Salt-tolerant β-glucosidases from Karamay saline soil for soybean isoflavone bioconversion

Abstract

β-Glucosidase (EC 3.2.1.21) hydrolyzes β-glycosidic bonds and is widely used in the food, fuel, and pharmaceutical industries. It holds significant potential particularly for converting glycosides, such as soybean isoflavone glycosides and ginsenosides, into their more bioactive aglycones. Here, metagenomic mining of saline soil from Karamay, Xinjiang, China, identified four novel GH1 β-glucosidase genes ( b0-bg30 , b0-bg32 , b0-bg36 , and b0-bg40 ). These genes were cloned and heterologously expressed in Escherichia coli DH5α. Subsequent screening using cellobiose and isoflavone substrates pinpointed two enzymes, B0-BG30 and B0-BG36, exhibiting high hydrolytic activity toward these glycosides. Both enzymes were purified, systematically characterized, and evaluated for their isoflavone-hydrolyzing capabilities. Additionally, molecular docking was employed to investigate their substrate recognition and catalytic mechanisms toward cellobiose and genistin (a representative isoflavone glycoside). Biochemical analysis revealed that B0-BG30 and B0-BG36 exhibited optimal temperatures of 40 °C and 55 °C, and optimal pH values of 5.6 and 5.0, respectively. B0-BG30 retained full activity after a 2 h incubation at 35 °C, demonstrated broad pH stability (pH 5.0–10.0), and displayed remarkable salt tolerance, with its activity enhanced (> 130%) in the presence of 0.5–3.5 M NaCl. Both enzymes showed high specific hydrolytic activity toward genistin (5.36±0.27 U/mg and 3.46±0.25 U/mg, respectively). Molecular docking indicated that Asn-414 (for B0-BG30) and Glu-166 (for B0-BG36) are key residues involved in substrate binding and catalysis. The formation of hydrogen-bond networks around the active sites stabilized the substrates, structurally explaining the catalytic properties of both enzymes toward cellobiose and genistin, which is consistent with the enzymatic data. Furthermore, high-performance liquid chromatography (HPLC) was employed to validate the feasibility of a high-throughput glucose oxidase-based method for screening enzymes with such hydrolytic capabilities. In summary, this study identified novel β-glucosidases from a saline environment, providing robust candidate biocatalysts for the green bioconversion of isoflavones into aglycones and laying a technical foundation for the high-throughput screening and mechanistic investigation of glycoside hydrolases.