Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with β-glucosidase, β-fucosidase and β-galactosidase activities

Cold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds.

BglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.

A bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and β-linked oligosaccharides, and had remarkably high β-galactosidase, β-glucosidase and β-fucosidase activities. Because of the lack of information about the usefulness of β-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for β-glucosidase or β-galactosidase. The BglMKg had maximal β-galactosidase and β-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for β-galactosidase activity was 6.5, whereas the optimum pH for β-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed β-glucosidase substrates than β-galactosidase ones. Conclusions: BglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.