title: [Residues affecting hydrolysis of soy isoflavone glycosides, stability and catalytic properties of Thermotoga maritime β-glucosidase].
abstract: The thermostable β-glucosidase A (TmBglA) from Thermotoga maritime is a promising biocatalyst for production of isoflavone aglycones. Use of enzymes with high specificity for soy isoflavone conjugates is however essential for efficient hydrolysis. The effect of the amino acids located in the aglycone binding pocket with non-conserved residues between specificity groups in family 1 glycoside hydrolase (GH1) was studied using wild-type TmBglA and 3 exchange mutants (MI-TmBglA, M2-TmBglA, M1M2-TmBglA). Three mutants were expressed in Escherichia coli, purified and characterized. They had shifts in both optimum tem- perature and thermal stability, and their narrowing pH-activity curve caused by removing the ionized side chain in mutation. All mutants demonstrated the decreased catalytic efficiency more effectively revealed with natural glycoside, salicin, than with artificial substrate, p-nitrophenyl-β-D-glucopyranoside, suggesting that' these amino acids are the key residues to determine aglycone specificity. A lower hydrolysis of genistin and daidzin for M2-TmBglA than M1-TmBglA indicated that L400, A407 and E408 being preferable to V170, A171, V173, G 174 and H180 residues of Tm-BglA could be essential for soy isoflavone glycoside binding and catalysis.
authors.forename: Y
authors.initials: Y
authors.lastname: Xue
authors.forename: X
authors.initials: X
authors.lastname: Song
authors.forename: H
authors.initials: H
authors.lastname: Sun
authors.forename: Z
authors.initials: Z
authors.lastname: Cao
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001592:Benzyl Alcohols / Q000737:chemistry*; D002384:Catalysis; D004795:Enzyme Stability / Q000235:genetics; D005960:Glucosides / Q000737:chemistry*; D007529:Isoflavones / Q000737:chemistry*; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013025:Glycine max / Q000737:chemistry*; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001592:Benzyl Alcohols; D005960:Glucosides; D007529:Isoflavones; D011994:Recombinant Proteins; C040641:genistin; C005696:salicin; C013908:daidzin; D001617:beta-Glucosidase