Improve ethanol tolerance of β-glucosidase Bgl1A by semi-rational engineering for the hydrolysis of soybean isoflavone glycosides

A β-glucosidase Bgl1A variant (A24S/F297Y) with improved ethanol tolerance was obtained by semi-rational engineering. At 30-40 °C, IC50 values (the amount required for inhibiting 50% enzyme activity) of the variant for ethanol were 17-30% (v/v), 1.4- to 2.4-fold of Bgl1A. When incubating in 15% (v/v) ethanol at 30 °C, the half-life of A24S/F297Y was 13 min; whereas Bgl1A lost all enzyme activity within 5 min. A24S/F297Y was more stable at pH 7.5 than at pH 6.5, and more than 50% of the original activity remained after incubation at 30 °C for 10 h. At 35 °C and pH 7.5, the half-life of A24S/F297Y was 80 min, 4.3 times longer than that of Bgl1A. When converting isoflavone glycosides to aglycones using A24S/F297Y as catalyst, the hydrolysis rates were 99% for daidzin and 98% for genistin. The concentrations of daidzein and genistein rapidly increased by 7.02 mM and 4.35 mM within 10 min, respectively. These results showed that A24S/F297Y was a promising candidate for the enzymatic hydrolysis of soybean isoflavone glycosides.