A NOVEL, SMALL ENDOGLUCANASE GENE, EGL5, FROM TRICHODERMA-REESEI ISOLATED BY EXPRESSION IN YEAST

A method is presented for the isolation of genes encoding hydrolytic e nzymes without any knowledge of the corresponding proteins. cDNA made from the organism of interest is cloned into a yeast vector to constru ct an expression library in the yeast Saccharomyces cerevisiae. Coloni es producing hydrolytic enzymes are screened by activity plate assays. In this work, we constructed a yeast expression library from the fila mentous fungus Trichoderma reesei and isolated a new beta-1,4-endogluc anase gene on plates containing beta-glucan. This gene, egl5, codes fo r a previously unknown small protein of 242 amino acids. Despite its s mall size, the protein contains two conservative domains found in Tric hoderma cellulases, namely the cellulose-binding domain (CBD) and the linker region that connects the CBD to the catalytic core domain. Mole cular modelling of the EGV CBD revealed some interesting structural di fferences compared to the CBD of the major cellulase CBHI from T. rees ei. The catalytic core of EGV is unusually small for a cellulase and r epresents a new family of cellulases (Family K) and of glycosyl hydrol ases (Family 45) together with the endoglucanase B of Pseudomonas fluo rescens and the endoglucanase V of Humicola insolens on the basis of h ydrophobic cluster analysis.