Screening and characterization of a novel esterase from a metagenomic library

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.     

Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library

A metagenomic library was constructed using total genomic DNA extracted from the mud in the west coast of Korea and was used together with a fosmid vector, pCC1FOS in order to uncover novel gene sources. One clone from approximately 30,000 recombinant Escherichia coli clones was identified that showed proteolytic activity. The gene for the proteolytic enzyme was subcloned into pUC19 and sequenced, and a database search for homologies revealed it to be a zinc-dependent metalloprotease. The cloned gene included the intact coding gene for a novel metalloproteinase and its own promoter. It comprised an open reading frame of 1,080 base pairs, which encodes a protein of 39,490 Da consisting of 359 amino acid residues. A His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent metalloproteases, was found in the deduced amino acid sequence of the gene, suggesting that the enzyme is a zinc-dependent metalloprotease. The purified enzyme showed optimal activity at 50°C for 1 h and pH 7.0. The enzyme activity was inhibited by metal-chelating reagents, such as EDTA, EGTA and 1,10-phenanthroline. The enzyme hydrolyzed azocasein as well as fibrin. Thus, the enzyme could be useful as a therapeutic agent to treat thrombosis. The sequence reported in this paper has been deposited in the GenBank database (Accession number: EF100137).     

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.     

Characterization of a new and thermostable esterase from a metagenomic library

A new gene encoding an esterase (designated as EstEP16) was identified from a metagenomic library prepared from a sediment sample collected from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 249 amino acid residues. It was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified to homogeneity. The monomeric EstEP16 presented a molecular mass of 51.7 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. When p-nitrophenyl butyrate was used as a substrate, recombinant EstEP16 exhibited highest activity at pH 8.0 and 60 °C. The recombinant enzyme retained about 80% residual activity after incubation at 90 °C for 6 h, which indicated that EstEP16 was thermostable. Homology modeling of EstEP16 was developed with the monoacylglycerol lipase from Bacillus sp. H-257 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of a typical catalytic triad. The activity of EstEP16 was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism.     

Isolation and characterization of a thermostable esterase from a metagenomic library

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₁₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K _m and k _cat of 0.33 mM and 36.21 s^?1, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.     

Isolation and characterization of a novel organic solvent-tolerant and halotolerant esterase from a soil metagenomic library

Soil metagenome conceals a great variety of unexploited genes for industrially important enzymes. To identify novel genes conferring lipolytic activity, one metagenomic library comprising of 200,000 transformants were constructed. Among the 48,000 clones screened, 19 clones which exhibited lipolytic activity were obtained. After sequence analysis, 19 different lipolytic genes were identified. One of these genes, designated as estWSD, consisted of 1152 nucleotides, encoding a 383-amino-acid protein. Multiple sequence alignment and phylogenetic analysis indicated that EstWSD and its closest homologues may constitute a new family of bacterial lipolytic enzymes. The best substrate for the purified EstWSD among the ρ-nitrophenol esters examined was ρ-nitrophenol butyrate. Recombinant EstWSD displayed a pH optimum of 7.0 and a temperature optimum of 50 °С. This enzyme retained 52% of maximal activity after incubation at 50 °C for 3 h. Furthermore, EstWSD also exhibited salt tolerance with over 51% of its initial activity in the presence of up to 4.5 M NaCl for 1 h. In particular, this enzyme showed remarkable stability in 15% and 30% dimethylsulfoxide, ρ-xylene, hexane, heptane, and octane even after incubation for 72 h. To our knowledge, it is the first report to find a novel esterase belonging to a new lipolytic family and possessing such variety of excellent features. All these characteristics suggest that EstWSD may be a potential candidate for application in industrial processes.     

