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-mo lecular-mass endo-beta-1,4-glucanase) gene from Trichoderma reesei QM9 414. The nucleotide sequence of the cDNA fragment was verified to cont ain a 702-bp open reading frame that encodes a 234-amino-acid propepti de. The deduced protein sequence has significant homologies with famil y H endo-beta-1,4-glucanases. The 16-amino-acid N-terminal sequence wa s shown to function as a leader peptide for possible secretion. Northe rn blot analysis showed that the EG III gene transcript, with a length of about 700 bp, was expressed markedly by cellulose but not by gluco se. The protein has been expressed as a mature form in Escherichia col i and as secreted forms in Saccharomyces cerevisiae and Schizosaccharo myces pombe under the control of tac, alcohol dehydrogenase (ADH1), an d human cytomegalovirus promoters, respectively. The S. cerevisiae and Schizosaccharomyces pombe recombinant strains showed strong celluloly tic activities on agar plates containing carboxymethyl cellulose. The E. 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 for E. coli, S. cerevisiae, and Schizosaccharomyces pombe, respectively, by immunoblot analysis following sodium dodecyl sulfate-polyacrylamide ge l electrophoresis. Parts of the yeast recombinant EG IIIs decreased th eir molecular masses to 25 kDa after treatment with endoglycosidase H and a-mannosidase, suggesting that they are N glycosylated at least pa rtly.