As a first step to exploit the potential of Trichoderma reesei to produce hemicellulases, we have purified two endo-β-1,4-xylanases (1,4-β-D-xylan xylanohydrolase, EC 220.127.116.11) and cloned their genes. The enzymes were isolated from culture filtrates of T. reesei C 30 grown on xylan as a carbon source, using two steps of cation exchange chromatography. They exhibited molecular weights of 19 (XYN I) and 21 (XYN II) kD, and isoelectric points of 5.2 and 9.0, respectively. These enzymes differed in their pH optimum for activity and affinity for xylan, and accounted for more than 90% of the total xylanolytic activity of the fungus. The purified enzymes were subjected to N-terminal sequence analysis, and after cleavage with trypsin and endoproteinase Glu-C the resulting peptides were sequenced. Oligonucleotides based on these sequences were used to clone gene fragments via PCR, and these were used as probes to isolate full-length copies of xyn1 and xyn2 from a lambda gene bank of T. reesei. The products of xyn1 and xyn2 share considerable homology, but the enzyme encoded by xyn2 appears to more closely resemble several other bacterial and fungal xylanases than does that of xyn1.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Biely, P. 1985. Microbial xylanolytic systems. Trends in Biotechnol. 3: 286–290.
Viikari, L., Ranua, M., Kantelinen, A., Sundqusit, J. and Linko, M. 1986. Bleaching with enzymes, p. 67–69. In: Proc. 3rd Internal. Conf. Biotechnol. in the Pulp and Paper Ind., Stockholm.
Chauvet, J.-M., Comtat, J. and Noe, P. 1987. Assistance in bleaching of never-dried pulps by the use of xylanases, consequences on pulp properties, Vol. II: 325–327. In: Proc. 4th Int. Congr. Wood and Pulping Chemistry, Paris.
Wong, K.K.Y., Tan, L.U.L. and Saddler, J.N. 1988. Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiol. Rev. 52: 305–317.
Poutanen, K., Rättö, M., Puls, J. and Viikari, L. 1987. Evaluation of different microbial xylanolytic systems. J. Biotechnol. 6: 49–60.
Huang, L., Hseu, T.-H. and Wey, T.T. 1991. Purification and characterization of an endoxylanase from Trichoderma koningii G-39. Biochem. J. 278: 329–333.
Dean, J.F.D. and Anderson, J.D. 1991. Ethylan biosynthesis inducing xylanase. II. Purification and physical characterization of the enzyme produced by Trichoderma viride. Plant Physiol. 95: 316–323.
Tan, L.U.L., Wong, K.K.Y., Yu, E.K.C. and Saddler, J.N. 1985. Purification and characterization of two β-D-xylanases from Trichoderma harzianum. Enzyme Microb. Technol. 7: 425–430.
Lappalainen, A. 1986. Purification and characterization of xylanolytic enzymes from Trichoderma reesei. Biotechnol. Appl. Biochem. 8: 437–448.
John, M. and Schmidt, J. 1988. Xylanases and beta-xylosidase of Trichoderma lignorum. Meth. Enzymol. 168: 662–671.
Rose, D.R., Birnbaum, G.I., Tan, L.U.L. and Saddler, J.N. 1987. Crystallization and preliminary X-ray diffraction study of a xylanase from Trichoderma harzianum. J. Mol. Biol. 194: 755–756.
Ujiie, M., Roy, C. and Yaguchi, M. 1991. Low-molecular weight xylanase from Trichoderma viride. Appl. Environ. Microbiol. 57: 1860–1862.
Royer, J.C. and Nakas, J.P. 1991. Purification and characterization of two xylanases from Trichoderma longibrachiatum. Eur. J. Biochem. 202: 521–529.
Yaguchi, M., Roy, C., Ujiie, M., Watson, D.C. and Wakarchuk, W. 1992. Amino acid sequence of the low molecular weight xylanase from Trichoderma viride. In: Xylans and Xylanases. J. Visser (Ed.). Elsevier, Amsterdam. In press.
Yaguchi, M., Roy, C., Watson, D.C., Rollin, F., Tan, L.U.L., Senior, D.J. and Saddler, N. 1992. The amino acid sequence of the 20 kD xylanase from Trichoderma harzianum E58. Ibid.
Teeri, T.T., Lehtovaara, P., Kauppinnen, S., Salovuori, I. and Knowles, J.K.C. 1987. Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. Gene 51: 43–52.
Penttilä, M., Lehtovaara, P., Nevalainen, H., Bhikhabhai, R. and Knowles, J.K.C. 1986. Homology between the cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. Gene 45: 253–263.
Chen, C.M., Gritzali, M. and Brown, R.D. Jr . 1987. Nucleotide sequence and deduced primary structure of cellobiohydrolase II of Trichoderma reesei. Bio/Technology 5: 274–278.
Arsdell, J.N., Van Kwock, S., Schweickart, V.L., Ladner, M.B., Gelfand, D.H. and Innis, M.A. 1987. Cloning, characterization and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei. Bio/Technology 5: 60–64.
