Prokaryotic Heterologous Expression,Bioinformatics Analysis And Characterization Of Hydroxy Fatty Acid Dehydrogenase From Lactobacillus Plantarum P-8

Conjugated linoleic acid(CLA) is an octadecadienoic acid with a conjugated double bond.It has physiological effects such as anti-cancer,anti-atherosclerosis and lipid-lowering.Lactic acid bacteria can convert linoleic acid(LA) to form CLA.The transformation process requires fatty acid hydratase(CLA-HY),hydroxyl fatty acid dehydrogenase(CLA-DH) and acetoacetate decarboxylase(CLA-DC) to participate in the metabolic process.Therefore,CLA-DH is one of the bottlenecks in enzymatic CLA production.In this experiment gene cloning,heterologous prokaryotic heterologous expression,bioinformatics analysis and enzymatic properties of hydroxy fatty acid dehydrogenase from Lactobacillus plantarum p-8 were completed.The results were as follows:Firstly,the genomic DNA of Lactobacillus plantarum p-8 was used as a template in PCR amplification of CLA-DH.Using Colony PCR,Enzyme digestion and sequence analysis,the pET-28a-CLA-DH was successfully constructed and transformed into E.coli Trans1-T1.Secondly,bioinformatics analysis of CLA-DH revealed that CLA-DH is a hexamer protein belonging to the typical SDR superfamily with conserved Rossman-fold.There is coenzyme binding site G-x-x-x-G-x-G in the N-terminal of CLA-DH.A conserved catalytic active site S-xn-Y-x-x-x-K lies between amino acid residues 140?160.CLA-DH consists of 286 amino acids,is a stable hydrophilic cytoplasmic protein,does not contain any transmembrane region and signal peptide.The molecular weight of CLA-DH is 32.0918 kDa,the isoelectric point is 5.15,instability index is 25.53,and average hydrophilicity(GRAVY) is-0.179.Thirdly,pET-28a-CLA-DH was transformed into E.coli Transetta,induced using IPTG The heterologous expression of CLA-DH was successful by SDS-PAGE and Western Blot testing.Fourthly,in order to increase the expression of soluble CLA-DH,the effects of induction temperature,inducer concentration and induction time on the expression of soluble CLA-DH were studied.The results showed that the expression level of soluble CLA-DH was the highest at the induction temperature of 16?,the final concentration of IPTG was 0.5 mmol/L,and the induction time was 12 h.Fifthly,study on the enzymatic properties of purified and concentrated CLA-DH under ricinoleic acid as substrate showed that the optimum reaction temperature of CLA-DH is 45?,the optimum reaction pH is 6?7,Vmax is 1.04 ?mol/L·min,Km is 6.72 mmol/L,and kcat is 3.36 min-1.CLA-DH is stable at 20? and pH 6-7.The Mg2+,Mn2+ and Fe3+ is activator of CLA-DH.The Cu2+,Zn2+and Fe2+ is inhibitor of CLA-DH.