共查询到20条相似文献,搜索用时 15 毫秒
1.
Yun-Hee Song Kyung-Tai Lee Jin-Young Baek Min-Ju Kim Mi-Ra Kwon Young-Joo Kim Mi-Rim Park Haesu Ko Jin-Sung Lee Keun-Sung Kim 《Folia microbiologica》2017,62(3):175-181
This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-β-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20–50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20–40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future. 相似文献
2.
Sophie Biver Aurore Stroobants Daniel Portetelle Micheline Vandenbol 《Journal of industrial microbiology & biotechnology》2014,41(3):479-488
New β-glucosidase activities were identified by screening metagenomic libraries constructed with DNA isolated from the topsoil of a winter wheat field. Two of the corresponding proteins, displaying an unusual preference for alkaline conditions, were selected for purification by Ni-NTA chromatography. AS-Esc6, a 762-amino-acid enzyme belonging to glycoside hydrolase family 3, proved to be a mesophilic aryl-β-glucosidase with maximal activity around pH 8 and 40 °C. A similar pH optimum was found for AS-Esc10, a 475-amino-acid GH1-family enzyme, but this enzyme remained significantly active across a wider pH range and was also markedly more stable than AS-Esc6 at pH greater than 10. AS-Esc10 was found to degrade cellobiose and diverse aryl glycosides, with an optimal temperature of 60 °C and good stability up to 50 °C. Unlike AS-Esc6, which showed a classically low inhibitory constant for glucose (14 mM), AS-Esc10 showed enhanced activity in the presence of molar concentrations of glucose. AS-Esc10 was highly tolerant to hydrogen peroxide and also to sodium dodecyl sulfate, this being indicative of kinetic stability. This unique combination of properties makes AS-Esc10 a particularly promising candidate whose potential in biotechnological applications is worth exploring further. 相似文献
3.
Xing Yan Alei Geng Jun Zhang Yongjun Wei Lei Zhang Changli Qian Qianfu Wang Shengyue Wang Zhihua Zhou 《Applied microbiology and biotechnology》2013,97(18):8173-8182
In this study, 341, 246, and 386 positive clones with endo-β-1,4-glucanase, β-glucosidase, and endo-β-1,4-xylanase activities, respectively, were identified by screening from a metagenomic fosmid library constructed from a biogas digester. Subsequently, pools of 4, 10, and 16 positive clones were subjected to 454 pyrosequencing in different subruns. In total, 21 unique glycosyl hydrolase (GH) genes were predicted by bioinformatic analysis, which showed similarities to their nearest neighbors from 39 % to 72 %. In addition to bioinformatics prediction, nine GH genes were expressed and purified to identify their activity with four kinds of substrates. The activities of the most expressed proteins were consistent with their annotation based on bioinformatics prediction; however, three GH genes belonging to the GH5 family showed different activities from their annotation. An efficient acidic cellulase En1 had an optimal condition at 55 °C, pH 5.5, with a specific activity toward carboxymethylcellulose at 118 U/mg and K m at 12.8 g/L. This study demonstrated that there are diverse GHs in the biogas digester system with potential industrial application in lignocellulose hydrolysis, and their activities should be investigated with different substrates before their application. Additionally, pool sequencing of positive fosmid clones might be a cost-effective approach to obtain functional genes from metagenomic libraries. 相似文献
4.
Paula Istvan Amanda Araújo Souza Aisel Valle Garay Debora Farage Knupp dos Santos Gideane Mendes de Oliveira Renata Henrique Santana Fabyano Alvares Cardoso Lopes Sonia Maria de Freitas João Alexandre Ribeiro Gonçalves Barbosa Ricardo Henrique Krüger 《Biotechnology letters》2018,40(9-10):1395-1406
Objective
To isolate putative lipase enzymes by screening a Cerrado soil metagenomic library with novel features.Results
Of 6720 clones evaluated, Clone W (10,000 bp) presented lipolytic activity and four predicted coding sequences, one of them LipW. Characterization of a predicted esterase/lipase, LipW, showed 28% sequence identity with an arylesterase from Pseudomonas fluorescens (pdb|3HEA) from protein database (PDB). Phylogenetic analysis showed LipW clustered with family V lipases; however, LipW was clustered in different subclade belonged to family V, suggesting a different subgroup of family V. In addition, LipW presented a difference in family V GH motif, a glycine replaced by a serine in GH motif. Estimated molecular weight and stokes radius values of LipW were 29,338.67–29,411.98 Da and 2.58–2.83 nm, respectively. Optimal enzyme activity was observed at pH 9.0–9.5 and at 40 °C. Circular dichroism analysis estimated secondary structures percentages as approximately 45% α-helix and 15% β-sheet, consistent with the 3D structure predicted by homology.Conclusion
Our results demonstrate the isolation of novel family V lipolytic enzyme with biotechnological applications from a metagenomic library.5.
