Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414

We have isolated the genomic and cDNA clones encoding EG III (a low-molecular-mass endo-β-1,4-glucanase) gene fromTrichoderma reesei QM9414. The nucleotide sequence of the cDNA fragment was verified to contain a 702-bp open reading frame that encodes a 234-amino-acid propeptide. The deduced protein sequence has significant homologies with family H endo-β-1,4-glucanases. The 16-amino-acid N-terminal sequence was shown to function as a leader peptide for possible secretion. Northern blot analysis showed that the EG III gene transcript, with a length of about 700 bp, was expressed markedly by cellulose but not by glucose. The protein has been expressed as a mature form inEscherichia coli and as secreted forms inSaccharomyces cerevisiae andSchizosaccharomyces pombe under the control oftac , alcohol dehydrogenase (ADH1 ), and human cytomegalovirus promoters, respectively. TheS. cerevisiae andSchizosaccharomyces pombe recombinant strains showed strong cellulolytic activities on agar plates containing carboxymethyl cellulose. TheE. coli strain expressed small amounts of EG III in an active form and large amounts of EG III in an inactive form. The molecular masses of the recombinant EG IIIs were estimated to be 25, 28, and 29 kDa forE. coli ,S. cerevisiae , andSchizosaccharomyces pombe , respectively, by immunoblot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Parts of the yeast recombinant EG IIIs decreased their molecular masses to 25 kDa after treatment with endoglycosidase H and α-mannosidase, suggesting that they are N glycosylated at least partly.