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
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea.
J Biotechnol. 2005 Apr 6;116(4):337-46. doi: 10.1016/j.jbiotec.2004.07.019.
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.
从嗜热栖热菌属（Thermus sp.）IB - 21的基因组文库中克隆出三种耐热乳糖水解酶，即两种β - 糖苷酶（bglA和bglB）和一种β - 半乳糖苷酶（bgaA）基因。bglA、bglB和bgaA分别由1311个碱基对（436个氨基酸残基）、1296个碱基对（431个氨基酸）和1938个碱基对（645个氨基酸）组成，预测分子量分别为49,066、48,679和72,714道尔顿。使用pET21b(+)载体系统在大肠杆菌BL21(DE3)中对这些酶进行了过量表达。通过热沉淀（70℃，40分钟）和Ni2+亲和层析将重组酶纯化至同质。通过SDS - PAGE估计的纯化酶分子量与其预测值一致。所有纯化的酶在pH约5.0 - 6.0时表现出最佳活性。相比之下，每种酶的活性温度曲线和热稳定性模式不同。BglBβ - 糖苷酶在三种酶中表现出最佳的乳糖水解活性，在70℃和pH 7.0条件下，高达200 mM乳糖时无底物抑制。在70℃和pH 7.0条件下，BglA、BglB和BgaA对138 mM乳糖的比活性（U/mg）分别为36.8、160.3和8.5。