Li-Qin Du Hao Pang Yuan-Yuan Hu Yu-Tuo Wei Ri-Bo Huang 《World journal of microbiology & biotechnology》2010,26(3):419-428
A gene encoding Uninv, an invertase active in the neutral pH range, was isolated from a metagenomic library and the enzyme was biochemically characterized. The enzyme had an optimum pH of 6.5 and an optimum temperature of 50°C. It showed maximal ~37% sequence identity to all invertases identified so far. Uninv displayed typical Michaelis–Menten kinetics with substrate inhibition of sucrose, but it retained 50% activity at sucrose concentrations up to 1,000 mM. Uninv had no transfructosylating activity even at high sucrose concentrations. These properties will make this enzyme useful in the food industry. 相似文献
6.
Western Ghats of India is recognized as one of the 12 mega diversity regions of the world and is the hot spot for unrevealed microbial diversity. To explore the diversity of polysaccharide degrading enzymes in that region, metagenomic library was constructed from forest soil of Southern Western Ghats region. Nine pectinolytic clones with the ability to degrade citrus pectin were isolated based on function based screening of the library. Sequence analysis of pg_4 clone containing revealed that it contained GH family 28 domain (pfam00295) belonging to polygalacturonase superfamily (PLN03003). Its amino acid sequence analysis showed 25–55 % identity to the other well-characterized polygalacturonases. Molecular modeling of pg_4 revealed that it comprised of three right handed-parallel β sheets, one anti-parallel β sheet and one α helix with three conserved catalytic residue D 2263, D 284-85 and H 312 at the C terminal end. The enzyme characterized was able to hydrolyze both apple and citrus pectin with K m values of 1.685 and 1.542 mg ml?1 and retained more that 80 % of activity at pH 5–9 and temperature 20–60 °C. 相似文献
7.
【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。 相似文献
8.
X. Gong R. J. Gruniniger R. J. Forster R. M. Teather T. A. McAllister 《Applied microbiology and biotechnology》2013,97(6):2423-2431
A metagenomic library was generated using microbial DNA extracted from the rumen contents of a grass hay-fed dairy cow using a bacterial artificial chromosome-based vector system. Functional screening of the library identified a gene encoding a potent glycoside hydrolase, xyn10N18, localised within a xylanolytic gene cluster consisting of four open-reading frames (ORFs). The ORF, xyn10N18, encodes an endo-β-1,4-xylanase with a glycosyl hydrolase family 10 (GH10) catalytic domain, adopts a canonical α8/ß8-fold and possesses conserved catalytic glutamate residues typical of GH10 xylanases. Xyn10N18 exhibits optimal catalytic activity at 35 °C and pH 6.5 and was highly stable to pH changes retaining at least 85 % relative catalytic activity over a broad pH range (4.0–12.0). It retained 25 % of its relative activity at both low (4 °C) and high (55 °C) temperatures, however the stability of the enzyme rapidly decreased at temperatures of >40 °C. The specific activity of Xyn10N18 is enhanced by the divalent cations Mn2+ and Co2+ and is dramatically reduced by Hg2+ and Cu2+. Interestingly, EDTA had little effect on specific activity indicating that divalent cations do not function mechanistically. The enzyme was highly specific for xylan containing substrates and showed no catalytic activity against cellulose. Analysis of the hydrolysis products indicated that Xyn10N18 was an endoxylanase. Through a combination of structural modelling and in vitro enzyme characterisation this study provides an understanding of the mechanism and the substrate specificity of this enzyme serving as a starting point for directed evolution of Xyn10N18 and subsequent downstream use in industry. 相似文献
9.
Arivaradarajan Preeti Devaraj Hemalatha Jeyaprakash Rajendhran Peter Mullany Paramasamy Gunasekaran 《Indian journal of microbiology》2014,54(3):284-292
The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes. 相似文献
10.
11.
One clone (ACPGA001) exhibiting penicillin G acylase (PGA) activity was screened from a metagenomic library by using a medium containing penicillin G. A novel PGA gene from the inserted fragment of ACPGA001 was obtained by sequencing. The amino acid sequence of ACPGA001 PGA exhibited <33 % similarity to PGAs retrieved from GenBank. This gene was expressed in Escherichia coli M15 and the recombinant protein was purified and characterized. The ACPGA001 PGA exhibited a maximum activity at 60 °C and showed high activity at pH 4–10 with an optimum pH of 8.0. This enzyme was stable at 40 °C for 70 min with a half-life of 60 min at 55 °C. These beneficial characteristics of ACPGA001 PGA provide some advantages for the potential application of ACPGA001 PGA in industry. 相似文献
12.
