Expression and characterization of a novel ß-glucosidase, with transglycosylation and exo-ß-1,3-glucanase activities, from Rhizomucor miehei.

ABSTRACT

A novel ß-glucosidase gene, designated RmBglu3B, was cloned from the thermophilic fungus, Rhizomucor miehei CAU432. Its 2196-bp open reading frame encoded 731 amino acids. Its deduced amino-acid sequence showed highest identity (66%) with a glycoside hydrolase family 3 ß-glucosidase from R. miehei NRRL5382. RmBglu3B was successfully expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity with 18.2-fold purification and 59% recovery yield. Molecular masses of 76.5 kDa, by SDS-PAGE, and 66.4 kDa, by gel filtration, suggested that it is a monomer. Optimal pH and temperature of the purified enzyme were 5.0 and 50°C, respectively. RmBglu3B exhibited a broad range of substrate specificity, catalyzing the cleavage of ß-1,2, ß-1,3, ß-1,4 and ß-1,6 linkages, in various oligosaccharides, to liberate glucose. RmBglu3B also showed relatively high activity (19.1 U/mg) toward laminaran and transglycosylation activity, enabling gentiobiose production. This enzyme is a potential candidate for several industrial applications.