植物乳杆菌P-8羟基脂肪酸脱氢酶的克隆表达及活性鉴定  

Cloning and activity identification of hydroxy fatty acid dehydrogenase from Lactobacillus plantarum P-8

作　　者：赵彤彤 赵微 王丹 赵国芬[1] 刘扬[1] 张和平[2] 包秋华[2] ZHAO Tongtong;ZHAO Wei;WANG Dan;ZHAO Guofen;LIU Yang;ZHANG Heping;BAO Qiuhua(College of Life Science,Inner Mongolia Agricultural University,Huhhot 010018,China;Key Laboratory of Dairy Biotechnology and Bioengineering,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,内蒙古呼和浩特010018 [2]内蒙古农业大学乳品生物技术与工程教育部重点实验室,内蒙古呼和浩特010018

出　　处：《食品与发酵工业》2019年第1期36-40,共5页Food and Fermentation Industries

基　　金：国家自然科学基金(31560443);内蒙古自然科学基金(2017MS0306)

摘　　要：以植物乳杆菌P-8的基因组DNA为模板扩增出其羟基脂肪酸脱氢酶基因,对其进行生物信息学分析,结果表明该基因共编码286个氨基酸,蛋白质理论分子质量约为32 k Da,理论等电点为5. 15,不含跨膜区,是1个亲水性的细胞质蛋白。Ser、Thr、Tyr磷酸化位点个数分别为3、3、7。蛋白质的二级结构主要构成方式为α螺旋、无规则卷曲和β折叠。将此基因连接到p ET-28a(+)质粒载体中构建重组质粒p ET28a-DH,并转化入大肠杆菌E. coli Transetta (DE3)中获得重组大肠杆菌。重组菌在16℃条件下,经0. 1 mmol/L IPTG诱导16 h有可溶性表达,经Western Blot验证确定为重组羟基脂肪酸脱氢酶。通过镍柱亲和层析,得到纯化的重组酶。重组酶经初步活性检测,结果显示重组酶可以转化10-羟基-顺-12-十八碳烯酸为10-氧代-顺-12-十八碳烯酸,本研究为进一步探明植物乳杆菌P-8转化共轭亚油酸的机制奠定了基础。The hydroxy fatty acid short chain dehydrogenase gene(CLA-DH) was amplified by PCR using the genomic DNA of Lactobacillus plantarum P-8 as a template.Bioinformatics analysis showed that this gene encoded 286 amino acids with a theoretical molecular mass of about 32 kDa,and a theoretical isoelectric point of 5.15.It was a hydrophilic cytoplasmic protein with no trans-membrane region.Its phosphorylation sites of serine,threonine,and tyrosine were 3,3,and 7,respectively.The secondary structure of the protein mainly composed of α-helices,random coils,and β-sheets.The recombinant plasmid pET28a- DH was constructed by inserting CLA-DH into pET-28a(+),and then transformed into E.coli Transetta to obtain a compounded E.coli for expression.The recombinant bacteria had soluble expression at 16 ℃ after 16h induction with 0.1mmol/L IPTG.The results from Western Blot confirmed that the expression was recombinant CLA-DH.The purified recombinant CLA-DH was obtained by nickel column affinity chromatography.The preliminary activity assay of the recombinant CLA-DH showed that 10-hydroxy-cis-12-octadecenoic acid could be converted into 10-oxo-cis-12-octadecenoic acid,which laid a foundation for further analysis of the mechanism of transforming conjugated linoleic acid by L.plantarum P-8.

关 键 词：植物乳杆菌P-8 羟基脂肪酸脱氢酶 生物信息学 共轭亚油酸 重组表达 

分 类 号：TS201.3[轻工技术与工程—食品科学]