文献类型：期刊文献

中文题名：大肠杆菌L-阿拉伯糖异构酶的克隆表达及D-塔格糖的制备

英文题名：Cloning and expression of L-arabinose isomerase from escherichia coli K12 and its application for D-tagatose conversion

作者：王栋[1];丁庆豹[1];欧伶[1];魏晓琨[2];许艳梅[2];孙丽云[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]上海斯贝生物科技有限公司,上海200235

年份：2010

卷号：21

期号：5

起止页码：87

中文期刊名：中国食品添加剂

外文期刊名：China Food Additives

收录：CSTPCD;;北大核心:【北大核心2008】；

基金：上海市初创期中小企业创新08DZ1190500;华东理工大学优秀青年教师科研基金:yf0157117

语种：中文

中文关键词：大肠杆菌;L-阿拉伯糖异构酶;D-塔格糖;表达;转化

外文关键词：escherichia coli; L-arabinose isomerase; D-tagatose; expression; transformation

摘要：将大肠杆菌K-12中的L-阿拉伯糖异构酶基因araA克隆到载体pET-28a上,并转化入大肠杆菌BL21(DE3)中表达。通过SDS-PAGE分析发现,重组菌株能表达出大量可溶性酶蛋白。以重悬菌液为酶源、D-半乳糖为底物,对酶转化D-塔格糖的条件进行测定。结果表明:D-塔格糖的最佳转化温度为60℃。在pH7～9的范围中,D-塔格糖的转化率均能达到30%～40%。加入Mn2+、Ca2+、Co2+和Mg2+均能够使D-塔格糖的转化率提高,EDTA处理后的酶明显不具备催化能力,加入Mn2+后能使酶液恢复催化能力,但并不随着Mn2+浓度的提高而增大。
The gene encoding L-arabinose isomerase from Escherichia coli K12 was cloned into expression vector pET-28a.Then the recombinant plasmid was transformed into the strain E.coli BL1（DE3）and enzyme was highly expressed after induction with IPTG.The expression product analyzed with SDS PAGE showed that target proteins were expressed in soluble form.To investigate the properties of L-arabinose isomerase for the conversion of D-galactose to D-tagatose,the resuspended cells was used as enzyme source and D-galactose as substrate.The results showed that the optimal temperature for the D-galactose isomerization reaction was 60℃.The conversion rate of D-galacose to D-tagatose could reach 30%～40% at pH 7～9.The addition of Mn2＋,Ca2＋,Co2＋and Mg2＋ions enhanced the conversion of D-galactose to D-tagatose.Enzyme treated with EDTA had no activity at all.The addition of Mn2＋ions to enzyme previously treated with EDTA could recover its activity.The recovered activity could not increase with the increasing of the concentration of Mg2＋ions.