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

Background: 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. Results: 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 beta-linked oligosaccharides, and had remarkably high beta-galactosidase, beta-glucosidase and beta-fucosidase activities. Because of the lack of information about the usefulness of beta-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for beta-glucosidase or beta-galactosidase. The BglMKg had maximal beta-galactosidase and beta-glucosidase activities at approximately 40 degrees C and 45 degrees C, respectively. The optimum pH for beta-galactosidase activity was 6.5, whereas the optimum pH for beta-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 beta-glucosidase substrates than beta-galactosidase ones. Conclusions: BglMKg is a small, monomeric, cold-active beta-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.