each protein were incubated with NaCl ranging from 0 to 1.0 M.

derma strains would allow us to obtain fungal strains or enzyme preparates with improved chitinolytic properties. Since Chit42 in particular has been shown to be important for the antifungal activity of T. harzianum, production of the ChBD-containing Chit42 in this fungus may result in better biocontrol strains. Acknowledgements We thank Dr. Anu Koivula and Dr. Markus Linder (VTT Biotechnology, Espoo, Finland) for advice on the binding assays. This work was ¢nancially supported by the grants from Junta de Andaluc|¨a PAI CVI 107, Universidad Internacional de Andaluc|¨a, CICYT BIO94-0289, BIO97-0521, IFD97-0668 and PTR94-0068. M.C.L. was recipient of a short term fellowship from the Spanish Ministry of Education and Culture. References [1] De la Cruz, J., Hidalgo-Gallego, A., Lora, J.M., Ben|¨tez, T., PintorToro, J.A. and Llobell, A. (1992) Isolation and characterization of three chitinases from Trichoderma harzianum. Eur. J. Biochem. 206, 859^867. [2] Garc|¨a, I., Lora, J.M., De la Cruz, J., Ben|¨tez, T., Llobell, A. and Pintor-Toro, J.A. (1994) Cloning and characterisation of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum. Curr. Genet. 27, 83^89. [3] Cohen-Kupiec, R. and Chet, I. (1998) The molecular biology of chitin digestion. Curr. Opin. Biotech. 9, 270^277. [4] Beintema, J.J. (1994) Structural features of plant chitinases and chitin-binding proteins. FEBS Lett. 350, 159^163. [5] Iseli, B., Boller, T. and Neuhaus, J.M. (1993) The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity. Plant Physiol. 103, 221^226.

