嗜酸乳酸杆菌ATCC 4356菌株异源二聚体b-半乳糖苷酶的克隆表达

Cloning, expression and characterization of a heterodimeric beta-galactosidase from Lactobacillus acidophilus ATCC 4356

摘要:

嗜酸乳酸杆菌（Lactobacillus acidophilus）的异源二聚体β-半乳糖苷酶属于糖苷水解酶2家族，由两个部分重叠、协同翻译的基因编码（lacL和lacM）。【目的】克隆表达该酶并测定其酶学特性。【方法】参照已全基因组测序的嗜酸乳酸杆菌NCFM菌株，以嗜酸乳酸杆菌ATCC 4356菌株基因组为模板，将lacL的RBS到lacM的终止子之间的序列（2834 bp）克隆到pQE31质粒上，并电转化JM109菌株。以下列步骤纯化表达产物：硫酸铵分级沉淀、阴离子交换、亲和层析和凝胶排阻层析。以凝胶排阻层析测定纯化酶的天然分子量，以邻硝基苯基半乳糖为底物测定其酶学特性。【结果】实现了该酶在JM109菌株中的可溶性表达。其氨基酸序列有一处不同于嗜酸乳酸杆菌NCFM菌株，即其大亚基（LacL）的第512位氨基酸不是组氨酸而是精氨酸。纯化酶比活力为226 U/mg蛋白，天然分子量为96.3±4.6 kDa，最适pH为7，最适温度为49℃，Km和Vmax值分别是：2.18±0.12 mmol/L， 273±5 U/mg蛋白。

Abstract:

Heterodimeric beta-galactosidase of Lactobacillus acidophilus belongs to glycoside hydrolase family 2, encoded by two overlapping and translational coupling genes, lacL and lacM. The lacL and lacM genes of the sequenced strain L. acidophilus NCFM encode polypeptides with calculated molecular masses of 73,253 and 35,817 Da, respectively. [objective] To clone, overexpress and characterize the enzyme in Escherichia coli. [Methods] We cloned the fragment (2834 bp) containing ribose-binding site (RBS) and coding regions of the lacLM genes from L. acidophilus ATCC 4356 into expression vector pQE31. RBS and HIS-Tag of pQE31 were substituted by inserted fragment. Recombinant plasmid was electrotransformed into E. coli JM109. Expression product was purified by ammonium sulphate fractionation, an-ion-exchange, affinity chromatography and gel permeation. Native molecular mass of homogenous enzyme was measured by gel permeation, and beta-galactosidase activity was determined by using o-nitrophenyl-b-D-galactopyranoside (ONPG) as the substrate. [Results] Overexpression of the soluble enzyme in E. coli was achieved. Amino acid residue 512 of re-combinant LacL was different from that of L. acidophilus NCFM. Homogenous enzyme was obtained by purification. The homogenous enzyme had a specific activity of 226 U/mg protein, a native molecular mass of 96.3±4.6 kDa, an optimum temperature at 49℃ and an optimum pH of 7. The Km and Vmax values of the enzyme were 2.18±0.12 mmol/L, 273±5 U/mg protein respectively.