Cloning and activity identification of hydroxy fatty acid dehydrogenase from Lactobacillus plantarum P-8
ZHAO Tongtong1,ZHAO Wei1,WANG Dan1,ZHAO Guofen1*, LIU Yang1,ZHANG Heping2,BAO Qiuhua2
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)
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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