A novel recombinant ethyl ferulate esterase from Burkholderia multivorans

AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND RESULTS: A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column. CONCLUSION: Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF). SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.     

Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima

A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4-C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 degrees C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 degrees C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family.     

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library

Aims:  The aim of this study was to demonstrate the application of environmental sample pre-enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s).
Methods and Results:  A positive recombinant clone (UVCL29), conferring an esterase phenotype was identified from a shotgun gene library. The complete sequence of the 3·0 kb DNA insert from the pUVCL29 recombinant plasmid was obtained using primer-walking strategies. Nucleotide sequence analysis revealed a complete 945 bp open reading frame (ORF1). Translational analysis of the ORF1 showed a protein of 314 amino acids (named EstAM) with a predicted molecular weight of 34 kDa. EstAM's primary structure showed a classical (–G–D–S–A–G–) motif, corresponding with the generally conserved (G–x–S–x–G) esterase signature motif. Identity searches indicated that EstAM has high sequence similarity with esterases from family IV. EstAM was successfully expressed in Escherichia coli in a biologically active form. Partial purification was achieved using a one-step Pro-PurTM IMAC column. Biochemical characterization revealed that EstAM has a temperature optimum of 40°C.
Conclusion:  Based on its substrate profile, EstAM was classified as a carboxylesterase because of its preference for short p -nitrophenyl ester substrates.
Significance and Impact of the Study:  This study is a demonstration of the successful application of environmental sample pre-enrichment technology in accessing novel esterases from a mining environment.     

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.     

Molecular cloning,over expression and characterization of thermoalkalophilic esterases isolated from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E.coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27.5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC₂) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5–10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.     

Screening and identification of a novel esterase EstPE from a metagenomic DNA library

Esterases represent a large family of hydrolases with broad substrate specificity and functional sequence space. Although many attempts to screen new esterases have been conducted, there have been few reports conducted to discriminate unique enzymes from typical ones based on novel structure and function. In this study, we discovered an esterase and a novel family through a successive assay of whole cells and crude lysates (oxidative open condition). The screened putative esterases from the metagenomic DNA of salted shrimp consisted of 753 bp encoding 27 kDa of polypeptide, namely PE esterase. Sequence analyses revealed that an identical gene was reported from whole genome sequencing of Stenotrophomonas maltophilia K279a. However, its biochemical and phylogenetic characteristics have not yet been evaluated. PE esterase was overexpressed only by the MBP fusion state in E. coli and was easily purified using an affinity column. This enzyme showed a typical spectrum of substrate specificity and possessed the consensus motifs, Ser-Asp-His and GXSXG, which are essential for most esterase/lipase superfamilies. Interestingly, the entire organization of the ORF and consensus sequence around the active site were distinct from the related enzymes, and its structure could be affected by a reducing agent, DTT.     

The Serratia marcescens bioH gene encodes an esterase

The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates. Three complete open reading frames (ORFs) were identified in the inserted DNA, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity. Interestingly, the ORF was 70% identical to a product of the E. coli bioH gene, which lies at a locus separated from the bioABFCD operon and acts in the early steps of the biotin synthetic pathway before pimeloyl-CoA synthesis. This gene complemented a bioH-deficient mutation of E. coli. From the sequence analysis, BioH is presumed to be a serine hydrolase, which belongs to the alpha/beta hydrolase-fold family comprising a wide variety of hydrolases including esterases. A catalytic triad composed of a nucleophilic residue (Ser80), an acidic residue (Asp206), and histidine (His234) was conserved in BioH, and the nucleophilic residue Ser, a catalytic center, was situated in the consensus sequence of G-X-S-X-G-G, a nucleophile elbow. Although the enzymatic function of BioH is not yet elucidated, the bioH gene products from S. marcescens and E. coli show esterase activity, which may imply the hydrolysis of a precursor leading to pimeloyl-CoA ester. The esterase activity of BioH and its CoA binding activity recently reported agree with a current hypothesis of pimeloyl-CoA ester synthesis from CoA and acylester derivatives including an acyl-carrier protein.     

Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when α‐naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub‐cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)‐derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional‐genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut. © 2009 Wiley Periodicals, Inc.     

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40°C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.     

A new esterase, belonging to hormone-sensitive lipase family, cloned from Rheinheimera sp. isolated from industrial effluent

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to class IV family. The purified enzyme worked optimally at 50 degrees C and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate (C?), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.     

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.     

A method for the estimation of esterase synthesis and degradation and its application to evaluate the influence of insulin and glucagon.

