植物乳杆菌p-8乙酰乙酸脱羧酶的原核表达及活性分析  

Prokaryotic Expression and Activity Analysis of Lactobacillus plantarum P-8 Acetoacetate Decarboxylase

作　　者：柳玫琪 赵微 秦雅丽 刘扬[1] 赵国芬 包秋华[1] 张和平[3] LIU Meiqi;ZHAO Wei;QIN Yali;LIU Yang;ZHAO Guofen;BAO Qiuhua;ZHANG Heping(College of Life Science,Inner Mongolia Agricultural University,Hohhot 010018,China;College of Food Science.Shanxi Normal University,Linfen 041004,China;College of Food Science and Engineering,Inner Mongolia Agricultural University,Hohhot 010018,China)

机构地区：[1]内蒙古农业大学生命科学学院,呼和浩特010018 [2]山西师范大学食品科学学院,临汾041004 [3]内蒙古农业大学食品科学与工程学院,呼和浩特010018

出　　处：《内蒙古农业大学学报（自然科学版）》2022年第1期40-47,共8页Journal of Inner Mongolia Agricultural University(Natural Science Edition)

基　　金：内蒙古自治区自然科学基金项目(2017MS0306);国家自然科学基金项目(31560443)。

摘　　要：利用PCR扩增出植物乳杆菌p-8的乙酰乙酸脱羧酶基因,构建原核表达载体pET-28a-CLA-DC,转化Trans BL21细胞,IPTG诱导表达,SDS-PAGE和Western Blot证明重组酶CLA-DC可大量表达且大部分为包涵体。在pET-28a-CLA-DC基础上将CLA-DC基因在133单位点、133和415两位点以及133、415和786 3个位点进行弱化同义突变,分别转化Trans BL21进行表达,结果显示3个同义突变体有效地提高了CLA-DC的可溶表达量;经Ni-IDA柱纯化后,CLA-DC的可溶表达量和比活力从大到小的顺序均为三位点突变体、两位点突变体和单点突变体。本研究为体外酶催化生产CLA和提高可溶性表达奠定基础。The acetoacetate decarboxylase gene of Lactobacillus plantarum p-8 was amplified by the PCR,the prokaryotic expression vector pET-28 a-CLA-DC was constructed,the Trans BL21 cells were transformed,and the expression was induced by the IPTG. The SDS-PAGE and Western Blot proved that the recombinase CLA-DC could be expressed in large quantities and most of them were inclusion bodies. On the basis of pET-28 a-CLA-DC,the CLA-DC gene was subjected to weaken synonymous mutations at the 133 unit points,two points 133 and 415,and three points 133,415 and 786,respectively,and transformed the Trans BL21 for expression.It was shown that the three synonymous mutants effectively increased the soluble expression level of CLA-DC,after the purification by the Ni-IDA column,the soluble expression level and specific activity of the CLA-DC in the order from the largest to the smallest were all three site mutants,two-point mutants,and single-point mutants. This study laid the foundation for the production of CLA in vitro enzymatically and the improvement of soluble expression.

关 键 词：共轭亚油酸 植物乳杆菌P-8 乙酰乙酸脱羧酶 原核表达 同义突变 

分 类 号：Q936[生物学—微生物学]