Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K _m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.     

Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library

A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K _m and k _cat of 39 μM and 25.8 s^?1, respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80–114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.     

Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment

To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50–57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.     

Structural and biochemical characterization of a metagenome‐derived esterase with a long N‐terminal extension

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC‐Est1～6, were isolated from a fosmid library of a leaf‐branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome‐derived enzymes, LC‐Est1 is characterized by the presence of a long N‐terminal extension (LNTE, residues 26–283) between a putative signal peptide (residues 1–25) and a C‐terminal esterase domain (residues 284–510). A putative esterase from Candidatus Solibacter usitatus (CSu‐Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC‐Est1. To examine whether LC‐Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC‐Est1 (residues 26–510), LC‐Est1C (residues 284–510), and LC‐Est1C* (residues 304–510) were overproduced in E. coli, purified, and characterized. LC‐Est1C* was only used for structural analysis. The crystal structure of LC‐Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC‐Est1C was lower than that of LC‐Est1 by 60%, although its substrate specificity was similar to that of LC‐Est1. LC‐Est1C was less stable than LC‐Est1 by 3.3°C. These results suggest that LNTE of LC‐Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.     

Extremely Stable and Versatile Carboxylesterase from a Hyperthermophilic Archaeon

We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (est_Pc) from strain VA1. est_Pc consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As est_Pc showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90°C), but also at ambient temperature (1,050 U/mg at 30°C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p-nitrophenyl caproate (C₆) the best substrate among the p-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.     

Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library

A metagenomic library was constructed from completely fermented compost using a fosmid vector. From a total of 23,400 clones, 19 esterase-positive clones were selected on LB plates containing 1% glyceryl tributyrate as the substrate. The esterase gene of an esterase-positive clone, est2K, was on an ORF of 1299 bp and encoded a protein of 432 amino acids. Est2K had a SMTK motif and was a family VIII esterase. Unlike most family VIII esterases, Est2K had a signal peptide of 27 amino acids. The molecular mass and pI of the mature Est2K was calculated to be 44,668 Da and 4.48, respectively. The amino acid sequence of Est2K showed 72% identity with that of EstC, an esterase of an uncultured bacterium from leachate. The purified Est2K was optimally active at pH 10.0 and 50 °C. Est2K was stable in the presence of 30% methanol and exhibited a 2.4-fold higher activity in the presence of 5% methanol than in the presence of 1% isopropanol. Est2K preferred short to medium length p-nitrophenyl esters, especially p-nitrophenyl butyrate, as the substrate. Est2K did not hydrolyze β-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC.     

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.     

A novel,versatile family IV carboxylesterase exhibits high stability and activity in a broad pH spectrum

Objectives

To investigate the properties of a novel metagenome-derived member of the hormone-sensitive lipase family of lipolytic enzymes.

Results

A forest soil metagenome-derived gene encoding an esterase (Est06) belonging to the hormone-sensitive lipase family of lipolytic enzymes was subcloned, heterologously expressed and characterized. Est06 is a polypeptide of 295 amino acids with a molecular mass of 31 kDa. The deduced protein sequence shares 61% similarity with a hypothetical protein from the marine symbiont Candidatus Entotheonella sp. TSY1. Purified Est06 exhibited high affinity for acyl esters with short-chain fatty acids, and showed optimum activity with p-nitrophenyl valerate (C5). Maximum enzymatic activity was at 50 °C and pH 7. Est06 exhibited high stability at moderate temperatures by retaining all of its catalytic activity below 30 °C over 13 days. Additionally, Est06 displayed high stability between pH 5 and 9. Esterase activity was not inhibited by metal ions or detergents, although organic solvents decreased activity.

Conclusions

The combination of Est06 properties place it among novel biocatalysts that have potential for industrial use including low temperature applications.

     

Screening for novel lipolytic enzymes from uncultured soil microorganisms

The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C₄) but not p-nitrophenyl palmitate (C₁₆).     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Cloning and Characterization of a Carboxylesterase from Bacillus coagulans 81-11

A genomic library of Bacillus coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27 528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50°C, although the enzyme displayed stability at temperatures up to 60°C.     

Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome

Functional screening for lipolytic enzymes at low temperatures resulted in the isolation of the novel cold-active esterases, EstM-N1 and EstM-N2, from a metagenomic DNA library of arctic soil samples. EstM-N1 and EstM-N2 were 395 and 407 amino acids in length, respectively, and showed the highest similarity to class C β-lactamases. However, they shared a relatively low level of sequence similarity (30%) with each other. Phylogenetic analysis of bacterial lipolytic enzymes confirmed that EstM-N1 and EstM-N2 belonged to family VIII of bacterial esterases/lipases. The (His)₆-tagged esterases were purified to about 99% homogeneity from the soluble fraction of recombinant Escherichia coli cultures. The purified EstM-N1 and EstM-N2 retained more than 50% of maximal activity in the temperature range of 0–35°C, with optimal temperatures of 20°C and 30°C, respectively. Both enzymes preferred the short acyl chains of p-nitrophenyl esters and exhibited very narrow substrate specificity, indicating that they are typical esterases. The β-lactamase activity of EstM-N1 and EstM-N2 was also detected and reached about 31% and 13% of the positive control enzyme, Bacillus cereus β-lactamase, respectively. These first cold-active esterases belonging to family VIII are expected to be useful for potential biotechnological applications as interesting biocatalysts.     

Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome,and its gene product

In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications.     

Identification and Characterization of Lipolytic Enzymes from a Peat-Swamp Forest Soil Metagenome

In this work, a metagenomic library was generated from peat-swamp forest soil obtained from Narathiwat Province, Thailand. From a fosmid library of approximately 15,000 clones, six independent clones were found to possess lipolytic activity at acidic pH. Analysis of pyrosequencing data revealed six ORFs, which exhibited 34–71% protein similarity to known lipases/esterases. A fosmid clone, designated LP8, which demonstrated the highest level of lipolytic activity under acidic conditions and demonstrated extracellular activity, was subsequently subcloned and sequenced. The full-length lipase/esterase gene, estPS2, was identified. Its deduced amino acid was closely related to a lipolytic enzyme of an uncultured bacterium, and contained the highly conserved motif of a hormone-sensitive family IV lipase. The EstPS2 enzyme exhibited highest activity toward p-nitrophenyl butyrate (C₄) at 37 °C at pH 5, indicating that it was an esterase with activity and secretion characteristics suitable for commercial development.     

Cloning,expression and characterization of a halotolerant esterase from a marine bacterium <Emphasis Type="Italic">Pelagibacterium halotolerans</Emphasis> B2<Superscript>T</Superscript>

An esterase PE10 (279 aa) from Pelagibacterium halotolerans B2^T was cloned and overexpressed in Escherichia coli Rosetta in a soluble form. The deduced protein was 29.91 kDa and the phylogenetic analysis of the deduced amino acids sequence showed it represented a new family of lipolytic enzymes. The recombinant protein was purified by Ni–NTA affinity chromatography column and the characterization showed its optimal temperature and pH were 45 °C and pH 7.5, respectively. Substrate specificity study showed PE10 preferred short chain p-nitrophenyl esters and exhibited maximum activity toward p-nitrophenyl acetate. In addition, PE10 was a halotolerant esterase as it was still active under 4 M NaCl. Three-dimensional modeling of PE10 suggested that the high negative electrostatic potential on the surface may relevant to its tolerance to high salt environment. With this halotolerance property, PE10 could be a candidate for industrial use.     

A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

Background

Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities.

Methodology/Principal Findings

Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220.

Conclusion

EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.     

Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon

We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (est(Pc)) from strain VA1. est(Pc) consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As est(Pc) showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90 degrees C), but also at ambient temperature (1,050 U/mg at 30 degrees C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p-nitrophenyl caproate (C(6)) the best substrate among the p-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.     

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. GMD509

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.     

Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119

More than 100 bacterial strains were isolated from composted polyester films and categorized into two groups, Actinomycetes (four genera) and Bacillus (three genera). Of these isolates, Thermobifida alba strain AHK119 (AB298783) was shown to possess the ability to significantly degrade aliphatic-aromatic copolyester film as well as decreasing the polymer particle sizes when grown at 50°C on LB medium supplemented with polymer particles, yielding terephthalic acid. The esterase gene (est119, 903 bp, encoding a signal peptide and a mature protein of 34 and 266 amino acids, respectively) was cloned from AHK119. The Est119 sequence contains a conserved lipase box (–G-X-S-X-G-) and a catalytic triad (Ser129, His207, and Asp175). Furthermore, Tyr59 and Met130 likely form an oxyanion hole. The recombinant enzyme was purified from cell-free extracts of Escherichia coli Rosetta-gami B (DE3) harboring pQE80L-est119. The enzyme is a monomeric protein of ca. 30 kDa, which is active from 20°C to 75°C (with an optimal range of 45 to 55°C) and in a pH range of 5.5 to 7.0 (with an optimal pH of 6.0). Its preferred substrate among the p-nitrophenyl acyl esters (C₂ to C₈) is p-nitrophenyl hexanoate (C₆), indicating that the enzyme is an esterase rather than a lipase.