Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization.
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.
Kang
Sang Kee
SK
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea.
Cho
Kwang Keun
KK
Ahn
Jong Kun
JK
Bok
Jin Duck
JD
Kang
Seung Ha
SH
Woo
Jung Hee
JH
Lee
Hong Gu
HG
You
Seung Kwon
SK
Choi
Yun Jaie
YJ
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
D007527:Isoenzymes; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; D001616:beta-Galactosidase; D007785:Lactose