深海沉积物微生物元基因组文库来源的新的酯酶基因的克隆、表达及酶学性质

【目的】从深海沉积物微生物元基因组文库中克隆新的酯酶基因,并进行酶学性质研究。【方法】利用含有三丁酸甘油酯的酯酶选择性筛选培养基,从深海沉积物微生物元基因组文库中筛选得到酯酶阳性Fosmid克隆。对筛选得到的fosmid FL10进行部分酶切构建亚克隆文库,筛选得到酯酶阳性亚克隆pFLS10。PCR扩增目的片段后与pET28a连接构建酯酶基因原核表达质粒,转化大肠杆菌(Escherichia coli)BL21。纯化表达产物并对其进行活性测定及酶学性质研究。【结果】序列分析显示该pFLS10亚克隆质粒含有一段924bp的ORF(Open Reading Frame),与一海洋元基因组文库中筛选出的酯酶ADA70030序列一致性为71%。该酶为一新的低温酯酶,对C4底物(对硝基苯丁酸酯)水解能力最强。该酶最适作用温度为20℃,最适作用pH为7.5,20℃时较为稳定,pH8-10的范围内有良好的pH稳定性,K+、Mg2+对该酶具有一定的激活作用,Mn2+等对其具有不同程度的抑制作用。【结论】应用元基因组技术筛选到了新的酯酶基因fls10并进行了克隆表达,该酶在低温及碱性条件下较为稳定且活力较高,对于工业化生产具有一定的应用潜力。关键词:深海沉积物;元基因组文库;低温酯酶;酶学特征     

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.     

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.     

Molecular cloning, overexpression and characterization of a novel feruloyl esterase from a soil metagenomic library

The gene estF27, encoding a protein with feruloyl esterase activity, was cloned through functional screening from a soil metagenomic library and expressed in Escherichiacoli BL21 (DE3) with high solubility. Sequence analysis showed that estF27 encoded a protein of 291 amino acids with a predicted molecular mass of 31.16 kDa. According to the substrate specificity, EstF27 was classified as a type A feruloyl esterase. EstF27 displayed optimal activity at 40°C and pH 6.8. This enzyme was stable in a broad pH range of 5.0-10.0 over 24 h, and retained more than 50% of its activity after 96 or 120 h incubation in the presence of 3 M KCl or 5 M NaCl. The enzyme activity was slightly enhanced by the addition of Mg(2+) and Fe(3+) at a low concentration, and completely inhibited by Cu(2+). In the enzymatic hydrolysis of destarched wheat bran, EstF27 could release ferulic acid from it in the presence of xylanase from Thermomyces lanuginosus. Given its alkalitolerance, halotolerance and highly soluble expression, EstF27 is a promising candidate for industrial applications.     

土壤宏基因组文库来源酯酶的鉴定与表征

【目的】利用宏基因组学技术挖掘土壤微生物来源的新型酯酶。【方法】构建土壤微生物宏基因组文库,利用三丁酸甘油酯平板法对所构建的文库进行筛选,并对阳性克隆中鉴定出的酯酶基因进行异源表达和生物化学特性分析。【结果】通过筛选文库中的12万个克隆,获得了一个阳性克隆,对克隆中的DNA片段进行序列分析,发现了一个可能的酯酶基因,通过研究其表达产物,确定其最适pH为9.0,最适反应温度为56°C,在90°C下仍可保持20%的酶活性;能专一性水解短链脂类,对长链脂类无水解作用;对一定浓度范围内的有机试剂如二甲基亚砜、甲醇、乙醇有较好的耐受性,尤其当二甲基亚砜含量为10%(体积比)时,相对酶活可提高44%。【结论】不依赖于微生物可培养性的宏基因组学技术可以发现新的活性酶,本研究获得的对高温、有机试剂有较好耐受性的酯酶ESTYN1具有在工业生产中应用的潜力。     

Identification and characterization of a novel salt‐tolerant esterase from a Tibetan glacier metagenomic library

A salt‐tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p‐nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three‐dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat‐resistant features. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:890–899, 2015     

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.     

