Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis.

ABSTRACT: The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vecto

SUBMITTER: Fernandez L 

PROVIDER: S-EPMC91993 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature