Molecular cloning and functional expression of extracellular Exo-β-(1,3)-Glucanase from Candida fermentati SI

An isoflavone glucosidase that catalyzes the hydrolysis of isoflavone glucosides into glucose and corresponding aglycones was purified from Candida fermentati SI. The N-terminal sequence was determined to be GLNCDYCN. We designed degenerate primers on the basis of these amino acid sequences and successfully cloned the full structural gene sequence of the isoflavone glucosidase using inverse PCR. The exo-β-(1,3)-glucanase gene consists of 1227 base-pair nucleotides, encoding a 408-amino-acid sequence that shares 41-96% amino acid homology with other yeast exo-β-(1,3)-glucanases belonging to glycoside hydrolase family 5. The recombinant exo-β-(1,3)-glucanase was expressed in Pichia pastoris X-33, using a pPICZA vector system, and further characterized. The molecular mass of the purified exo-β-(1,3)-glucanase was estimated by SDS-PAGE to be 47 kDa. The optimal pH and temperature were pH 4.5 and 40°C, respectively. The K_m values of the purified exo-β-(1,3)-glucanase for daidzinfland genistin were 0.12 mM and 0.14 mM, respectively. The V_max values of the purified iso avone glucosidase were 945.03 U/mg for daidzinfland 835.92 U/mg and for genistin.