Research On Phenyllactic Acid Production By Lactate Dehydrogenases From Pediococcus Species Of Lactic Acid Bacteria

Phenyllactic acid （PLA）, a kind of organic acid, can be produced by differentmicroorganisms, especially lactic acid bacteria. It was proved as an ideal antimicrobialcompound with broad and effective antimicrobial activity against both bacteria and fungi.Authough there are many reports about PLA produced by lactic acid bacteria （LAB）especially lactobacillus because of its security, few researches on the PLA production bypediococcus species. In this study, we firstly studied the PLA production ability bypediococcus acidilactici （P. ac） and pediococcus pentosaceus （P. pe）. When P. ac and P. pewere cultivated in MRS broth for36h at30oC, phenyllactic acid yield reached0.65and0.27mmol/L respectively. In addition, the possible pathway of the PLA production by P. ac wasstudied and discussed. During the strain fermentation process, PLA and4-hydroxyphenyllactic acid （HO-PLA） production could be significantly promoted by thesupplement of the corresponding amino acid （phenylalanine and tyrosine） and ketoacid（phenylpyruvic acid and4-hydroxyphenylpyruvic acid） precursors, but inhibited by thenon-corresponding precursors. Promotion and inhibitory effect were both strengthened whensupplement amounts increased. Ketoacids precursors showed more promotion effect on thecorresponding2-hydroxy acids production than amino acid precursors, however, amino acidprecursors showed more inhibitory effect on non-corresponding2-hydroxy acids productionthan ketoacids precursors.The putative D-lactate dehydrogenase （D-LDH） gene in P. ac and P. pe were synthesizedand inserted into the pET-22b（+） plasmid to get the reconstructed plamids, pET-PaLdhD andpET-PpLdhD, respectively. Then the reconstructed plasmids were transformed into E. coliBL21（DE3）, and the recombinant enzymes were expressed, purified, and characterized toconvert phenylpyruvic acid into D-PLA. The PacLDH showed acitivty maximally at30°Cand pH5.5with a specific activity of140and422.1U/mg for PPA and pyruvate, respectively.The Km, turnover number （kcat）, and catalytic efficiency （kcat/Km） for PPA were2.92mM,305s^-1, and105（mmol/L）^-1s^-1, respectively. By comparison, the optimum pH and temperature forP. pe D-LDH were45°C and pH5.5. The Km, kcat, and kcat/Kmfor substrate phenylpyruvatewere estimated to be1.73mM,173s^-1, and100（mmol/L）^-1s^-1, respectively.In addition, the cDNA gene coding for formate dehydrogenase （FDH） from Ogataeaparapolymorpha DL-1was cloned and expressed in E. coli. The recombinant enzyme showedwide pH optimum of catalytic activity from pH6.0–7.0. It had relatively high optimumtemperature at65°C, and retained93%,88%,83%, and71%of its initial activity after4h ofexposure at40,50,55, and60°C, respectively, suggesting that this enzyme had promisingthermal stability. The Km, kcat, and kcat/Kmfor substrate sodium formate were estimated to be 5.98mmol/L,15.6s^-1, and2.6（mmol/L）^-1s^-1, respectively. Due to the wide pH optimum andpromising thermostability, O. parapolymorpha FDH was used as an NADH regenerationcatalyst during D-PLA production by P. pe D-LDH. The optimized conditions of reaction areas follows: optimum temperature at50oC, optimum pH at6.0, and both the concentrations ofP. pe D-LDH and O. parapolymorpha FDH at0.4μmol/L.