Exogenous Expression And Bioinformatics Analysis Of Lactobacillus Plantarum P-8 Acetoacetate Decarboxylase

The process of producing Conjugated linoleic acid(CLA)by Lactobacillus plantarum p-8 requires fatty acid hydratase(CLA-HY)and hydroxy fatty acid dehydrogenase(CLA-DH)Together with the three enzymes of Acetoacetate decarboxylase(CLA-DC),this study carried out prokaryotic heterologous expression,eukaryotic heterologous expression and bioinformatics analysis of CLA-DC of Lactobacillus plantarum p-8,Lay the foundation for in vitro enzymatic production of CLA.The results of the study are as follows:(1)The colony PCR identification,enzyme digestion identification and sequence comparison analysis showed that the prokaryotic expression vector pET-28a-CLA-DC was successfully constructed.(2)After IPTG induction,SDS-PAGE and Western Blot detection,it is proved that pET-28a-CLA-DC can express the recombinase CLA-DC in BL21 cells,and most of them are inclusion bodies.(3)In order to increase the prokaryotic soluble expression of CLA-DC,the CLA-DC gene was subjected to weakening synonymous mutations,and three mutants were designed.The results showed that the three mutants effectively increased the soluble expression of CLA-DC.(4)After the prokaryotic soluble CLA-DC expressed by the codon weakening synonymous mutation was purified by the Ni-IDA prepacked column,with the increase of weakening sites,the soluble expression level and the specific activity gradually increased.(5)The colony PCR identification,enzyme digestion identification and sequence comparison analysis showed that the eukaryotic expression vector pPICZ?A-CLA-DC was successfully constructed.(6)The pPICZ?A-CLA-DC was electroporated into Pichia pastoris GS115,the yeast transformants were identified by PCR,the positive transformed strains were screened,and the expression was induced by methanol.Western Blot proved that the recombinase CLA-DC was successfully expressed and was extracellularly secreted Express.After purification by Ni-IDA prepacked column,the specific activity is 0.018U/mg.(7)Through the bioinformatics analysis of CLA-DC,it is found that the content of alanine and leucine in CLA-DC protein is the highest.It is a hydrophilic protein,which may bind to the inner membrane and is stable.Cytoplasmic soluble protein with 22 phosphorylation sites.