Saloheimo, M., Lehtovaara, P., Penttilä, M., Teeri, T.T., Stahlberg, J., Johansson, G., Petterson, G.L., Claeyssens, M., Tomme, P. and Knowles, J.K.C. 1988. EG III, a new endoglucanase from Trichoderma reesei: characterization of both gene and enzyme. Gene 63: 11–21.
Eckhardt, T., Strickler, J., Gorniak, L., Burnett, W.V. and Fare, L. 1987. Characterization of the promoter, signal sequence and amino terminus of a secreted β-galactosidase from Streptomyces lividans. J. Bacteriol. 169: 4249–4256.
Barnett, C.C., Berka, R.M. and Fowler, T. 1991. Cloning and amplification of the gene encoding an extracellular β-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates. Bio/Technology 9: 562–567.
Von Heijne, G. 1984. How signal sequences maintain cleavage specificity. J. Mol. Biol. 173: 243–251.
Von Heijne, G. 1986. A new method for predicting signal sequence cleavage sites. Nucl. Acids Res. 14: 4683–4690.
Gavel, Y. and Von Heijne, G. 1990. Sequence differences between glycosylated and nonglycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Engineering 3: 433–442.
Lehle, L. 1992. Protein glycosylation in yeast. Antonie Van Lleuwenhoek 61: 133–134.
Calmes, T.P.G., Martin, E., Durand, H. and Tiraby, G. 1991. Proteolytic events in processing of secreted proteins in fungi. J. Biotechnol. 17: 51–56.
Shareck, F., Roy, C., Yaguchi, M., Morosoli, R. and Kluepfel, D. 1991. Sequences of three genes specifying xylanases in Streptomyces lividans. Gene 107: 75–82.
Zappe, H., Jones, W.A. and Woods, D.R. 1990. Nucleotide sequence of a Clostridium acetobutylicum P262 xylanase gene (xynB). Nucl. Acids Res. 18: 2179.
Fukusaki, F., Panbangrcd, W., Shinmyo, A. and Okada, H. 1984. The complete nucleotide sequence of the xylanase (xynA) of Bacillus pumilus. FEBS Letts. 171: 197–201.
Paice, M.G., Bourbonnais, R., Desrochers, M., Jurasek, L. and Yaguchi, M. 1986. A xylanase gene from Bacillus subtilis: nucleotide sequence and comparison with B. pumilus gene. Arch. Microbiol. 144: 201–202.
Yang, R.C.A., MacKenzie, C.R. and Narang, S.A. 1988. Nucleotide sequence of a Bacillus circulams xylanase gene. Nucl. Acids Res. 16: 7187.
Chou, P.Y. and Fasman, G.D. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol. 47: 45–145.
Garnier, J., Osguthorpe, D.J. and Robson, B. 1978. Analysis of accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120: 97–120.
Bissett, J. 1984. A revision of the genus Trichoderma. I. Section Longibrachiatum sect. nov. Can. J. Bot. 62: 924–931.
Mandels, M. and Andreotti, R.E. 1978. The cellulose to cellulase fermentation. Proc. Biochem. 6: 6–13.
Laemrnli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Miller, G.L. 1959. Use of the dinitro salicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426–428.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275.
Kalkkinen, N. and Tilgrnann, C. 1988. A gas-pulsed liquid-phase sequence constructed from a Beckman 890 instrument by using applied systems delivery and cartridge blocks. J. Prot. Chem. 7: 242–243.
Morrisey, W.N. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117: 307–310.
Gruber, F., Visser, J., Kubicek, C.P. and De Graaff, I.H. 1990. The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG negative strain. Curr. Genet. 18: 71–76.
Morawetz, R., Gruber, F., Messner, R. and Kubicek, C.P. 1992. Presence, transcription and translation of cellobiohydrolase genes in several Trichoderma species. Curr. Genet. 21: 31–36.
Teeri, T.T., Kumar, V., Lehtovaara, P. and Knowles, J.K.C. 1987. Construction of cDNA libraries by blunt-end ligation: high frequency cloning of long cDNAs from filamentous fungi. Anal. Biochem. 164: 60–67.
Sambrook, J., Frisch, E.F. and Maniatis, T. 1989. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York.
Gruber, F., Visser, J., Kubicek, C.P. and De Graaff, L. 1990. Cloning of the Trichoderma reesei pyrG gene and its use as a homologous marker for a high-frequency transformation system. Curr. Genet. 18: 447–451.
Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.
About this article
Biotechnology for Biofuels (2017)
Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains
Applied Microbiology and Biotechnology (2017)
Microbial Cell Factories (2016)
Improvement of thermostability and activity of Trichoderma reesei endo-xylanase Xyn III on insoluble substrates
Applied Microbiology and Biotechnology (2016)
The effects of extracellular pH and of the transcriptional regulator PACI on the transcriptome of Trichoderma reesei
Microbial Cell Factories (2015)