• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.315-325
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• 2016

A novel esterase gene, est7K, was isolated from a compost metagenomic library. The gene encoded a protein of 411 amino acids and the molecular mass of the Est7K was estimated to be 44,969 Da with no signal peptide. Est7K showed the highest identity of 57% to EstA3, which is an esterase from a drinking water metagenome, when compared with the enzymes with reported properties. Est7K had three motifs, SMTK, YSV, and WGG, which correspond to the typical motifs of family VIII esterases, SxxK, Yxx, and WGG, respectively. Est7K did not have the GxSxG motif in most lipolytic enzymes. Three additional motifs, LxxxPGxxW, PLGMxDTxF, and GGxG, were found to be conserved in family VIII enzymes. The results of the phylogenetic analysis and the alignment study suggest that family VIII enzymes could be classified into two subfamilies, VIII.1 and VIII.2. The purified Est7K was optimally active at 40ºC and pH 10.0. It was activated to exhibit a 2.1-fold higher activity by the presence of 30% methanol. It preferred short-length p-nitrophenyl esters, particularly p-nitrophenyl butyrate, and efficiently hydrolyzed glyceryl tributyrate. It did not hydrolyze β-lactamase substrates, tertiary alcohol esters, glyceryl trioleate, fish oil, and olive oil. Est7K preferred an S-enantiomer, such as (S)-methyl-3-hydroxy-2-methylpropionate, as the substrate. The tolerance to methanol and the substrate specificity may provide potential advantage in the use of the enzyme in pharmaceutical and other biotechnological processes.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1484-1489
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• 2014

A novel esterase gene, est01, was successfully unearthed from a biogas digester microbiota metagenomic library. The 1,194 bp est01 gene encodes a protein of 44,804 Da (designated Est01). The amino acid sequence of Est01 shows only moderate (33%) identity to a lipase/esterase. Phylogenetic analysis and biochemical characterization confirmed that Est01 is a new member of family VIII esterases. The purified Est01 from recombinant Escherichia coli BL21 (DE3) showed high hydrolytic activity against short-chain fatty acid esters, suggesting that it is a typical carboxylesterase rather than a lipase. Furthermore, the Est01 was even active at $10^{\circ}C$ (43% activity remained), with the optimal temperature at $20^{\circ}C$, and had a broad pH range from 5.0 to 10.0, with the optimal pH of 8.0. These properties suggest that Est01 is a cold-adaptive esterase and could have good potential for low-temperature hydrolysis application.

• Journal of Microbiology and Biotechnology
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• v.24 no.9
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• pp.1260-1268
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• 2014

Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative ${\beta}$-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Ser-x-x-Lys motif that is conserved among all ${\beta}$-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme is a serine protein and was active against p-nitrophenyl esters of $C_2$, $C_4$, $C_8$, and $C_{10}$. The optimum pH and temperature for enzyme activity were pH 9.0 and $30^{\circ}C$, respectively. Relative activity of 55% remained at up to $5^{\circ}C$ with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, and $Hg^{2+}$ ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.