Bioconversion of phenylpyruvate to phenyllactate: gene cloning, expression, and enzymatic characterization of D- and L1-lactate dehydrogenases from Lactobacillus plantarum SK002.
Two DNA fragments containing the entire coding sequences of lactate dehydrogenase (LDH; ldhL1 and ldhD), whose enzymes have high activity for bioconversion of phenylpyruvate (PPA) to phenyllactate (PLA), were amplified from Lactobacillus plantarum SK002 using PCR. Sequencing showed open reading frames of 963 bp (ldhL1) and 999 bp (ldhD) encoding putative proteins of 320 and 332 amino acid residues, respectively. The LDH genes were cloned into an expression vector pET-22b(+) and expressed in Escherichia coli BL21(DE3). The purified recombinant L1-LDH and D-LDH had approximate (SDS-PAGE) molecular weights of 35 and 40 kDa, respectively. L1-LDH and D-LDH had PPA bioconversion specific activities of 71.06 and 215.84 U/mg with K (m) values of 3.96 and 5.4 mM, respectively. The rL1-LDH and rD-LDH showed maximum enzyme activity at 30 and 40 degrees C while both had optimum activity at pH 6.0. L1-LDH exhibited a higher pH and temperature stability than D-LDH. The results show that the his-tagged L. plantarum SK002 D- and L1-LDHs are efficient catalysts for bioconversion of PPA to PLA.
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China.
Jianghua
J
Jia
Wanmeng
W
Mu
Tao
T
Zhang
Bo
B
Jiang
D055162:Biocatalysis; D001711:Biotransformation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism*; D048191:Lactiplantibacillus plantarum / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010667:Phenylpyruvic Acids / Q000378:metabolism*; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D007773:Lactates; D010667:Phenylpyruvic Acids; C017648:3-phenyllactic acid; D007770:L-Lactate Dehydrogenase; C031606:phenylpyruvic acid