Rational engineering and application of the β-glucosidase Bgl2A:V224D with high stability

Abstract:

[Background] β-glucosidase (EC 3.2.1.21) has important application values in food and medicine industries. Bgl2A:V224D, a mutant of a marine microorganism-derived β-glucosidase, is potent in the conversion of soybean isoflavones in the deep processing of soybean yellow slurry water. However, the low stability and low ethanol tolerance limit the application of Bgl2A:V224D. [Objective] To enhance the application potential of Bgl2A:V224D, we adopted a computation-assisted rational design to improve the stability and organic solvent tolerance of this enzyme in the application environment. [Methods] We employed three complementary computational methods to design stable mutants of Bgl2A:V224D by seeking overlapping mutations. Then, sequence coevolution and conservation analyses were performed to determine candidate mutants. Finally, the target mutant enzymes were selected by experimental assays. After the stable mutant enzymes were identified, their stability was further enhanced by combinational mutation. Finally, the application performance of the mutant enzymes for the conversion of soybean isoflavones in the deep processing of soybean yellow slurry water was evaluated. [Results] Several single-point and multi-point mutants with improved stability and maintained or increased enzyme activity were obtained, among which S360K/E408Y had the highest stability, with the half-life at 35 ℃ and pH 6.5 about 6.7 times that of the starting enzyme. Moreover, S360K/E408Y was more tolerant to ethanol than the starting enzyme. The former had the relative activity of about 35% after 20 min of incubation with 20% ethanol at 35 ℃, while the latter had the relative activity of only about 5% after 10 min of incubation under the same conditions. S360K/E408Y was then applied to hydrolyze soybean isoflavones, and its efficiency was significantly increased compared with that of the starting enzyme Bgl2A:V224D. Specifically, the amount of substrate that could be hydrolyzed by the former enzyme was two times that of the latter enzyme under the same conditions. The amount of S360K/E408Y required for converting soybean isoflavones in the deep processing of soybean yellow slurry water was about 1/3 less than that of the starting enzyme. [Conclusion] The stability-enhancing engineering strategy for improving the enzyme tolerance to organic solvents is applicable to β-glucosidases, and the application potential of β-glucosidases in various fields can be enhanced by this strategy.

Key words:β-glucosidase;stability;organic solvent tolerance;rational design;soybean isoflavones