title: Effect of mutations to amino acid A301 and F361 in thermostability and catalytic activity of the β-galactosidase from Bacillus subtilis VTCC-DVN-12-01.
abstract: Beta-galactosidase (EC 3.2.1.23), a commercially important enzyme, catalyses the hydrolysis of β-1,3- and β-1,4-galactosyl bonds of polymer or oligosaccharidesas well as transglycosylation of β-galactopyranosides. Due to catalytic properties; β-galactosidase might be useful in the milk industry to hydrolyze lactose and produce prebiotic GOS. The purpose of this study is to characterize β-galactosidase mutants from B. subtilis.
  
  
  
  Using error prone rolling circle amplification (epRCA) to characterize some random mutants of the β-galactosidase (LacA) from B. subtilisVTCC-DVN-12-01, amino acid A301 and F361 has been demonstrated significantly effect on hydrolysis activity of LacA. Mutants A301V and F361Y had markedly reduced hydrolysis activity to 23.69 and 43.22 %, respectively. Mutants the site-saturation of A301 reduced catalysis efficiency of LacA to 20-50 %, while the substitution of F361 by difference amino acids (except tyrosine) lost all of enzymatic activity, indicating that A301 and F361 are important for the catalytic function. Interestingly, the mutant F361Y exhibited enhanced significantly thermostability of enzyme at 45-50 °C. At 45 °C, LacA-361Y retained over 93 % of its original activity for 48 h of incubation, whereas LacA-WT and LacA-301Vwere lost completely after 12 and 24 h of incubation, respectively. The half-life times of LacA-361Y and LacA-301 V were about 26.8 and 2.4 times higher, respectively, in comparison to the half-life time of LacA-WT. At temperature optimum 50 °C, LacA-361Y shows more stable than LacA-WT and LacA-301 V, retaining 79.88 % of its original activities after 2 h of incubation, while the LacA-WT and LacA-301 V lost all essential activities. The half-life time of LacA-361Y was higher 12.7 and 9.39 times than that of LacA-WT and LacA-301 V, respectively. LacA-WT and mutant enzymes were stability at pH 5-9, retained over 90 % activity for 72 h of incubation at 30 °C. However, LacA-WT showed a little bit more stability than LacA-301 V and LacA-361Y at pH 4.
  
  
  
  Our findings demonstrated that the amino acids A301V and F361 play important role in hydrolysis activity of β -galactosidase from B. subtilis. Specially, amino acid F361 had noteworthy effect on both catalytic and thermostability of LacA enzyme, suggesting that F361 is responsible for functional requirement of the GH42 family.
authors.affiliation: Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600, Hanoi, Vietnam.
authors.forename: Thao Thi
authors.initials: TT
authors.lastname: Nguyen
authors.affiliation: Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600, Hanoi, Vietnam.
authors.forename: Hanh Van
authors.initials: HV
authors.lastname: Vu
authors.affiliation: Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600, Hanoi, Vietnam.
authors.forename: Nhung Thi Hong
authors.initials: NT
authors.lastname: Nguyen
authors.affiliation: Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600, Hanoi, Vietnam.
authors.forename: Tuyen Thi
authors.initials: TT
authors.lastname: Do
authors.affiliation: Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600, Hanoi, Vietnam. nslthanh@ibt.ac.vn.
authors.forename: Thanh Sy Le
authors.initials: TS
authors.lastname: Nguyen
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D001616:beta-Galactosidase
keywords: Bacillus subtilis; Catalytic activity; Error prone rolling circle amplification; Escherichia coli; Mutants; β-Galactosidase