Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization

• Published in: Journal of biotechnology Vol. 116; no. 4; pp. 337 - 346
• Main Authors: Kang, Sang Kee, Cho, Kwang Keun, Ahn, Jong Kun, Bok, Jin Duck, Kang, Seung Ha, Woo, Jung Hee, Lee, Hong Gu, You, Seung Kwon, Choi, Yun Jaie
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 06.04.2005; Amsterdam Elsevier; New York, NY
• Subjects: Amino Acid Sequence; beta-Galactosidase - chemistry; beta-Galactosidase - genetics; beta-Galactosidase - isolation & purification; beta-Galactosidase - metabolism; beta-Glucosidase - chemistry; beta-Glucosidase - genetics; beta-Glucosidase - isolation & purification; beta-Glucosidase - metabolism; Biological and medical sciences; Biotechnology; Cloning, Molecular - methods; Enzyme Activation; Enzyme Stability; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial - physiology; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Isoenzymes - analysis; Isoenzymes - biosynthesis; Isoenzymes - chemistry; Isoenzymes - genetics; Kinetics; Lactose - chemistry; Lactose - metabolism; Lactose intolerance; Lactose-hydrolase; Low-lactose milk; Molecular Sequence Data; Protein Engineering - methods; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Temperature; Thermostability; Thermus; Thermus sp; β-Galactosidase; β-Glycosidase; Thermostability; Thermus sp; Lactose-hydrolase; Low-lactose milk; Lactose intolerance; β-Galactosidase; β-Glycosidase; Thermus; Enzyme; Thermal stability; Characterization; Intolerance; Lactose; Glycosidases; Bacteria; Hydrolases; O-Glycosidases; Milk
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2004.07.019

Three thermostable lactose-hydrolases, namely, two β-glycosidases ( bglA and bglB) and one β-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 (431aa), 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 °C, 40 min) and a Ni 2+-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 β-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70 °C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70 °C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.