Cloning and overexpression of Lactobacillus helveticusd‐lactate dehydrogenase gene in Escherichia coli

Abstract

NAD+‐dependent d‐lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N‐terminal sequence and amino acid residues 220–215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong‐Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499–8513], a 0.6‐kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the d‐lactate dehydrogenase gene. Both plasmids overexpressed d‐lactate dehydrogenase (> 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticusd‐lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of d‐2‐hydroxy‐acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong‐Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60–66]. The physicochemical and catalytic properties of recombinant d‐lactate dehydrogenase were identical to those of the wild‐type enzyme, e.g. α2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2‐oxo‐acid substrates. The kinetic profiles of 2‐oxo‐acid substrates showed some marked differences from that of l‐lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity. Copyright © 1992, Wiley Blackwell. All rights reserved