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 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.

Physiological role of S-formylglutathione hydrolase in C1 metabolism of the methylotrophic yeast Candida boidinii

“…In the central section, Ser156 of C. boidinii is in the consensus sequence GXSXG around the catalytic serine, typical of all lipases and esterases (Fernandez et al, 2000). Near the C-terminus, conserved His273 is found, which is predicted to be the catalytic histidine residue (Cygler et al, 1993).…”

Alicyclobacillus acidocaldariusThermophilic Esterase EST2's Activity in Milk and Cheese Models

“…EstA is probably a key enzyme in the development of food flavor through the degradation of esters and lipids and the synthesis of esters and thioesters (38). Studies using an EstAnegative mutant partially confirmed this (12). EstA has an optimal temperature of 30°C and exhibits maximal activity at pH 7.5 with p-nitrophenyl (pNP) esters having acyl chains that are four to six carbon atoms long.…”

“…EstA has an optimal temperature of 30°C and exhibits maximal activity at pH 7.5 with p-nitrophenyl (pNP) esters having acyl chains that are four to six carbon atoms long. This enzyme exhibits sequence homology to S-formyl glutathionine hydrolase, which can explain its thioesterase activity (12). EstA belongs to a superfamily of phylogenetically related proteins with representatives in the domains Eukarya, Bacteria, and Archaea (18,20,24,31).…”

Biochemical and Structural Analysis of Hormone-sensitive Lipase Homolog EstE7: Insight into the Stabilized Dimerization of HSL-Homolog Proteins

“…The enzyme activity of the EstE7 proteins in various oligomeric states was measured using p-nitrophenyl butyrate. 12 The esterase activity was measured by spectrophotometric assay at pH 7.5 and 30 o C for 5 min using 5 mM p-nitrophenyl butyrate as the substrate. Larger aggregates (void volume in size exclusion chromatography), aggregated, dimer and monomer EstE7 proteins were concentrated to 3 mg/mL and then used for subsequent analyses.…”