title: Improve ethanol tolerance of β-glucosidase Bgl1A by semi-rational engineering for the hydrolysis of soybean isoflavone glycosides.
abstract: 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 13min; whereas Bgl1A lost all enzyme activity within 5min. 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 10h. At 35°C and pH 7.5, the half-life of A24S/F297Y was 80min, 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 7.02mM and 4.35mM within 10min, respectively. These results showed that A24S/F297Y was a promising candidate for the enzymatic hydrolysis of soybean isoflavone glycosides.
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230601, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei, Anhui 230601, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Fang
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230601, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230601, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei, Anhui 230601, China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230601, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei, Anhui 230601, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yin
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230601, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei, Anhui 230601, China.
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Chemistry & Biochemistry, Florida International University, Miami, FL 33199, United States.
authors.forename: Xiaotang
authors.initials: X
authors.lastname: Wang
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230601, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei, Anhui 230601, China. Electronic address: zemin_fang@163.com.
authors.forename: Zemin
authors.initials: Z
authors.lastname: Fang
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230601, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei, Anhui 230601, China. Electronic address: yzxiao@ahu.edu.cn.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yazhong
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects*; D000595:Amino Acid Sequence; D004795:Enzyme Stability / Q000187:drug effects; D000431:Ethanol / Q000633:toxicity*; D005818:Genetic Engineering / Q000379:methods*; D005947:Glucose / Q000494:pharmacology; D006027:Glycosides / Q000378:metabolism*; D006860:Hydrogen Bonding / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007529:Isoflavones / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis / Q000187:drug effects; D050505:Mutant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013025:Glycine max / Q000378:metabolism*; D040681:Structural Homology, Protein; D013696:Temperature; D013997:Time Factors; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006027:Glycosides; D007529:Isoflavones; D050505:Mutant Proteins; D000431:Ethanol; D001617:beta-Glucosidase; D005947:Glucose
keywords: Ethanol tolerance; Semi-rational engineering; Soybean isoflavones; β-Glucosidase