Heterologous expression and characterization of endoglucanase I (EGI) fromTrichoderma viride HK-75

Endoglucanase I (EGI) secreted from Trichoderma viride HK-75 has a unique t ransglycosylation activity. The genomic and cDNA clones encoding EGI (egl1) of T. viride HK-75 were isolated and characterized. The coding region of e gl1, composed of 1392 bp, was found to encode a polypeptide of 464 amino ac ids that has extensive similarity (93.8%) with EGI of T. reesei. Expression of the egl1 gene in E. coli as a fusion protein (with N-terminal thioredox in and C-terminal histidine tag) led to a large production of a nonglycosyl ated protein of 62.5 kDa. However, it formed an insoluble inclusion body. U pon denaturation with 8 M urea followed by dialysis and successive purifica tion, the enzymatically active recombinant EGI (rEGI) was obtained at a lev el as high as 18.3 mg/l of 1,000 mi of culture. The rEGI had 67.8% activity for carboxymethyl cellulose (CMC), compared to native EGI (nEGI). The opti mum pH and optimum temperature of rEGI were lower than those of nEGI by 0.5 and 5 degrees C, respectively. The rEGI also had narrower CMCase ranges th an nEGI in pH and temperature stabilities. However, the catalytic and trans glycosylation abilities against cellotriose of rEGI were comparable to thos e of nEGI. These results suggest that the glycosylation is important for th e stabilities of EGI but not critical for the essential enzymatic capacity.