Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia

AIMS: To screen and clone a novel enzyme with specific activity for the resolution of (R)-beta-acetylmercaptoisobutyrate (RAM) from (R,S)-beta-acetylmercaptoisobutyrate [(R,S)-ester]. METHODS AND RESULTS: A micro-organism that produces a novel esterase was isolated and identified as the bacterium Burkholderia cepacia by using the analysis of cellular fatty acids, Biolog automated microbial identification/characterization system, and 16S rRNA gene sequence analysis. A novel esterase gene was cloned from the chromosomal DNA of B. cepacia and was designated as cpoA. The cpoA encodes a polypeptide of 273 amino acids which shows a strong sequence homology with many bacterial nonhaeme chloroperoxidases. In addition, a typical serine-hydrolase motif, Gly-X-Ser-X-Gly, and the highly conserved catalytic triad, Ser95, Asp224, and His253, were identified in the deduced amino acid sequence of cpoA by multiple sequence alignment. CONCLUSION: The cpoA cloned from B. cepacia encodes a novel esterase which is highly related to the nonhaeme chloroperoxidases. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes the isolation and cloning of a serine esterase gene from B. cepacia, which is useful in the chiral resolution of (R,S)-ester. The cloned gene will allow additional research on the bifunctionality of the enzyme with esterase and chloroperoxidase activity at the structural and functional levels.     

A feruloyl esterase derived from a leachate metagenome library

A feruloyl esterase encoding gene (designated fae6), derived from a leachate metagenomic library, was cloned and the nucleotide sequence of the insert DNA determined. Translational analysis revealed that fae6 consists of a 515 amino acid polypeptide, encoding a 55 kDa pre-protein. The Fae6 primary structure contained the G-E-S-A-G sequence, which corresponds well with a typical catalytic serine sequence motif (G-x-S-x-G). The fae6 gene was successfully over-expressed in E. coli and the recombinant protein was purified to 8.4 fold enrichment with 17% recovery. The K(M) data showed Fae6 has a high affinity to methyl sinapate while thermostability data indicated that fae6 was thermolabile with a half life (T(1/2)) < 30 min at 50°C. High affinity for Fae6 against methyl sinapate, methyl ferulate and ethyl ferulate suggest that the enzyme can be useful in hydrolyzing ferulated polysaccharides in a biorefinery process.     

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite(Coptotermes formosanus) gut

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and 37oC, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.     

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.     

Enzymatic synthesis and characterization of novel feruloylated lipids in selected organic media

A facile enzymatic synthesis approach to prepare novel feruloylated lipids through the lipase-catalyzed transesterification reaction of ethyl ferulate (EF) with tributyrin (TB) in toluene was investigated. The nuclear magnetic resonance (NMR) and electrospray ionization-mass spectroscopy (ESI-MS) analysis confirmed the formation of two major products, 1(3)-feruloyl-monobutyryl-glycerol (FMB) and 1(3)-feruloyl-dibutyryl-glycerol (FDB). The influences of different enzyme source, organic solvent, molar ratio, reaction temperature, agitation speed and water activity on the total conversion of reaction, distribution of FMB and FDB and selectivity of these two novel derivatives of FA were analyzed systematically. Under the optimal conditions, the highest conversion of feruloylated lipids achieved was 73.6%, which was composed of FMB 58.3% and FDB 13.1%, respectively. The enzyme can be reused for 14 runs without evident loss in activity and stability.     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL “Amano” from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50°C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25°C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.     

Characteristics of batch culture of a recombinant bacillus brevis excreting a foreign esterase

The productivity of extracellular enzyme was evaluated in batch culture using a protein hyperexcreting host, Bacillus brevis HPD31(5) harboring pHSC131, which carried a gene (est) encoding esterase activity from Bacillus stearother mophilus. Optimum temperature and pH for the bacterial growth and the production of extracellular esterase were found to be 35 degrees C and pH 6.5, by using the standard medium (GPY) containing neomycin as a selective pressure, Under the cultivation condition employed, cell growth reached 5 g dry cell weight/L, while the extracellular esterase activity amounted to 4.5 U/mL. Most (79%-92%) of the esterase produced was excreted into the medium. pHSC131 was stably retained in the host cell during cultivation in the presence of neomycin. However, in the absence of neomycin, the plasmid was completely lost from the host after 12-h cultivation accompanied by decreases in both esterase activity and production of total extracellular protein. The copy number of the plasmid was estimated to be approximately 7 throughout the cultivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the excreted proteins showed the presence of a protein having an apparent molecular weight of 32,000, which equals to the value predicted from the DNA sequence of the est gene.     

Application of Two Newly Identified and Characterized Feruloyl Esterases from Streptomyces sp. in the Enzymatic Production of Ferulic Acid from Agricultural Biomass

Ferulic acid (FA), a component of hemicellulose in plant cell walls, is a phenolic acid with several potential applications based on its antioxidant properties. Recent studies have shown that feruloyl esterase (FAE) is a key bacterial enzyme involved in FA production from agricultural biomass. In this study, we screened a library of 43 esterases from Streptomyces species and identified two enzymes, R18 and R43, that have FAE activity toward ethyl ferulate. In addition, we characterized their enzyme properties in detail. R18 and R43 showed esterase activity toward other hydroxycinnamic acid esters as well, such as methyl p-coumarate, methyl caffeate, and methyl sinapinate. The amino acid sequences of R18 and R43 were neither similar to each other, nor to other FAEs. We found that R18 and R43 individually showed the ability to produce FA from corn bran; however, combination with other Streptomyces enzymes, namely xylanase and α-l-arabinofuranosidase, increased FA production from biomass such as corn bran, defatted rice bran, and wheat bran. These results suggest that R18 and R43 are effective FAEs for the enzymatic production of FA from biomass.     

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.     

Burkholderia cepacia胆固醇酯酶及其分子伴侣基因的串联表达及酶学性质的研究

为实现胆固醇酯酶的可溶性表达,以Burkholderia cepacia ST-200胆固醇酯酶为研究对象,将酶及其分子伴侣通过人工添加的SD序列在大肠杆菌中进行串联表达。通过SDS-PAGE电泳分析,胆固醇酯酶成功获得可溶性表达,摇瓶水平的总酶量为1 077 U/L。经过镍柱纯化之后可以获得一条相对分子量约为37 kDa的单一条带。利用圆二色谱解析胆固醇酯酶的二级结构,并测定Tm值,从最适温度、最适pH、热稳定性、pH稳定性及有机溶剂耐受性等方面对胆固醇酯酶进行研究,结果表明该酶在pH为7.0,45℃条件下表现出最大活力。     

Expression and biochemical characterization of two novel feruloyl esterases derived from fecal samples of Rusa unicolor and Equus burchelli

Two novel genes (tvms10a, tvmz2a) were identified in the metagenomic DNA of Rusa unicolor and Equus burchelli fecal samples. The amplified PCR product of tvms10a is composed of 917bp and the gene was found to encode a protein containing 165 amino acids, while the tvmz2a PCR product was 1053bp long encoding 298 amino acid proteins. The gene has 72% primary sequence identity with Clostridiales sp. These amplified PCR products which can encode FAE were cloned into pGEMT Easy TA cloning vector and then sub-cloned into the EcoRI site of pET32a expression vector to generate pET32-tvms10a and pET32-tvmz2a, which was then transformed into Escherichia coli BL21. The recombinants were grown in LB medium and gene expression was induced with IPTG for 6h. Purified recombinant Tvms10a and Tvmz2a proteins showed molecular masses of 18.6 and 31.2kDa respectively, and displayed hydrolytic activity towards substrate ethyl ferulate. The activities of Tvms10a and Tvmz2a produced in E. coli were 15 and 9U/min respectively, and their specific activities 16.6 and 10.4U/mg protein respectively. The optimal pH is between 5.0 and 8.0 and the optimal temperature is 37°C for enzyme reaction. Unusually, these proteins were found to be capable of releasing ferulic acid (FA) and diferulic acid (diFA) from untreated crude plant cell wall materials. The substrate utilization preferences and sequence similarity of these clones place it in the type-D sub-class of FAE.     

Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomea

Aims: A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 ‐bp open reading frame encoding 314 amino acids with a molecular weight of 35·5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser–His–Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C‐terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long‐chain acyl esterases at pH 7 and 30°C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4°C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry. Conclusion: We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities. Significance and Impact of the Study: This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level.     

Decreasing the level of ethyl acetate in ethanolic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter(-1). Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using alpha-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40 degrees C. The Km and Vmax for alpha-naphthyl acetate were 18 microM and 48.1 micromol. min(-1). mg of protein(-1), respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 micromol. min(-1). mg of protein(-1)), followed by ethyl acetate (66 micromol. min(-1). mg of protein(-1)). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter(-1).     

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.     

Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidium pullulans

The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60 degrees C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 micro mol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-[5-O-[(E)-feruloyl]-alpha-L-arabinofuranosyl]-(1,3)-O-beta-D-xylopyranosyl-(1,4)-D-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (k(cat)/K(m)) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.     

A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to beta-lactamases and DD-peptidases

The gene (estB) encoding for a novel esterase (EstB) from Burkholderia gladioli (formerly Pseudomonas marginata) NCPPB 1891 was cloned in Escherichia coli. Sequence analysis showed an open reading frame encoding a polypeptide of 392 amino acid residues, with a molecular mass of about 42 kDa. Comparison of the amino acid sequence with those of other homologous enzymes indicated homologies to beta-lactamases, penicillin binding proteins and DD-peptidases. The serine residue (Ser(75)) which is located within a present class A beta-lactamase motif ([F,Y]-X-[L,I,V,M,F,Y]-X-S-[T,V]-X-K-X-X-X-X-[A,G,L]-X-X-[L,C]) was identified by site-directed mutagenesis to represent the active nucleophile. A second serine residue (Ser(149)) which is located within a G-x-S-x-G motif which is typically found in esterases and lipases was demonstrated not to play a significant role in enzyme function. The estB gene was overexpressed in E. coli using a tac promoter-based expression system. Investigation of EstB protein with respect to the ability to hydrolyse beta-lactam substrates clearly demonstrated that this protein has no beta-lactamase activity. The recombinant enzyme is active on triglycerides and on nitrophenyl esters with acyl chain lengths up to C6. The preference for short chain length substrates indicated that EstB is a typical carboxylesterase. As a special feature EstB esterase was found to have high deacetylation activity on cephalosporin derivatives.     

Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications

Aims:  The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications.
Methods and Results:  The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6·5 and at 50–60°C. Furthermore, EstA remained stable at pH 6–8 and below 50°C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP).
Conclusion:  The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P.   equi was demonstrated to efficiently release FA from various natural substrates.
Significance and Impact of the Study:  Recombinant EstA produced in an industrial enzyme producer, T. reesei , was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.     

Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile

The cellulolytic system of the thermophilic fungus Sporotrichum thermophile contains a recently discovered esterase that may hydrolyze the ester linkage between the 4- O -methyl- d -glucuronic acid of glucuronoxylan and lignin alcohols. The glucuronoyl esterase named St GE1 was purified to homogeneity with a molecular mass of M _r 58 kDa and pI 6.7. The enzyme activity was optimal at pH 6.0 and 60 °C. The esterase displayed a narrow pH range stability at pH 8.0 and retained 50% of its activity after 430 and 286 min at 50 and 55 °C, respectively. The enzyme was active on substrates containing glucuronic acid methyl ester, showing a lower catalytic efficiency on 4-nitrophenyl 2- O -(methyl-4- O -methyl-α- d -glucopyranosyluronate)-β- d -xylopyranoside than its mesophilic counterparts reported in the literature, which is typical of thermophilic enzymes. St GE1 was proved to be a modular enzyme containing a noncatalytic carbohydrate-binding module. LC-MS/MS analysis provided peptide mass and sequence information that facilitated the identification and classification of St GE1 as a family 15 glucuronoyl esterase that showed the highest homology with the hypothetical glucuronoyl esterase CHGG_10774 of Chaetomium globosum CBS 148.51. This work represents the first example of the purification and identification of a thermophilic glucuronoyl esterase from S. thermophile .