A novel endoglucanase gene, mgcel44, was isolated from a mangrove soil metagenomic library by functional-based screening. It encodes a 648-aa peptide with a catalytic domain of glycosyl hydrolase family 44. The deduced amino acid sequence of mgcel44 shares less than 50 % identity with endoglucanases in GenBank database. mgcel44 was cloned and overexpressed in Escherichia coli. The recombinant enzyme, MgCel44, has a molecular mass of 70.8 kDa as determined by SDS-PAGE. Its optimal pH and temperature for activity were 6 and 45 °C, respectively. It was highly active at 25–45 °C and pH 5–8. Its activity was enhanced in 0.5 M NaCl by >1.6-fold and stable up to 1.5 M NaCl. MgCel44 was resistant to several organic solvents and had high activity at 15 % (v/v) solvent after incubating for 24 h at 25 °C. 相似文献
13.
A cel44C-man26A gene of endophytic Paenibacillus
polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains 总被引:2,自引:0,他引:2
Cho KM Hong SY Lee SM Kim YH Kahng GG Kim H Yun HD 《Applied microbiology and biotechnology》2006,73(3):618-630
A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu91 and Glu222 may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A. 相似文献
14.
Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organism 总被引:1,自引:0,他引:1
Aims: The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro‐organisms. Methods and Results: The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni‐nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35·6 kDa. Typical residues essential for lipolytic activity such as penta‐peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487 ) from Pseudomonas mendocina ymp and esterase (31%, AAY45707 ) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7·5 and 10°C. At thermal stability analysis, lipo1 was more unstable at 40°C than 10°C. Conclusions: An activity based strategy has been an effective method for fishing out a low‐temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone‐sensitive lipase family due to the enzyme’s oxyanion hole by the sequence HGGG. Significance and Impact of the Study: Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry. 相似文献
15.
Tanzeem Akbar Cheema Kanya Jirajaroenrat Theerapol Sirinarumitr 《Animal biotechnology》2013,24(4):261-277
Ruminants are capable of hydrolyzing lignocellulosic residues to absorbable sugars by virtue of the microbial communities residing in their rumen. However, large sections of such microbial communities are not yet culturable using conventional laboratory techniques. Therefore in the present study, the metagenomic DNA of swamp buffalo (Bubalus bubalis) rumen contents was explored using culture-independent techniques. The consensus regions of glycosyl hydrolase 5 (GH5) family of cellulases were used as primers for PCR amplification. A full-length metagenomic cellulase gene, Umcel5B29, with a complete open reading frame (ORF) of 1611 bp was identified. The similarity search analysis revealed that Umcel5B29 is closely related to the cellulases (73% to 98% similarity) of ruminal unculturable microorganisms, indicating its phylogenetic origin. Further analysis indicated that Umcel5B29 does not contain a carbohydrate binding module (CBM). Subsequently, Umcel5B29 was overexpressed in Escherichia coli. The recombinant enzyme worked optimally at pH 5.5 and 45°C, a condition similar to the buffalo's rumen. However, the enzyme retained more than 70% of its maximal activity after incubation at pH 4–7 and more than 50% maximal activity after incubation at 30–60°C for 30 min. These characteristics render Umcel5B29 as a potential candidate for the bio-stoning process of denim. 相似文献
16.
K. J. Rashamuse D. F. Visser F. Hennessy J. Kemp M. P. Roux-van der Merwe J. Badenhorst T. Ronneburg R. Francis-Pope D. Brady 《Current microbiology》2013,66(2):145-151
Ruminant digestive tract microbes hydrolyse plant biomass, and the application of metagenomic techniques can provide good coverage of their glycosyl hydrolase enzymes. A metagenomic library of circa 70,000 fosmids was constructed from bacterial DNA isolated from bovine rumen and subsequently screened for cellulose hydrolysing activities on a CMC agar medium. Two clones were selected based on large clearance zones on the CMC agar plates. Following nucleotide sequencing, translational analysis and homology searches, two cellulase encoding genes (cel5A and cel5B) belonging to the glycosyl hydrolyse family 5 were identified. Both genes encoded pre-proteins of about 62 kDa, containing signal leader peptides which could be cleaved to form mature proteins of about 60 kDa. Biochemical characterisation revealed that both enzymes showed alkaline pH optima of 9.0 and the temperature optima of 65 °C. Substrate specificity profiling of the two enzymes using 1,4-β-d-cello- and xylo-oligosaccharides revealed preference for longer oligosaccharides (n ≥ 3) for both enzymes, suggesting that they are endo-cellulases/xylanases. The bifunctional properties of the two identified enzymes render them potentially useful in degrading the β-1,4 bonds of both the cellulose and hemicellulose polymers. 相似文献
17.
《Bioscience, biotechnology, and biochemistry》2013,77(9):1848-1854
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. 相似文献
18.
Zongqing Huang Xiaoshuang Liu Shaowei Zhang Ziduo Liu 《Journal of industrial microbiology & biotechnology》2014,41(1):65-74
A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K m and k cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s?1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K m and k cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s?1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K m and k cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s?1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall. 相似文献
19.
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. 相似文献
20.
Yanyu Zhao Kun Meng Huiying Luo Huoqing Huang Tiezheng Yuan Peilong Yang Bin Yao 《World journal of microbiology & biotechnology》2013,29(2):327-334
A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K m and V max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone. 相似文献