Isolation and characterization of a family VII esterase derived from alluvial soil metagenomic library

A novel esterase gene, estDL30, was isolated from an alluvial metagenomic library using function-driven screening. estDL30 consisted of 1,524 nucleotides and encoded a 507-amino acid protein. Sequence analysis revealed that EstDL30 is similar to many type B carboxylesterases, containing a G-E-S-A-G pentapeptide with a catalytic Ser residue. Phylogenetic analysis suggested that EstDL30 belongs to the family VII lipases, together with esterases from Bacillus subtilis (P37967), Streptomyces coelicolor A3(2) (CAA22794), and Arthrobacter oxydans (Q01470). Purified EstDL30 showed its highest catalytic efficiency toward p-nitrophenyl butyrate, with a k _cat of 2293 s⁻¹ and k _cat/K _m of 176.4 s⁻¹mM⁻¹; however, little activity was detected when the acyl chain length exceeded C₈. Biochemical characterization of EstDL30 revealed that it is an alkaline esterase that possesses maximal activity at pH 8 and 40° C. The effects of denaturants and divalent cations were also investigated. EstDL30 tolerated well the presence of methanol and Tween 20. Its activity was strongly inhibited by 1 mM Cu²⁺ and Zn²⁺, but stimulated by Fe²⁺. The unique properties of EstDL30, its high activity under alkaline conditions and stability in the presence of organic solvents, may render it applicable to organic synthesis.     

从植物共生菌宏基因组文库筛选新的生物催化酶基因

【目的】通过建立宏基因组文库的高通量保存与基于探针洗脱的多次膜杂交筛选方法,从植物共生菌宏基因组文库筛选具有生物催化潜力的新酶基因。【方法】首先根据滴度将初始文库噬菌体包装颗粒感染到EPI300-T1R E.coli,过夜培养后对应保存于96孔板;提取粘粒进行文库的杂交筛选。【结果】描述的洗脱条件可完全去除尼龙膜上与靶DNA结合的探针,并且尼龙膜上的靶DNA至少可用于7次探针杂交,从而明显提高宏基因组文库的筛选效率。【结论】以Enoate reductase(ER)和短链脱氢酶(SDR)的同源基因片段为探针,运用该方法经两轮筛选获得候选单克隆并进行了部分粘粒的测序,发现了新的ER和SDR同源基因,并克隆到相应的全长基因序列用于后续的表达与酶化学研究。     

Isolation and characterization of a novel cyanophycin synthetase from a deep-sea sediment metagenomic library

Cyanophycin is non-ribosomally synthesized protein-like copolymer. Synthesis of cyanophycin is catalyzed by cyanophycin synthetase (CphA). In this study, a novel cyanophycin synthetase CphA49 belonging to NOR5 clade of Gammaproteobacteria was identified with primer-based screening from a deep-sea sediment metagenomic library. The cphA49 gene contained an open reading frame of 2,637 bp and encoded a protein with a predicted molecular mass of 100 kDa. A recombinant CphA49 was obtained by the functional expression of cphA49 in Escherichia coli BL21 (DE3). The biochemical properties of the purified CphA49 were determined. The optimum pH and temperature of the recombinant CphA49 were 9.0 and 40 °C, respectively. The enzyme was stable at temperatures below 40 °C. The recombinant CphA49 exhibited strict primer dependency and broad substrate specificities. Cyanophycin catalyzed by CphA49 exhibited homogenous molecular mass. The amino acid composition of cyanophycin was determined and constitutes arginine, aspartic acid, and lysine.     

云南土壤宏基因组文库中替加环素耐药基因的筛选与分析

[背景]随着抗生素的广泛应用以及滥用,出现了多重耐药的"超级细菌",并且在临床上已出现对治疗"超级细菌"感染的最后一线抗生素——替加环素耐药的细菌.[目的]对土壤环境中存在的替加环素耐药基因进行筛选调查,探讨替加环素耐药机制,为临床抗生素治疗提供借鉴.[方法]利用功能宏基因组学技术对土壤宏基因组文库进行替加环素耐药阳性...     

Isolation of antibiotics turbomycin a and B from a metagenomic library of soil microbial DNA

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.