[6] Morimoto, K., Karita, S., Kimura, T., Sakka, K. and Ohmita, K. (1997) Cloning, sequencing, and expression of the gene encoding Clostridium paraputri¢cum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain. J. Bacteriol. 179, 7306^7314. [7] Watanabe, T., Ito, Y., Yamada, T., Hashimoto, M., Sekine, S. and Tanaka, H. (1994) The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J. Bacteriol. 176, 4465^4472. [8] Svitil, A. and Kirchman, D.L. (1998) A chitin-binding domain in a marine bacterial chitinase and other microbial chitinases : implications for the ecology and evolution of 1,4-L-glycanases. Microbiology 144, 1299^1308. [9] Raikhel, N.V. and Lee, H.I. (1993) Structure and function of chitinbinding proteins. Annu. Rev. Plant Physiol. Mol. Biol. 44, 591^615. [10] Zeltins, A. and Schrempf, H. (1997) Speci¢c interaction of the Streptomyces chitin-binding protein CHB1 with K-chitin. The role of individual tryptophan residues. Eur. J. Biochem. 246, 557^564. [11] Linder, M., Lindeberg, G., Reinikainen, T., Teeri, T. and Pettersson, G. (1995) The di¡erence in a¤nity between two fungal cellulose-binding domains is dominated by a single amino-acid substitution. FEBS Lett. 372, 96^98. [12] Linder, M., Mattinen, M.-L., Kontteli, M., Lindeberg, G., Sta®hlberg, J., Drakenberg, T., Reinikainen, T., Petterson, G. and Annila, A. (1995) Identi¢cation of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I. Protein Sci. 4, 1056^1064. [13] Reinikainen, T., Teleman, O. and Teeri, T.T. (1995) E¡ects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei. Proteins 22, 392^403. [14] Din, N., Forsythe, I., Burtnick, L., Gilkes, N.R., Miller, J.C., Warren, R.A.J. and Kilburn, D.G. (1994) The cellulose-binding domain of endoglucanase A (Cen A) from Cellulomonas ¢mi evidence for the involvement of tryptophan residues in binding. Mol. Microbiol. 11, 747^755. [15] Xhu, G.Y., Ong, E., Gilkes, N., Kilburn, D., Muhandiram, D.R., Harris-Brandts, M., Carver, J., Kay, L. and Harvey, T. (1995) Solution structure of a cellulose-binding domain from Cellulomonas ¢mi by nuclear magnetic resonance spectroscopy. Biochemistry 34, 6993^ 7009. [16] Abuja, P.M., Schmuck, M., Pilz, I., Tomme, P., Claeyssens, M. and Esterbauer, H. (1988) Structural and functional domains of cellobiohydrolase I from Trichoderma reesei. Eur. Biophys. J. 15, 339^342. [17] Langsford, M.L., Gilkes, N.R., Singh, B., Moser, B., Miller Jr., R.C., Warren, R.A.J. and Kilburn, D.G. (1987) Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains. FEBS Lett. 25, 163^167. [18] Tomme, P., van Tilbeurgh, H., Petterson, G., Van Damme, J., Vandekerckhove, J., Knowles, J., Teeri, T.T. and Clayssens, M. (1988) Studies of the cellulolytic system of Trichoderma reesei QM 9414. Eur. J. Biochem. 170, 575^581. [19] Srisodsuk, M., Reinikainen, T., Penttila«, M. and Teeri, T. (1993) Role of the interdomain linker peptide of Trichoderma reesei cellobiohydrolase I in its interaction with crystalline cellulose. J. Biol. Chem. 268, 20756^20761. [20] Wilson, D., Spezio, M., Irwin, D., Karplus, A. and Taylor, J. (1995) Comparison of enzymes catalysing the hydrolysis of insoluble polysaccharides. ACS Symp. Ser. 618, 1^12. [21] Goldstein, M., Takagi, M., Hashida, S., Shoseyov, O., Doi, R. and Segel, I. (1993) Characterisation of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. J. Bacteriol. 175, 5762^5768. [22] Kilburn, D.G., Assouline, Z., Din., N., Gilkes, N.R., Ong, E., Tomme, P. and Warren, R.A.J. (1993) Cellulose binding domains: properties and applications. In: Proceedings of the 2nd TRICEL Symposium on Trichoderma reesei Cellulases and other Hydrolases

FEMSLE 9884 18-4-01

M.C. Limo¨n et al. / FEMS Microbiology Letters 198 (2001) 57^63

[23]

[24]

[25] [26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

(Suominen, P. and Reinikainen, T., Eds.), pp. 281^290. Foundation for Biotechnological and Industrial Fermentation Research, Espoo, Finland. Linder, M., Salovuori, I., Ruohonem, L. and Teeri, T.T. (1996) Characterization of a double cellulose-binding domain. Synergistic high a¤nity binding to crystalline cellulose. J. Biol. Chem. 271, 21268^ 21272. Ong, E., Gilkes, N., Miller, R.C., Warren, R.A.J. and Kilburn, D.J. (1993) The cellulose-binding domain (CBDCex) of an exoglucanase from Cellulomonas ¢mi: production in Escherichia coli and characterisation of the polypeptide. Biotechnol. Bioeng. 42, 401^409. Blackwell, J. (1988) Physical methods for the determination of chitin structure and conformation. Methods Enzymol. 161, 435^442. van Tilbeurgh, H., Tomme, P., Claeyssens, M., Bhikhabhai, R. and Petterson, G. (1986) Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei. FEBS Lett. 204, 223^227. Gilkes, N.R., Warren, R.A.J., Miller Jr., R.C. and Kilburn, D.G. (1988) Precise excision of the cellulose binding domains from two Cellulomonas ¢mi cellulases by a homologous protease and the e¡ect on catalysis. J. Biol. Chem. 263, 10401^10407. Poole, D.M., Durrant, A.J., Hazlewood, G.P. and Gilbert, H.J. (1991) Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains. Biochem. J. 279, 787^792. Maglione, G., Matsushita, O., Russell, J.B. and Wilson, D.B. (1992) Properties of a genetically reconstructed Prevotella ruminicola endoglucanase. Appl. Environ. Microbiol. 58, 3593^3597. Reinikainen, T., Ruohonem, L., Nevanen, T., Laaksonen, L., Kraulis, P., Jones, T.A., Knowles, J.K.C. and Teeri, T.T. (1992) Investigation of the function of the mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I. Proteins 14, 475^482. Kruus, K., Lua, A., Demain, A. and Wu, J.H.D. (1995) The anchorage function of CipA (CelL), a sca¡olding protein of the Clostridium thermocellum cellulosome. Proc. Natl. Acad. Sci. USA 92, 9254^ 9258. Blaak, H. and Schrempf, H. (1995) Binding and substrate speci¢cities of a Streptomyces olivaceoviridis chitinase in comparison with its proteolytically processed form. Eur. J. Biochem. 229, 132^139. Dawson, C., Belloch, C., Garc|¨a-Lo¨pez, M.D. and Uruburu, F. (1990) Catalogue of Strains, Spanish Type Culture Collection. Universidad de Valencia, Valencia, Spain.

