1，3-丙二醇氧化还原酶同工酶基因yqhD的克隆与原核表达.pdfVIP

维普资讯 学兔兔 化 _T 进 展 2008年第27卷第4期 CHEMICAL INDUSTRY AND ENGINEERING PROGRESS ·527· 1，3．丙二醇氧化还原酶同工酶基因yqhD的克隆与原核表达 李红梅，陈 佳，李 琳，徐 斐 (}：海理工人学食品与牛物技术研究所，上海 200093) 摘 要：大肠杆菌1，3一丙二醇氧化还原酶同工酶被yqhD的基因编码，能催化羟基丙醛生成1，3一丙二醇。采用PCR 方法从大肠杆菌中扩增到yqhD，测序结果显示该片段大小为1164bp。将目标片段克隆到原核表达载体pET28(0【+) 中并转化到大肠杆菌Novablue，经IPTG诱导后，SDS—PAGE电泳鉴定，结果表明克隆得到的yqhD基因能在大 肠杆菌Novablue—pET28中实现蛋白表达；在25℃条件下，IPTG诱导12 h后，1，3一丙二醇氧化还原酶同工酶的酶 活力达到124 U／mg，而野生型的酶活力仅为1．4 U／mg；重组菌全细胞发酵结果显示，产物1，3一丙二醇的产率能达 到65％，而野生型仅为9．5％。 关键词：1，3一丙二醇氧化还原酶同工酶；克隆；原核表达；yqhD基因 中图分类号：TQ 923 文献标识码：A 文章编号：1000—6613(2008)04—0527—04 Cloning and expression of 1,3-propenediol oxidoreductase isoenzyme gene yqhd from Escherichia coli LIHongmei，CHENJia，LILin， Fei (Insititute ofFood and Biotechnology，University of Shanghai for Science and Technology，Shanghai 200093，C~na) Abstract：1,3一propenediol oxidoreductase isoenzyme is one of the key enzymes in biotransformation systhesis of 1,3一propenediol，and is encoded by the yqhD gene．YqhD gene was amplified from E coli by polymerase chain reaction(PCR)．The fragment was inserted into plasmid pET28 cut by two restriction enzymes BamHI and NheI．The yqhD gene after recombinant and transformation was induced and expressed by IPTG in E．coli at 25 c for 12 h．The expression product was checked by SDS—PAGE． The cloned yqhD gene was obtained and protein expression was realized．The catalytic activity of 1,3一propenediol oxidoreductase isoenzyme was improved by 88 fold．The yield of 1,3一propenediol in