title: Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization.
abstract: Three thermostable lactose-hydrolases, namely, two beta-glycosidases (bglA and bglB) and one beta-galactosidase (bgaA) genes were cloned from the genomic library of Thermus sp. IB-21. The bglA, bglB, and bgaA consisted of 1311 bp (436 amino acid residues), 1296 bp (431 aa), and 1938 bp (645 aa) of nucleotides with predicted molecular masses of 49,066, 48,679, and 72,714 Da, respectively. These enzymes were overexpressed in Escherichia coli BL21(DE3) using pET21b(+) vector system. The recombinant enzymes were purified to homogeneity by a heat precipitation (70 degrees C, 40 min) and a Ni2+-affinity chromatography. The molecular masses of the purified enzymes estimated by SDS-PAGE agreed with their predicted values. All the purified enzymes showed their optimal pH at around 5.0-6.0. In contrast, the temperature profiles for activity and thermostability patterns were different for each enzyme. BglB beta-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70 degrees C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70 degrees C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.
authors.affiliation: School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Sang Kee
authors.initials: SK
authors.lastname: Kang
authors.forename: Kwang Keun
authors.initials: KK
authors.lastname: Cho
authors.forename: Jong Kun
authors.initials: JK
authors.lastname: Ahn
authors.forename: Jin Duck
authors.initials: JD
authors.lastname: Bok
authors.forename: Seung Ha
authors.initials: SH
authors.lastname: Kang
authors.forename: Jung Hee
authors.initials: JH
authors.lastname: Woo
authors.forename: Hong Gu
authors.initials: HG
authors.lastname: Lee
authors.forename: Seung Kwon
authors.initials: SK
authors.lastname: You
authors.forename: Yun Jaie
authors.initials: YJ
authors.lastname: Choi
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D007785:Lactose / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; D001616:beta-Galactosidase; D007785:Lactose