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. 2005 Feb;71(2):817-25.
doi: 10.1128/AEM.71.2.817-825.2005.

New Thermophilic and Thermostable Esterase With Sequence Similarity to the Hormone-Sensitive Lipase Family, Cloned From a Metagenomic Library

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New Thermophilic and Thermostable Esterase With Sequence Similarity to the Hormone-Sensitive Lipase Family, Cloned From a Metagenomic Library

Jin-Kyu Rhee et al. Appl Environ Microbiol. .
Free PMC article

Abstract

A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30 degrees C up to 95 degrees C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C(4) to C(16)) examined was p-nitrophenyl caproate (C(6)), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.

Figures

FIG. 1.
FIG. 1.
Amino acid sequence blocks conserved in the deduced amino acid sequences of EstE1 and homologous lipases and esterases. (A) Multiple amino acid sequence alignments of EstE1 and its homologs. The accession numbers of the aligned sequences are for the following organisms: 1EVQ_A, carboxylesterase Est2 from Alicyclobacillus acidocaldarius; ZP_00095860, esterase/lipase from Novosphingobium aromaticivorans; AAC38151, lipase from Pseudomonas sp. strain B11-1; ZP_00170489, hypothetical protein from Ralstonia eutropha JMP134; ZP_00215452, hypothetical protein from Burkholderia cepacia R18194; AAC12774, brefeldin A esterase from Bacillus subtilis; CAA37862, triacylglycerol lipase from Moraxella sp.; AAC73578, putative lipase (EC 3.1.1.) from E. coli K-12; NP_005348, hormone-sensitive lipase from Homo sapiens (human); BAB59879, carboxylesterase from Thermoplasma volcanium; BAC06606, esterase from Pyrobaculum calidifontis; NP_343862, lipase (LipP-2) from S. solfataricus; NP_070544, carboxylesterase (EstA) from Archaeoglobus fulgidus DSM 4304; NP_343839, lipase (LipP-1) from S. solfataricus; NP_375919, 303-amino-acid hypothetical esterase from S. tokodaii; NP_243114, lipase (esterase) from Bacillus halodurans C-125; NP_947767, putative lipase/esterase from Rhodopseudomonas palustris CGA009; ZP_00057664, esterase/lipase from Thermobifida fusca; ZP_00215124, hypothetical protein from Burkholderia cepacia R18194; NP_960379, hypothetical protein from Mycobacterium avium subsp. paratuberculosis strain k10; ZP_00214276, hypothetical protein from Burkholderia cepacia R18194; AAC41424, lipase-like enzyme from Wautersia eutropha; ZP_00028572, esterase/lipase from Burkholderia fungorum; ZP_00216007, hypothetical protein from Burkholderia cepacia R18194; ZP_00223864, hypothetical protein from Burkholderia cepacia R1808; AAO17429, unknown protein from Pseudomonas aeruginosa; BAA82510, esterase HDE from Oleomonas sagaranensis. (B) Amino acid sequence alignment of EstE1 with putative esterases identified from Sagasso Sea environmental genomes. The accession numbers are indicated to the left of the amino acid sequences. Identical residues have a gray background. Symbols: •, amino acids forming a catalytic triad; ○, amino acids involved in oxyanion hole formation.
FIG. 2.
FIG. 2.
Phylogenetic tree of HSL family homologues to EstE1. The phylogenetic tree was constructed by the neighbor-joining method with MEGA, version 2.1, software. The accession numbers of the aligned sequences shown in parentheses are the same as those described in the legend to Fig. 1A. The numbers associated with the branches refer to bootstrap values (confidence limits) resulting from 1,000 replicate resamplings. Only bootstrap values higher than 50% are shown. The scale represents the number of amino acid substitutions per site.
FIG. 3.
FIG. 3.
Expression and purification of recombinant EstE1. (A) Proteins recovered during the various purification steps were separated by SDS-12% polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Lane M, molecular weight standards; lane 1, total lysate of E. coli BL21(DE3) transformed with an empty vector; lane 2, proteins as described above, but with the EstE1 expression vector; lane 3, proteins after thermal denaturation; lane 4, purified fraction from Ni-NTA column; lane 5, purified fraction after GFC. (B) Western blot analysis of purification steps probed with an anti-penta-His antibody. The samples are the same as those described for panel A. (C) Silver staining of the purified esterase (1 μg) from Ni-NTA affinity chromatography of the pooled fractions from GFC. Protein size markers are indicated in kilodaltons on the left, and recombinant EstE1 proteins are indicated by the arrowheads on the right.
FIG. 4.
FIG. 4.
pH activity profile of EstE1. Est1 activity was measured at different pH values by a standard assay. The buffers used were sodium acetate (•) (pH 3.0 to 5.5), MES (○) (pH 5.5 to 7.0), HEPES (▪) (pH 7.0 to 7.5), and glycine (□) (pH 7.5 to 9.5).
FIG. 5.
FIG. 5.
Temperature dependence and thermostability of EstE1. (A) The effect of temperature on the esterase activity of EstE1 was determined at different temperatures by a standard assay. The logarithms of specific activities were plotted against 1,000/T according to an Arrhenius plot. The correlation coefficient was 0.993. (B) The enzyme (6.0 μM), in 20 mM potassium phosphate buffer (pH 7.0), was incubated at 80°C (•), 85°C (○), 90°C (▴), and 95°C (▵) for the indicated times. The residual activity was measured by a standard assay. The activity of a nonincubated sample was defined as 100%.

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