63

[34] Teeri, T.T., Lehtovaara, P., Kauppinen, S., Salovuori, I. and Knowles, J. (1987) Homologous domains in Trichoderma reesei cellulolytic enzymes: gene expression of cellobiohydrolase II. Gene 51, 43^52. [35] Neuhaus, J-M., Ahl-Goy, P., Hinz, U., Flores, S. and Meins Jr., F. (1991) High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection. Plant Mol. Biol. 16, 141^151. [36] van Buuren, M., Neuhaus, J.-M., Shinshi, H., Ryals, J. and Meins Jr., F. (1992) The structure and regulation of homologous tobacco endochitinase genes of Nicotiana sylvestris and N. tomentosiformis origin. Mol. Gen. Genet. 232, 460^469. [37] Shinshi, H., Neuhaus, J.-H., Ryals, J. and Meins Jr., F. (1990) Structure of a tobacco endochitinase gene: evidence that di¡erent chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain. Plant Mol. Biol. 14, 357^368. [38] Pace, N., Vajdos, F., Fee, L., Grimsley, G. and Gray, T. (1995) How to measure and predict the molar absorption coe¤cient of a protein. Protein Sci. 4, 2411^2423. [39] Fleet, G. and Pha¡, H.J. (1974) Glucanases in Schizosaccharomyces. Isolation and properties of the cell wall associated L-1,3-glucanase. J. Biol. Chem. 249, 1717^1728. [40] Jeuniaux, C. (1966) Chitinases. Methods Enzymol. 8, 644^650. [41] Molano, J., Dura¨n, A. and Cabid, E. (1997) A rapid and sensitive assay for chitinase using tritiated chitin. Anal. Biochem. 83, 648^656. [42] Boller, T. and Mauch, F. (1988) Colorimetric assay for chitinase. Methods Enzymol. 161, 430^435. [43] Reissig, J.L., Strominger, J.L. and Leloir, L.F. (1955) A modi¢ed colorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem. 217, 959^966. [44] Imoto, T. and Yagishita, K. (1971) A simple activity measurement of lysozyme. Agric. Biol. Chem. 35, 1154^1156. [45] Kyriacou, A., Neufeld, R. and MacKenzie, C.J. (1988) E¡ect of physical parameters on the adsorption characteristics of fractionated Trichoderma reesei cellulase components. Enzym. Microb. Technol. 10, 675^681. [46] Golovchensko, N.P., Kataeva, I.A. and Akimenko, V.K. (1992) Elucidation of the role of hydrophobic interactions in the adsorption of endo-1,4-L-glucanases on polysaccharides. Enzym. Microb. Technol. 14, 327^331. [47] Gooday, G.W. (1990) The ecology of chitin degradation. Adv. Microb. Ecol. 11, 387^430.

FEMSLE 9884 18-4-01