Cloning, purification, and characterization of GH3 -glucosidase, MtBgl85, from DAU221.

BACKGROUND

-Glucosidases have attracted considerable attention due to their important roles in various biotechnological processes such as cellulose degradation to make energy and hydrolysis of isoflavone. () is isolated from deep-sea sediment and has not been researched much yet. As a potential candidate for a variety of biotechnological industries, -glucosidases from the novel bacterial species should be researched extensively.

METHODS

-Glucosidase, MtBgl85, from DAU221 was purified by His-tag affinity chromatography and confirmed by SDS-PAGE and zymogram. Its biochemical and physiological properties, such as effects of temperature, pH, metal ions, and organic solvents, substrate specificity, and isoflavone hydrolysis, were investigated.

RESULTS

DAU221 showed -glucosidase activity in a marine broth plate containing 0.1% esculin and 0.25% ammonium iron (III) citrate. The -glucosidase gene, , was isolated from the whole genome sequence of DAU221. The -glucosidase gene was 2,319 bp and encoded 772 amino acids. The deduced amino acid sequence had a 43% identity with OaBGL84 from and 35% and 32% identity with to CfBgl3A and CfBgl3C from among bacterial glycosyl hydrolase family 3, respectively. The optimal temperature of MtBgl85 was 50 °C and the optimum pH was 7.0. MtBgl85 activity was strongly reduced in the presence of Hg and Cu ions. As a result of measuring the activity at various concentrations of NaCl, it was confirmed that the activity was maintained up to the concentration of 1 M, but gradually decreased with increasing concentration. MtBgl85 showed higher enzyme stability at non-polar solvents (high Log ) than polar solvents (low Log ). The hydrolyzed products of isoflavone glycosides and arbutin were analyzed by HPLC.