Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis.
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 vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.
Fernández
L
L
NIZO Food Research, Ede, The Netherlands.
Beerthuyzen
M M
MM
Brown
J
J
Siezen
R J
RJ
Coolbear
T
T
Holland
R
R
Kuipers
O P
OP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009561:Nisin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism*
D014280:Triglycerides; D009561:Nisin; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C005830:tributyrin