Bioconversion Of Phenylpyruvate To Phenyllactate: Gene Cloning, Expression And Characterization Of Lactate Dehydrogenases

Phenyllactic acid (PLA) is a novel antimicrobial compound. Lactate dehydrogenase (LDH) is the main enzyme for converting phenylpyruvate (PPA) to PLA in lactic acid bacteria (LAB). According to the genome of Lactobacillus plantarum WCFS1, there are three lactate dehydrogenases genes: ldhL1, ldhL2, ldhD which products are L1-LDH (EC:1.1.1.27) , L2-LDH ( EC:1.1.1.27 ) and D-LDH (EC:1.1.1.28) , respectively. .In this work, we designed the primers according to the lactate dehydrogenases genes of L. plantarum WCFS1. Using the genome of L. plantarum SK002 as the template, we cloned the three genes by PCR. The three genes were then ligated to pMD-19-T simple vector. The following sequencing step showed that in L. plantarum SK002 ldhL1(Gene bank accession number: FJ712707)is 963 bp; ldhL2 (Gene bank accession number: FJ392647) is 930 bp and ldhD (Gene bank accession number: FJ712706) is 999 bp.After the three genes were ligated to the expressed plasmids, and transferred into E. coli BL21(DE3), we got the recombinant bacteria. The recombinant LDHs were succesfully purified. The molecular weight of L1-LDH, L2-LDH, D-LDH were 35, 34, 40 kDa respectively shown by SDS-PAGE. The activity of rLDHs were determined at pH 6.5, 30 C. The specific activity of D-LDH on PPA was 215.84 U/mg. While that of L1-LDH was 71.06 U/mg which was over 1000 times that of L2-LDH (0.06 U/mg ). Furthermore, the characters of L1-LDH and D-LDH were studied. The optimum pH for both enzymes was 6.0. The optimum temperature for two enzymes were different: 40 C for L1-LDH and 30 C for D-LDH. More studies showed that both the pH stability and thermal stability of L1-LDH is much better than D-LDH. The Km values for L1-LDH were 0.23 and 3.96 mM using pyruvate and PPA as substrates, respectively. For D-LDH, the Km values were 0.06 and 5.4 mM.