研究报告

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

  • 赵彤彤 ,
  • 赵微 ,
  • 王丹 ,
  • 赵国芬 ,
  • 刘扬 ,
  • 张和平 ,
  • 包秋华
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  • 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)
硕士研究生(赵国芬教授为通讯作者,E-mail:guofenzhao@126.com)。

收稿日期: 2018-07-03

  网络出版日期: 2019-02-01

基金资助

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

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

  • ZHAO Tongtong ,
  • ZHAO Wei ,
  • WANG Dan ,
  • ZHAO Guofen ,
  • LIU Yang ,
  • ZHANG Heping ,
  • BAO Qiuhua
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  • 1(College of Life Science,Inner Mongolia Agricultural University,Huhhot 010018,China)
    2(Key Laboratory of Dairy Biotechnology and Bioengineering,Inner Mongolia Agricultural University,Hohhot 010018,China)

Received date: 2018-07-03

  Online published: 2019-02-01

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 kDa,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pET-28a(+)质粒载体中构建重组质粒pET28a-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。

本文引用格式

赵彤彤 , 赵微 , 王丹 , 赵国芬 , 刘扬 , 张和平 , 包秋华 . 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[J]. 食品与发酵工业, 2019 , 45(1) : 36 -40 . DOI: 10.13995/j.cnki.11-1802/ts.018180

Abstract

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 16 h induction with 0.1 mmol/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.

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