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. 2000 Apr;66(4):1360-8.
doi: 10.1128/AEM.66.4.1360-1368.2000.

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

L Fernández  1 , M M Beerthuyzen, J Brown, R J Siezen, T Coolbear, R Holland, O P Kuipers
Affiliations

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

L Fernández et al. Appl Environ Microbiol. 2000 Apr.

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 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.

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Figures

FIG. 1
FIG. 1
Nucleotide sequence of the L. lactis B1014 tributyrin esterase gene. The predicted amino acid sequence is given below the nucleotide sequence in the standard one-letter code. The stop codon is marked by an asterisk. The transcription initiation site mapped by primer extension is marked by a vertical arrow (nt 62). The putative Shine-Dalgarno site (SD), the conserved TG dinucleoside, and the −35 and −10 regions are underlined. The lipase/esterase consensus sequence at positions 119 to 123 is double underlined.
FIG. 2
FIG. 2
Multiple-sequence alignment of (putative) proteins with sequence homology to EstA of L. lactis B1014 (LL EstA), Caldocellum saccharolyticum acetyl esterase XynC (CS XynC), Clostridium thermocellum XynZ (CT XynZ), Mycobacterium scrofulaceum α-antigen (MS A-α), Mycobacterium avium antigen 85B (MA A85B), Mycobacterium leprae antigen 85C (ML A85C), Corynebacterium glutamicum PS1 protein (CG PS1), human esterase D (HS EstD), Anabaena azollae FGH (AA FGH), an Arabidopsis thaliana T32G6.5 hypothetical gene product (AT T32G6.5), Saccharomyces cerevisiae FGH (SC YJL068c), P. denitrificans FGH (PD FGH), an H. influenzae 0184 hypothetical gene product (HI 0184), a Synechocystis sp. hypothetical gene product (SS ?), and E. coli YeiG and YaiM hypothetical gene products (EC YeiG and EC YaiM). Only relevant regions of highest homology are shown. The numbering at the top is that of EstA, while numbers before and after each sequence indicate the numbers of additional N-terminal and C-terminal residues, respectively. The consensus sequence shows fully conserved residues in uppercase and highly conserved residues in lowercase letters. The putative catalytic Ser, Asp, and His residues are indicated in boldface and by asterisks.
FIG. 3
FIG. 3
Expression levels of estA in different L. lactis strains and constructs. B1014 and MG1363 are wild-type tributyrin esterase producer strains, NZ9000 and NZ9800 are nisin-sensitive strains for induction of gene expression, and NZ9340 is an esterase-deficient mutant. Plasmids pNZ9308 (B1014 estA) and pNZ9330 (MG1363 estA) are derivatives of pNZ8020 (transcriptional fusion vector), while pNZ9310 (B1014 estA) and pNZ9331 (MG1363 estA) are derivatives of pNZ8030 (translational fusion vector). The esterase activity was determined in cell extracts after induction with 3 ng of nisin A ml−1 and in the absence of nisin A, and it is expressed as micromoles of p-nitrophenol liberated from p-NP-butanoate per minute per milligram of protein.
FIG. 4
FIG. 4
Phospholipase activity of purified EstA from L. lactis B1014 on the chromogenic substrates diC6dithioPC (short-chain phospholipid substrate) and diC12dithioPC (long-chain phospholipid substrate). The enzyme activity is expressed as micromoles of thiol groups released from the substrate per minute per milligram of protein.
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