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1.
Jeon JH Kim JT Kim YJ Kim HK Lee HS Kang SG Kim SJ Lee JH 《Applied microbiology and biotechnology》2009,81(5):865-874
To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount
and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and
the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed
the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase
together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and
25°C, respectively, and rEML1 displayed more than 50% activity at 5°C. The activation energy for the hydrolysis of olive oil
was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols
acyl-group chains with long chain lengths of ≥8 carbon atoms and displayed hydrolyzing activities toward various natural oil
substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example
which developed a new cold-active lipase from a deep-sea sediment metagenome. 相似文献
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Jikun Du Li Li Xian Ding Haiyan Hu Yongjun Lu Shining Zhou 《Applied microbiology and biotechnology》2013,97(19):8619-8628
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. 相似文献
4.
Sohyeon Seo Young-Seok Lee Sang-Hong Yoon Soo-Jin Kim Jae Youl Cho Bum-Soo Hahn Bon-Sung Koo Chang-Muk Lee 《World journal of microbiology & biotechnology》2014,30(3):879-886
A functional screen of a metagenomic library from “Upo” swamp sediment in Korea identified a gene EstL28, the product of which displayed lipolytic properties on a tributyrin-supplemented medium. The EstL28 sequence encodes a 290 amino acid protein (designated as EstL28), with a predicted molecular weight of 31.3 kDa. The encoded EstL28 protein exhibited the highest sequence similarity (45 %) to a hydrolase found in Streptococcus sanguinis. Phylogenetic analysis indicated that EstL28 belongs to a currently uncharacterized family of esterases. Within the conserved α/β-hydrolase 6 domain, the EstL28 retains the catalytic triad Ser103–Asp248–His268 that is typical of esterases. The Ser103 residue in the catalytic triad is located in the consensus pentapeptide motif GXSXG. The purified EstL28 enzyme worked optimally at 35 °C and pH 8.5 and remained stable at temperatures lower than 20 °C. The catalytic activity of EstL28 was maximal with p-nitrophenyl butyrate, indicating that it was an esterase. This enzyme also exhibited stable activity in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, the level of stability in organic solvents and cold temperature suggests that EstL28 has potential for many biotechnological applications. 相似文献
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Yu EY Kwon MA Lee M Oh JY Choi JE Lee JY Song BK Hahm DH Song JK 《Applied microbiology and biotechnology》2011,90(2):573-581
Functional screening for lipolytic enzymes at low temperatures resulted in the isolation of the novel cold-active esterases,
EstM-N1 and EstM-N2, from a metagenomic DNA library of arctic soil samples. EstM-N1 and EstM-N2 were 395 and 407 amino acids
in length, respectively, and showed the highest similarity to class C β-lactamases. However, they shared a relatively low
level of sequence similarity (30%) with each other. Phylogenetic analysis of bacterial lipolytic enzymes confirmed that EstM-N1
and EstM-N2 belonged to family VIII of bacterial esterases/lipases. The (His)6-tagged esterases were purified to about 99% homogeneity from the soluble fraction of recombinant Escherichia coli cultures. The purified EstM-N1 and EstM-N2 retained more than 50% of maximal activity in the temperature range of 0–35°C,
with optimal temperatures of 20°C and 30°C, respectively. Both enzymes preferred the short acyl chains of p-nitrophenyl esters and exhibited very narrow substrate specificity, indicating that they are typical esterases. The β-lactamase
activity of EstM-N1 and EstM-N2 was also detected and reached about 31% and 13% of the positive control enzyme, Bacillus cereus β-lactamase, respectively. These first cold-active esterases belonging to family VIII are expected to be useful for potential
biotechnological applications as interesting biocatalysts. 相似文献
6.
Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome 总被引:1,自引:0,他引:1
Lee CM Yeo YS Lee JH Kim SJ Kim JB Han NS Koo BS Yoon SH 《Biochemical and biophysical research communications》2008,370(2):322-326
4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3 kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4 kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific β-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium. 相似文献
7.
Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome 总被引:2,自引:0,他引:2
Park HJ Jeon JH Kang SG Lee JH Lee SA Kim HK 《Protein expression and purification》2007,52(2):340-347
A metagenomics approach is an efficient method of isolating novel and useful genes from uncultured microorganisms in diverse environments. In this research, a gene encoding a new esterase (EM2L8) was cloned and characterized from the metagenomic DNA library of a deep-sea sediment. The gene consisted of 804bp encoding a polypeptide of 267 amino acids with a molecular mass of 28,952. The deduced amino acid sequence showed similarities with the BioH of Kurthia, the 3-oxoadipate enol-lactonase of Haloarcula and the acyltransferase of Thermoanaerobacter, which feature identities of 38%, 32%, and 33%, respectively. Residues essential for esterase activity, such as pentapeptide (GXSXG) and catalytic triad sequences, were uncovered. While the protein was overproduced mainly as inclusion body at 37 degrees C, it was mainly produced as a soluble active enzyme at 18 degrees C. A zymogram analysis revealed that purified EM2L8 taken from the soluble fraction could hydrolyze tributyrin substrate. Furthermore, the protein from the inclusion body fraction also showed strong activity on gel, thus indicating that the protein was refolded during SDS-gel electrophoresis and the ensuing incubation period. When the inclusion body was mixed with some anionic detergent solutions and diluted with a non-detergent buffer, the insoluble EM2L8 refolded rapidly and recovered its full esterase activity. Although EM2L8 had an optimum temperature of 50-55 degrees C, its activation energy in the range of 10-40 degrees C was 8.34kcal/mol, indicating that it is a cold-adapted enzyme. Moreover, it was found to have an optimum pH of 10-11, thus revealing that it is an alkaline enzyme. In this paper, the new esterase EM2L8 buried in a deep-sea sediment became known on the surface and was characterized biochemically. 相似文献
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Serinicoccus profundi MCCC 1A05965(T) was isolated from deep-sea sediment collected from the Indian Ocean. It was a Gram-positive, moderately halophilic, aerobic bacterium. Here, we describe the 3.4-Mbp draft genome sequence of S. profundi MCCC 1A05965(T). 相似文献
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Gong Cheng Yongfei Hu Na Lu Jing Li Zhiyun Wang Quanze Chen Baoli Zhu 《Biotechnology letters》2013,35(2):273-278
A soil metagenomic library was constructed and screened for clones that conferred fosfomycin resistance. A novel protein with 46 % identity to UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Desulfuromonas acetoxidans DSM 684 (GenBank accession number: ZP_01311756) was identified. Multiple sequence alignment revealed that the novel protein was a natural MurA, in which an aspartic acid instead of a cysteine was located in the active site. An Asp120Cys mutant of Escherichia coli was constructed from the subclone through site-specific mutagenesis, and minimum inhibitory concentration of fosfomycin for the resistant subclone and its mutant were determined. These results showed that fosfomycin resistance was a result of the aspartic acid in the active site. Analysis of all existing MurA sequences revealed that MurAs with an active site aspartic acid that can confer fosfomycin resistance occur in ~14 % of bacteria. 相似文献
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Jeon JH Lee HS Kim JT Kim SJ Choi SH Kang SG Lee JH 《Applied microbiology and biotechnology》2012,93(2):623-631
To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island
in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones
of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed
50–57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were
identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes.
The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif,
conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In
particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl
(or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded
the diversity of lipolytic enzyme-encoding genes. 相似文献
15.
Perret A Lechaplais C Tricot S Perchat N Vergne C Pellé C Bastard K Kreimeyer A Vallenet D Zaparucha A Weissenbach J Salanoubat M 《PloS one》2011,6(8):e22918
Background
Bacteria are key components in all ecosystems. However, our knowledge of bacterial metabolism is based solely on the study of cultivated organisms which represent just a tiny fraction of microbial diversity. To access new enzymatic reactions and new or alternative pathways, we investigated bacterial metabolism through analyses of uncultivated bacterial consortia.Methodology/Principal Findings
We applied the gene context approach to assembled sequences of the metagenome of the anaerobic digester of a municipal wastewater treatment plant, and identified a new gene which may participate in an alternative pathway of lysine fermentation.Conclusions
We characterized a novel, unique aminotransferase that acts exclusively on Coenzyme A (CoA) esters, and proposed a variant route for lysine fermentation. Results suggest that most of the lysine fermenting organisms use this new pathway in the digester. Its presence in organisms representative of two distinct bacterial divisions indicate that it may also be present in other organisms. 相似文献16.
Identification of two novel esterases from a marine metagenomic library derived from South China Sea
The demand for novel biocatalysts is increasing in modern biotechnology, which greatly stimulates the development of powerful tools to explore the genetic resources in the environment. Metagenomics, a culture independent strategy, provides an access to valuable genetic resources of the uncultured microbes. In this study, two novel esterase genes designated as estA and estB, which encoded 277- and 328-amino-acid peptides, respectively, were isolated from a marine microbial metagenomic library by functional screening, and the corresponding esterases EstA and EstB were biochemically characterized. Amino acid sequence comparison and phylogenetic analysis indicated that EstA together with other putative lipolytic enzymes was closely related to family III, and EstB with its relatives formed a subfamily of family IV. Site-directed mutagenesis showed that EstA contained classical catalytic triad made up of S146-D222-H255, whereas EstB contained an unusual catalytic triad which consisted of S-E-H, an important feature of the subfamily. EstA exhibited habitat-specific characteristics such as its high level of stability in the presence of various divalent cations and at high concentrations of NaCl. EstB displayed remarkable activity against p-nitrophenyl esters and was highly stable in 30% methanol, ethanol, dimethylformamide, and dimethyl sulfoxide, making EstB a potential candidate for industrial applications. 相似文献
17.
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. 相似文献
18.
Elend C Schmeisser C Leggewie C Babiak P Carballeira JD Steele HL Reymond JL Jaeger KE Streit WR 《Applied and environmental microbiology》2006,72(5):3637-3645
The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to beta-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50 degrees C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47 degrees C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications. 相似文献
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Dominic W. S. Wong Victor J. Chan Hans Liao Mary J. Zidwick 《Journal of industrial microbiology & biotechnology》2013,40(3-4):287-295
A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae2) was identified as a type C feruloyl esterase. The RuFae2 alone released ferulic acid from rice bran, wheat bran, wheat-insoluble arabinoxylan, corn fiber, switchgrass, and corn bran in the order of decreasing activity. Using a saturating amount of RuFae2 for 100 mg substrate, a maximum of 18.7 and 80.0 μg FA was released from 100 mg corn fiber and wheat-insoluble arabinoxylan, respectively. Addition of GH10 endoxylanase (EX) synergistically increased the release of FA with the highest level of 6.7-fold for wheat bran. The synergistic effect of adding GH11 EX was significantly smaller with all the substrates tested. The difference in the effect of the two EXs was further analyzed by comparing the rate in the release of FA with increasing EX concentration using wheat-insoluble arabinoxylan as the substrate. 相似文献