The irreversible reaction between liver esterases and the active-site-directed inhibitor bis(4-nitrophenyl)phosphate can be used in vivo both for the estimation of the esterase contents and for the measurement of the esterase degradation rates. A method based on this reaction is described which allows the simultaneous estimation of the rate constants of degradation and synthesis of esterases during a period of change in protein concentration. Rat liver was found to contain about 1 mg of organophosphate-binding esterases per g of fresh tissue while the microsomal fraction contains about 30 mg of esterases per g of microsomal protein. Esterase degradation and de novo synthesis were shown to remain in equilibrium for a period of at least five days following the injection of 10 mg bis(4-nitro-[14C]phenyl)phosphate per kg. The decrease of the relative amount of labeled esterases with time was found to follow first-order kinetics yielding an average esterase degrading constant of 0.0165 h-1 which corresponds to a half-life of 42 h. These data were confirmed by an independent experiment using one of the standard procedures for the estimation of degradation rates: [14C]leucine was incorporated and one of the esterases was subsequently isolated by immuno-precipitation. Using isoelectric focussing and dodecyl sulfate electrophoretic methods, the various esterase isoenzymes appeared to have very similar, if not identical turnover rates. This method for the estimation of the turnover characteristics was applied to evaluate hormone effects on liver esterases. The time course of the contents and the turnover of liver esterases was measured under the influence of glucagon treatment in diabetic rats and under the influence of high doses of insulin. The esterase content decreased faster than the average content of microsomal protein under the influence of glucagon. The reverse effect was observed with insulin-treated rats. Both insulin and glucagon apparently reduced the intracellular esterase turnover in rat liver. Kinetic analysis of the results revealed that insulin mainly lowered the esterase degradation rate, though the rate of esterase synthesis might also have been restricted. In the glucagon-treated rats the de novo synthesis of esterases was strongly reduced.     

Cloning and characterization of thermostable esterase from <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa. It also showed repeating units G-X-S-X-G (GHSLG) (residues 86 approximately 90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Asp198 and His226. The esterase activity test with various acyl chain length of rho-nitrophenol resulted that Est-AF showed highest specific activity with rho-nitrophenylbutyrate (pNPC4) and rapidly decrease with rho-nitrophenyl ester contain more than 8 carbon chain. These results represent that cloned enzyme is verified as a carboxylesterase but not a lipase because esterase activity is decreased with rho-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with rho-nitrophenylbutyrate (pNPC4) was 80 degrees C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70 degrees C, and 10% of activity still remains after 3 h of incubation at 90 degrees C. This result represents Est-AF has high thermostability with comparison of other esterases that have been reported. However, Est-AF showed low enantioselectivity with ketoprofen ethyl ester. Optimum pH of Est-AF is between pH 7.0 and pH 8.0. Km value of ketoprofen ethyl ester is 1.6 mM and, Vmax is 1.7 micromole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.     

SSoNΔ and SSoNΔlong: two thermostable esterases from the same ORF in the archaeon Sulfolobus solfataricus?

Previously, we reported from the Sulfolobus solfataricus open reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we named SSoNDelta. Searching the recently reported Sulfolobus acidocaldarius genome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, named SSoNDeltalong, was 15-fold more active with the substrate p-nitrophenyl hexanoate than SSoNDelta. Furthermore, SSoNDeltalong and SSoNDelta displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship between S. solfataricus and S. acidocaldarius suggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression of SSoNDeltalong under specific metabolic conditions could be hypothesized.     

Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea

A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.     

Purification and molecular properties of rabbit liver esterase ES-1A

1. The isolation of esterase ES-1A from rabbit liver microsomes/lysosomes is reported. The purification as measured by methylbutyrate-hydrolysing activity, was about 27-fold with a recovery of 2.4%. 2. The resulting product is apparently homogeneous by polyacrylamide (gradient) gel electrophoresis and sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis after protein staining. The enzyme exhibits heterogeneity after staining for esterase activity and in isoelectric focusing. 3. The molecular mass of the native protein was found to be about 183 kDa (determined by gel filtration and polyacrylamide gel electrophoresis) with a subunit mass of about 63 kDa, indicating a trimeric structure of the enzyme, with subunits of equal size. 4. ES-1A is a glycoprotein and is classified as a carboxylesterase (EC 3.1.1.1). 5. The high degree of similarity of the properties of rabbit ES-1A with those of mouse ES-6A and rat ES-10 suggests that these three esterases may have a common evolutionary origin.     

The feruloyl esterase system of Talaromyces stipitatus: production of three discrete feruloyl esterases, including a novel enzyme, TsFaeC, with a broad substrate specificity

Several extracellular feruloyl esterases were produced by the mesophilic fungus Talaromyces stipitatus when grown on selective carbon sources in liquid media. Type-A and Type-B feruloyl esterases, as defined by their substrate specificity against methyl hydroxycinnamates, were produced during growth on wheat bran and sugar beet pulp, respectively. In addition, Tal. stipitatus produced a new type of esterase (TsFaeC) during growth on sugar beet pulp with a broader spectrum of activity (Type-C) against the (hydroxy)cinnamate esters than those previously described. All three enzymes were purified and N-terminal amino acid sequences and internal peptide sequences determined. The TsFaeC sequences were used to amplify a gene fragment from Tal. stipitatus genomic DNA. The flanking sequences were identified with the aid of RACE-RTPCR, and a full-length clone constructed. The faeC gene is present as a single copy and contains a single intron. The complete cDNA fragment contains an ORF of 1590bp, faeC, which is predicted to encode a 530 amino acid pre-protein, including a 25-residue signal peptide, and to produce a mature protein of M(R) 55 340Da. There was no evidence for a carbohydrate-binding domain in TsFaeC.