β-半乳糖苷酶的筛选、克隆表达、酶学性质及其酶法合成低聚半乳糖

王欣，吴斌，何冰芳

(南京工业大学生物与制药工程学院，江苏南京211800)

摘要：采用人工底物邻硝基苯酚-β-D-半乳糖苷(oNPG)为筛选标记,从耐有机溶剂微生物菌库中,筛选出具有较高水解活性的β-半乳糖苷酶产生菌，再以乳糖为底物考察菌株低聚半乳糖的合成性能，筛选得到1株产β-半乳糖苷酶的Erwinia billingiae WX1。根据GenBank中相同属种的基因组序列推测β-半乳糖苷酶基因，克隆得到β-半乳糖苷酶基因gal，并在大肠杆菌中实现了来源于Erwinia billingiae菌β-半乳糖苷酶的克隆表达。该基因的开放阅读框(ORF)为1 428 bp，编码475个氨基酸，理论相对分子质量为5.2×10`4。镍柱法分离纯化得到电泳纯的β-半乳糖苷酶GAL，其酶学性质研究表明最适催化温度55 ℃，最适pH 7.0；Mg`(2+)、Mn`(2+)对该酶起较强促进作用， EDTA对该酶抑制作用较强。利用β-半乳糖苷酶GAL的转糖基作用，以乳糖为底物合成低聚半乳糖，初步优化的反应条件：底物乳糖质量浓度400 g/L，每克乳糖添加酶量 1.0 U，在40 ℃反应16 h后，低聚半乳糖合成率达到34%(质量分数)，显示了较好的开发前景。

关键词：Erwinia billingiae；β-半乳糖苷酶；克隆表达；酶法合成；低聚半乳糖

doi:10.3969/j.issn.1672-3678.2015.06.006

收稿日期：2014-04-04

基金项目：高等学校博士学科点专项基金(20123221130001)；国家自然科学基金(21376119)

作者简介：王欣(1988—)，女，辽宁营口人，硕士研究生，研究方向：生物化工；何冰芳(联系人)，教授，E-mail:bingfanghe@njtech.edu.cn

中图分类号：Q93

文献标志码：A

文章编号：1672-3678(2015)06-0030-06

Abstract：Strains were screened from organic solvent tolerant bacteria,though the hydrolysis of o-nitrophenyl-β-D-galactopyranoside (oNPG).After rescreened by lactose as using lactose as substrate,strain Erwinia billingiae WX1 showed high transgalactosidation.Based on the nucleotide sequence of the β-galactosidase from Erwinia billingiae,the gene encoding the β-galactosidase GAL was cloned and expressed in Escherichia coli. The gene consists of 1 428 bp,and the translated protein encodes 475 amino acids and its molecular mass is approximately 5.2×10`4. After purification,the purity of this enzyme showed that the optimal pH and temperature were 7.0 and 55 ℃,respectively. Its activity was notably promoted by Mg`(2+),Mn`(2+),whereas EDTA had high inhibition to the enzyme. The enzyme synthesis of galacto-oligosachorides was studied by using its transgalactosylation. The optimum synthesis condition of galacto-oligosachorides was as follows:400 g/L lactose,1.0 U β-galactosidase GAL,reaction with temperature 40 ℃ for 16 h,under the conditons,the production rate of galacto-oligosachorides was 34%. Our findings demonstrate that the recombinant β-galactosidase has good development prospects.

Keywords：Erwinia billingiae; β-galactosidase; cloning expression; enzymatic synthesis; galacto-oligosachorides

Screening,cloning,expression,characterization of β-galactosidase and enzymatic synthesis of galacto-oligosaccharides

WANG Xin,WU Bin,HE Bingfang

(College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211800,China)

低聚半乳糖(galacto-oligosaccharide,GOS)是在乳糖分子的半乳糖基一侧连接上1～4个半乳糖基生成的低聚糖类，属于葡萄糖和半乳糖组成的杂低聚糖。……