Dataset Information

Enzymological characterization of a novel d-lactate dehydrogenase from Lactobacillus rossiae and its application in d-phenyllactic acid synthesis.

ABSTRACT: A novel lactate dehydrogenase gene, named lrldh, was cloned from Lactobacillus rossiae and heterologously expressed in Escherichia coli. The lactate dehydrogenase LrLDH is NADH-dependent with a molecular weight of approximately 39 kDa. It is active at 40 °C and pH 6.5 and stable in a neutral to alkaline environment below 35 °C. The kinetic constants, including maximal reaction rate (V _max), apparent Michaelis-Menten constant (K _m), turnover number (K _cat) and catalytic efficiency (K _cat/K _m) for phenylpyruvic acid were 1.95 U mg^-1, 2.83 mM, 12.29 s^-1, and 4.34 mM^-1 s^-1, respectively. Using whole cells of recombinant E. coli/pET28a-lrldh, without coexpression of a cofactor regeneration system, 20.5 g l^-1 d-phenyllactic acid with ee above 99% was produced from phenylpyruvic acid in a fed-batch biotransformation process, with a productivity of 49.2 g l^-1 d^-1. Moreover, LrLDH has broad substrate specificity to a range of ketones, keto acids and ketonic esters. Taken together, LrLDH is a promising biocatalyst for the efficient synthesis of d-phenyllactic acid and other fine chemicals.

SUBMITTER: Luo X

PROVIDER: S-EPMC7005236 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Enzymological characterization of a novel d-lactate dehydrogenase from Lactobacillus rossiae and its application in d-phenyllactic acid synthesis.

Luo Xi X Zhang Yingying Y Yin Fengwei F Hu Gaowei G Jia Qiang Q Yao Changsheng C Fu Yongqian Y

3 Biotech 20200206 3

A novel lactate dehydrogenase gene, named lrldh, was cloned from Lactobacillus rossiae and heterologously expressed in Escherichia coli. The lactate dehydrogenase LrLDH is NADH-dependent with a molecular weight of approximately 39 kDa. It is active at 40 °C and pH 6.5 and stable in a neutral to alkaline environment below 35 °C. The kinetic constants, including maximal reaction rate (V max), apparent Michaelis-Menten constant (K m), ...[more]

PMID: 32099742

Similar Datasets

Project description:The Lactobacillus sakei strain LK-145 isolated from Moto, a starter of sake, produces potentially large amounts of three D-amino acids, D-Ala, D-Glu, and D-Asp, in a medium containing amylase-digested rice as a carbon source. The comparison of metabolic pathways deduced from the complete genome sequence of strain LK-145 to the type culture strain of Lactobacillus sakei strain LT-13 showed that the L- and D-amino acid metabolic pathways are similar between the two strains. However, a marked difference was observed in the putative cysteine/methionine metabolic pathways of strain LK-145 and LT-13. The cystathionine ?-lyase homolog gene malY was annotated only in the genome of strain LT-13. Cystathionine ?-lyase is an important enzyme in the cysteine/methionine metabolic pathway that catalyzes the conversion of L-cystathionine into L-homocysteine. In addition to malY, most genome-sequenced strains of L. sakei including LT-13 lacked the homologous genes encoding other putative enzymes in this pathway. Accordingly, the cysteine/methionine metabolic pathway likely does not function well in almost all strains of L. sakei. We succeeded in cloning and expressing the malY gene from strain LT-13 (Ls-malY) in the cells of Escherichia coli BL21 (DE3) and characterized the enzymological properties of Ls-MalY. Spectral analysis of purified Ls-MalY showed that Ls-MalY contained a pyridoxal 5'-phosphate (PLP) as a cofactor, and this observation agreed well with the prediction based on its primary structure. Ls-MalY showed amino acid racemase activity and cystathionine ?-lyase activity. Ls-MalY showed amino acid racemase activities in various amino acids, such as Ala, Arg, Asn, Glu, Gln, His, Leu, Lys, Met, Ser, Thr, Trp, and Val. Mutational analysis revealed that the ?-amino group of Lys233 in the primary structure of Ls-MalY likely bound to PLP, and Lys233 was an essential residue for Ls-MalY to catalyze both the amino acid racemase and ?-lyase reactions. In addition, Tyr123 was a catalytic residue in the amino acid racemase reaction but strongly affected ?-lyase activity. These results showed that Ls-MalY is a novel bifunctional amino acid racemase with multiple substrate specificity; both the amino acid racemase and ?-lyase reactions of Ls-MalY were catalyzed at the same active site.

Dataset Information

Enzymological characterization of a novel d-lactate dehydrogenase from Lactobacillus rossiae and its application in d-phenyllactic acid synthesis.

Publications

Enzymological characterization of a novel d-lactate dehydrogenase from <i>Lactobacillus rossiae</i> and its application in d-phenyllactic acid synthesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets