A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36G37, a conserved pentapeptide G103HSLG107 and a conserved catalytic His265 and Asp237. The EstR sequence shared 64-70 and 44-48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees C and pH range 7.5-9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.     

Molecular cloning and characterization of a novel acetylalginate esterase gene in alg operon from Sphingomonas sp. MJ-3

A novel acetylalginate esterase (AcAlgE) gene was cloned and characterized from the genomic DNA library of Sphingomonas sp. MJ-3. A putative gene encoding AcAlgE protein of 292-residue precursor protein with 20-amino acid signal peptide was identified in the alg operon. The deduced AcAlgE protein has GDSL-like consensus motif and shares a highest sequence identity (51 %) with GDSL family lipolytic protein from Pseudoxanthomonas suwonensis. Enzymatic assays with bacterial acetylalginate as the substrate showed that the recombinant AcAlgE protein possesses deacetylation activity. The optimal temperature and pH for the AcAlgE were 22 °C and pH 6.5 (citrate buffer), respectively. The recombinant AcAlgE protein catalyzed deacetylation of acetylalginate with release of acetate. The resulting de-acetylated alginate was readily degraded by alginate lyases, indicating that the recombinant AcAlgE enhanced the subsequent degradation of acetylalginate by alginate lyases. The recombinant AcAlgE can play an important role in the degradation of acetylated alginate such as mucoidal acetylalginate in cystic fibrosis patient.     

Mining bacterial genomes for novel arylesterase activity

One hundred and seventy-one genes encoding potential esterases from 11 bacterial genomes were cloned and overexpressed in Escherichia coli; 74 of the clones produced soluble proteins. All 74 soluble proteins were purified and screened for esterase activity; 36 proteins showed carboxyl esterase activity on short-chain esters, 17 demonstrated arylesterase activity, while 38 proteins did not exhibit any activity towards the test substrates. Esterases from Rhodopseudomonas palustris (RpEST-1, RpEST-2 and RpEST-3), Pseudomonas putida (PpEST-1, PpEST-2 and PpEST-3), Pseudomonas aeruginosa (PaEST-1) and Streptomyces avermitilis (SavEST-1) were selected for detailed biochemical characterization. All of the enzymes showed optimal activity at neutral or alkaline pH, and the half-life of each enzyme at 50°C ranged from < 5 min to over 5 h. PpEST-3, RpEST-1 and RpEST-2 demonstrated the highest specific activity with pNP-esters; these enzymes were also among the most stable at 50°C and in the presence of detergents, polar and non-polar organic solvents, and imidazolium ionic liquids. Accordingly, these enzymes are particularly interesting targets for subsequent application trials. Finally, biochemical and bioinformatic analyses were compared to reveal sequence features that could be correlated to enzymes with arylesterase activity, facilitating subsequent searches for new esterases in microbial genome sequences.     

Structural and Functional Characterisation of TesA - A Novel Lysophospholipase A from Pseudomonas aeruginosa

TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.     

Identification and Characterization of Three Novel Lipases Belonging to Families II and V from Anaerovibrio lipolyticus 5ST

Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14).     

Genomic architecture of three newly isolated unclassified Butyrivibrio species elucidate their potential role in the rumen ecosystem

One cellulose-degrading strain CB08 and two xylan-degrading strains XB500–5 and X503 were isolated from buffalo rumen. All the strains were designated as putative novel species of Butyrivibrio based on phylogeny, phylogenomy, digital DNA-DNA hybridization, and average nucleotide identity with their closest type strains. The draft genome length of CB08 was ~3.54 Mb, while X503 and XB500-5 genome sizes were ~3.24 Mb and ~3.27 Mb, respectively. Only 68.28% of total orthologous clusters were shared among three genomes, and 40–44% of genes were identified as hypothetical proteins. The presence of genes encoding diverse carbohydrate-active enzymes (CAZymes) exhibited the lignocellulolytic potential of these strains. Further, the genome annotations revealed the metabolic pathways for monosaccharide fermentation to acetate, butyrate, lactate, ethanol, and hydrogen. The presence of genes for chemotaxis, antibiotic resistance, antimicrobial activity, synthesis of vitamins, and essential fatty acid suggested the versatile metabolic nature of these Butyrivibrio strains in the rumen environment.     

An acid and highly thermostable xylanase from <Emphasis Type="Italic">Phialophora</Emphasis> sp. G5

An endo-β-1,4-xylanase gene, designated xyn10G5, was cloned from Phialophora sp. G5 and expressed in Pichia pastoris. The 1,197-bp full-length gene encodes a polypeptide of 399 amino acids consisting of a putative signal peptide at residues 1–20, a family 10 glycoside hydrolase domain, a short Gly/Thr-rich linker and a family 1 carbohydrate-binding module (CBM). The deduced amino acid sequence of XYN10G5 shares the highest identity (53.4%) with a putative xylanase precursor from Aspergillus terreus NIH2624. The purified recombinant XYN10G5 exhibited the optimal activity at pH 4.0 and 70 °C, remained stable at pH 3.0–9.0 (>70% of the maximal activity), and was highly thermostable at 70 °C (retaining ~90% of the initial activity for 1 h). Substrate specificity studies have shown that XYN10G5 had the highest activity on soluble wheat arabinoxylan (350.6 U mg⁻¹), and moderate activity to various heteroxylans, and low activity on different types of cellulosic substrates. Under simulated gastric conditions, XYN10G5 was stable and released more reducing sugars from soluble wheat arabinoxylan; when combined with a glucanase (CelA4), the viscosity of barley–soybean feed was significantly reduced. These favorable enzymatic properties make XYN10G5 a good candidate for application in the animal feed industry.     

Characterization and Expression of a GDSL-Like Lipase Gene from Brassica napus in Nicotiana benthamiana

The GDSL esterase and lipase families play important roles in abiotic stress, pathogen defense, seed development and lipid metabolism. Identifying the lipase activity of the putative GDSL lipase is the prerequisite for dissecting its function. According to the sequence similarity and the conserved domains, we cloned the Brassica napus BnGLIP gene, which encodes a GDSL-like protein. We failed to identify the BnGLIP lipase activity in the bacterium and yeast expression systems. In this paper, we expressed the BnGLIP gene by fusing a 6× His tag in Nicotiana benthamiana and purified the recombinant protein. The extraction buffer contained 1 % (v/v) n-caprylic acid and was able to remove most of the protein impurities. About 50 μg of recombinant BnGLIP was obtained from 1 g of N. benthamiana leaves. The lipase activity was tested with the purified BnGLIP and the maximum enzyme activity reached 17.7 mM/mg. In conclusion, this study found that the recombinant protein BnGLIP expressed in tobacco system was effectively purified and was detected as a GDSL lipase.     

Isolation of a gene encoding endoglucanase activity from uncultured microorganisms in buffalo rumen

A gene, umcel5N, was isolated from a metagenomic library constructed from the contents of buffalo rumen. Its putative product belongs to the glycosyl hydrolase family 5 and is most closely related to an endoglucanase (ABN54006.1) from Clostridium thermocellum with 44% identity and 60% similarity. Gene umcel5N was heterologously expressed in Escherichia coli. The purified recombinant Umcel5N hydrolyzed carboxymethyl cellulose with a rapid decrease in the viscosity of the solution but with little release of reducing sugars, suggesting an endo mode of action. The enzyme exhibited optimal activity toward p-nitrophenyl β-d-cellobioside at pH 5.5 and 55°C, and had a Km of 1.56 mM and a Vmax of 285.6 U/mg. Two glutamic acids (E144 and E285) of the wild-type Umcel5N were predicted as a proton donor and a nucleophile, respectively. Site-directed mutagenesis confirmed that they were required for the enzyme’s activity.     

Cloning,expression, and characterization of serine protease from thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.     

Cloning,expression and characterization of a new lipase from <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Bioinformatic analysis of the Yarrowia lipolytica CLIB122 genome has revealed 18 putative lipase genes all of which were expressed in Escherichia coli and screened for hydrolyzing activities against p-nitrophenyl-palmitate. One positive transformant containing an ORF of 1,098 bp encoding a protein of 365 amino acids was obtained. To characterize its enzymatic properties, the lipase gene was functionally expressed in Pichia pastoris. The resulting lipase exhibited the highest activity towards p-NP-decanoate at pH 7 and 35°C. In addition, the new lipase had a lower optimal temperature and pH compared to other Y. lipolytica lipases. It was noticeably enhanced by Ca²⁺, but was inhibited by PMSF, Hg²⁺ and Ni²⁺. The new lipase displayed the 1,3-specificity for triolein.     

Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol

The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.     

A new xylanase from Streptomyces megasporus DSM 41476 with high yield of xylobiose

A new xylanase gene, xynBM4, was cloned from Streptomyces megasporus DSM 41476 and expressed in Pichia pastoris. The full-length gene consists of 1,443 bp and encodes 480 amino acids including a putative 49-residue signal peptide. The deduced amino acid sequence of xynBM4 shows the highest identity of 66.3% to the xylanase Xys1L from Streptomyces halstedii JM8. The purified recombinant XYNBM4 had a high specific activity of 350.7 U mg^-1 towards soluble wheat arabinoxylan, exhibited optimal activity at pH 6.0 and 57°C, showed broad pH adaptability (>75% of the maximum activity at pH 2.5–9.0), was resistant to neutral proteases and most chemicals, and produced simple products. The hydrolysis products of birchwood xylan and corncob xylan were predominantly xylobiose (76.9 and 90.8%, respectively) and no xylose. These characteristics suggest that XYNBM4 has potential in various applications, especially in the food industry.     

Cloning of a Gene Encoding Acetate Kinase from Methanosarcina mazei 2-P Isolated from a Japanese Paddy Field Soil

The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His₆-tagged acetate kinase purified from E. coli JM109 was optimal at 35°C. Received: 28 January 2002 / Accepted: 27 February 2002     

4株羊瘤胃功能性细菌的筛选

通过DNS法测定羊瘤胃源功能性细菌产生的纤维素酶和淀粉酶的活力,福林酚法测定产生的蛋白酶的活力,检测细菌产生酶的特性。同时检测菌株的发酵液对大肠埃希菌(ATCC25922)、副溶血弧菌(ATCC17802)、藤黄八叠球菌(HY78)和产气杆菌(AS1489)等指示菌的抑制能力,分析它们的抑菌活性。结果表明,羊瘤胃源细菌C13产生的纤维素酶活力最高,产酶量也最高;而细菌C5产淀粉酶活力和蛋白酶活力最高,产生淀粉酶和蛋白酶的能力也最高。抑菌活性检测发现,细菌C9对副溶血弧菌(ATCC17802)有很高的抑制作用,而细菌C12对大肠埃希菌(ATCC25922)的抑制能力最明显。     

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.     

Syntrophococcus sucromutans sp. nov. gen. nov. uses carbohydrates as electron donors and formate,methoxymonobenzenoids or Methanobrevibacter as electron acceptor systems

Most probable number (MPN) estimates indicated that a mean of 4.3×10⁷ and 5×10⁶ bacteria per ml of rumen fluid from a predominantly alfalfa hay-fed steer demethoxylated ferulate and syringate, respectively. After further enrichment from an MPN tube of the highest dilution showing demethoxylation of syringate, strain S195 was isolated using roll tubes with syringate as an added energy source. S195 was an anaerobic, Gram-negative, nonmotile coccus, 1 to 1.3 m in diameter, and was unique in using various carbohydrates as electron donor with acetate as the sole organic product. One of the following electron acceptor systems allowed growth (organic products in parentheses): Methanobrevibacter simithii (CH₄), formate (acetate), 3,4,5-trimethoxybenzoate and syringate (acetate and gallate), vanillate (acetate and protocatechuate), vanillin (acetate, protocatechuic aldehyde and protocatechuate), ferulate (acetate, caffeate and hydrocaffeate), caffeate (hydrocaffeate). Strain S195 required 30% (v/v) rumen fluid in the medium for good growth. S195 was placed in a new genus and species, Syntrophococcus sucromutans, of the family Veillonellaceae.Abbreviations G+C Guanine plus cytosine - MPN most probable number - OD optical density     

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 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.     

Esterases of the ox adrenal: their histochemical and biochemical distribution

Summary Esterases were demonstrated in the ox adrenals histochemically. The substrates used were -naphthyl acetate, and naphthyl acetate AS on fresh and fixed tissue. The highest activity was in the zona glomerulosa, the adrenaline-storing cells and the ganglia. The other parenchymal cells showed a moderate activity. The cytological localization of the enzyme was both diffuse and particulate and highly polarized in the adrenaline-storing cells. The particulate form persisted after formaldehyde fixation. The following inhibitors were used to differentiate between the various esterases histochemically and biochemically: DFP, E600, eserine, p-chloromercuribenzoate and 62C47.The cortex, adrenaline storing- and noradrenaline-storing cells were separated by dissection. Suitable marker were used to assess contamination. Total esterase, carboxylesterase, arylesterase, acetylesterase, acetylcholinesterase and cholinesterase were assayed biochemically in conjunction with the inhibitors. Acetylesterase showed the highest activity and was fairly evenly distributed as was carboxylesterase, although at a lesser level of activity. Aryl esterase was more abundant in the cortex than the medulla. The reverse was found for acetylcholinesterase and at a lower level of activity cholinesterase. The results were compared with the histochemical findings.