GDSL Esterase Research Articles

Isolation and Characterization of a GDSL Esterase from the Metagenome of a Marine Sponge-associated Bacteria

Using a metagenome library constructed from a bacterial associated with a marine sponge Hyrtios erecta, we identified a novel esterase that belongs to the SGNH hydrolase superfamily of esterases. The substrate specificity of EstHE1 was determined using p-nitrophenyl (pNP) ester (C2: acetate, C4: butylate, C6: caproate, C12: laurate, C16: palmitate). EstHE1 exhibited activity against C2 (5.6 U/mg), C4 (5.1 U/mg), and C6 (2.8 U/mg) substrates. The optimal temperature for EstHE1 esterase activity of the pNP acetate substrate was 40 degrees C, and EstHE1 retained 60% of its enzymatic activity in the 30-50 degrees C range. This esterase showed moderate thermostability, retaining 58% of its activity even after preincubation for 12 h at 40 degrees C. EstHE1 also maintained activity in high concentrations of NaCl, indicating that this esterase is salt-tolerant. Thus, EstHE1 has the thermal stability and salt tolerance necessary for use as an industrial enzyme.

Mutation in the RGD motif decreases the esterase activity of Xcc_est

Five truncated constructs of Xcc_est GDSL esterase from Xanthomonas campestris were heterologously expressed and purified. The truncated constructs with a RGD motif had higher specific activities than those without the motif. The specific activity of wild-type Xcc_est was 32.5 +/- 2.7 U/mg, while the RGD mutant was 12.5 +/- 4.9 U/mg. Moreover, we expressed mature forms of the Xcc_est protein and the RGD mutant as inclusion bodies and, after refolding, there was no significant difference between the two constructs in specific activity. These results suggest that the RGD motif affects the esterase-domain folding in vivo during the translocation process.

Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system

Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.

Identification and characterization of a GDSL esterase gene located proximal to the swr quorum-sensing system of Serratia liquefaciens MG1.

Serratia liquefaciens MG1 employs the swr quorum-sensing system to control various functions, including production of extracellular enzymes and swarming motility. Here we report the sequencing of the swr flanking DNA regions. We identified a gene upstream of swrR and transcribed in the same direction, designated estA, which encodes an esterase that belongs to family II of lipolytic enzymes. EstA was heterologously expressed in Escherichia coli, and the substrate specificity of the enzyme was determined in crude extracts. With the aid of zymograms visualizing EstA on polyacrylamide gels and by the analysis of a transcriptional fusion of the estA promoter to the promoterless luxAB genes, we showed that expression of the esterase is not regulated by the swr quorum-sensing system. An estA mutant was generated and was found to exhibit growth defects on minimal medium containing Tween 20 or Tween 80 as the sole carbon source. Moreover, we show that the mutant produces greatly reduced amounts of N-acyl-homoserine lactone (AHL) signal molecules on Tween-containing medium compared with the wild type, suggesting that under certain growth conditions EstA may be important for providing the cell with precursors required for AHL biosynthesis.

Esterase EstE from Xanthomonas vesicatoria ( Xv_EstE) is an outer membrane protein capable of hydrolyzing long-chain polar esters.

A new esterase gene from Xanthomonas vesicatoria (formerly X. campestris) DSM 50861 was identified, cloned from a chromosomal gene library and overexpressed in Escherichia coli. The corresponding DNA fragment contains an ORF of 1,818 bp, encoding a hydrolase of the GDSL esterase family. A protein of about 67 kDa, named Xv_EstE, was expressed from this fragment. A N-terminal signal peptide was processed under low-expression conditions, yielding a 63-kDa mature protein. The predicted amino acid sequence showed distinct homology to esterases of the GDSL family. Based on homology, a catalytic triad Gly-Asp-Ser could be defined. Amino acid sequence alignments and computer-assisted structure prediction indicated the presence of a carboxyl-terminal beta-barrel membrane domain which might facilitate binding of Xv_EstE to the outer membrane. This could be verified by differential cell fractionation experiments, in which Xv_EstE was exclusively found in the outer membrane fraction. Xv_EstE showed preferential hydrolytic activity on short chain (up to C(8)) and para-substituted nitrophenylesters as substrates. However, only long-chain 1-hydroxy-pyrene-3,6,8-trisulfonic acid (HPTS)-fatty acid esters were hydrolyzed. Xv_EstE was also found to be active on a series of substrates of industrial interest, such as 1-methylprop-2-ynyl acetate, for which an enantioselectivity up to 93% ee could be recognized.