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Lipase metagenomic

Amélie Dukunde, Dominik Schneider, Mingji Lu, Silja Brady, Rolf Daniel
OBJECTIVES: To investigate the properties of a novel metagenome-derived member of the hormone-sensitive lipase family of lipolytic enzymes. RESULTS: A forest soil metagenome-derived gene encoding an esterase (Est06) belonging to the hormone-sensitive lipase family of lipolytic enzymes was subcloned, heterologously expressed and characterized. Est06 is a polypeptide of 295 amino acids with a molecular mass of 31 kDa. The deduced protein sequence shares 61% similarity with a hypothetical protein from the marine symbiont Candidatus Entotheonella sp...
January 2, 2017: Biotechnology Letters
Rakesh Kumar, Linga Banoth, Uttam Chand Banerjee, Jagdeep Kaur
In the present study, efficient enzymatic methods were developed using a recombinant metagenomic lipase (LipR1) for the synthesis of corresponding esters by the transesterification of five different pharmaceutically important secondary alcohols. The recombinant lipase (specific activity=87m6U/mg) showed maximum conversion in presence of ionic liquid with Naphthyl-ethanol (eeP=99%), Indanol and Methyl-4 pyridine methanol (eeS of 98% and 99%) respectively in 1h. Vinyl acetate was found as suitable acyl donor in transesterification reactions...
February 2017: International Journal of Biological Macromolecules
Rajesh Kumar Sahoo, Mohit Kumar, Lala Behari Sukla, Enketeswara Subudhi
Screening of metagenomic library from Taptapani Hot Spring (Odisha) yielded a positive lipase clone (pUC-lip479). Sequence analysis showed an ORF (RK-lip479) of 416 amino acid residues which was overexpressed in Escherichia coli BL21 (DE3). Optimum pH and temperature of purified lipase RK-lip479 were 8.0 and 65 °C, respectively, and found to be stable over a pH range of 7.0-9.0 and temperatures 55-75 °C. RK-lip479 could hydrolyse a wide range of 4-nitrophenyl esters (4-nitrophenyoctanoate, 4-nitrophenyldodecanoate, 4-nitrophenylpalmitate, 4-nitrophenylmyristate and 4-nitrophenylstearate), and maximum activity was observed with 4-nitrophenyldodecanoate...
November 28, 2016: Environmental Science and Pollution Research International
Mahejibin Khan, Amit Kumar
The understanding of the 3-dimensional enzyme structure is important for the point of protein engineering and applications. Computer-based molecular modelling is a vital tool for theoretical predication of enzyme activities and finding their substrates and inhibitors. SMlipA lipase was cloned from forest soil metagenome and characterized as broad spectrum enzyme with high stability in various organic solvents. In the present study, to understand the mechanism of SMlipA lipase and to identify the key residues involved in enzyme-substrate interaction, three dimensional-computational model of SMlipA has been generated and validated for stereo-chemical and amino-acid environment quality using appropriate programs, and further validation of the active-site architecture was achieved by performing docking studies with different ligand...
November 2016: Journal of Molecular Graphics & Modelling
Lada E Petrovskaya, Ksenia A Novototskaya-Vlasova, Sultan Sh Gapizov, Elena V Spirina, Ekaterina V Durdenko, Elizaveta M Rivkina
Siberian permafrost is a unique environment inhabited with diverse groups of microorganisms. Among them, there are numerous producers of biotechnologically relevant enzymes including lipases and esterases. Recently, we have constructed a metagenomic library from a permafrost sample and identified in it several genes coding for potential lipolytic enzymes. In the current work, properties of the recombinant esterases obtained from this library are compared with the previously characterized lipase from Psychrobacter cryohalolentis and other representatives of the hormone-sensitive lipase family...
October 18, 2016: Bioengineered
Haseong Kim, Kil Koang Kwon, Wonjae Seong, Seung-Goo Lee
The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library(1). We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database...
2016: Journal of Visualized Experiments: JoVE
Jing Huang, Ying-Yi Huo, Rui Ji, Siyun Kuang, Chaoneng Ji, Xue-Wei Xu, Jixi Li
Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate...
2016: Scientific Reports
Stephan Thies, Sonja Christina Rausch, Filip Kovacic, Alexandra Schmidt-Thaler, Susanne Wilhelm, Frank Rosenau, Rolf Daniel, Wolfgang Streit, Jörg Pietruszka, Karl-Erich Jaeger
DNA derived from environmental samples is a rich source of novel bioactive molecules. The choice of the habitat to be sampled predefines the properties of the biomolecules to be discovered due to the physiological adaptation of the microbial community to the prevailing environmental conditions. We have constructed a metagenomic library in Escherichia coli DH10b with environmental DNA (eDNA) isolated from the microbial community of a slaughterhouse drain biofilm consisting mainly of species from the family Flavobacteriaceae...
2016: Scientific Reports
Tayvich Vorapreeda, Chinae Thammarongtham, Kobkul Laoteng
Lipid-degrading or lipolytic enzymes have gained enormous attention in academic and industrial sectors. Several efforts are underway to discover new lipase enzymes from a variety of microorganisms with particular catalytic properties to be used for extensive applications. In addition, various tools and strategies have been implemented to unravel the functional relevance of the versatile lipid-degrading enzymes for special purposes. This review highlights the study of microbial lipid-degrading enzymes through an integrative computational approach...
July 2016: World Journal of Microbiology & Biotechnology
Shelly Goomber, Rakesh Kumar, Ranvir Singh, Neelima Mishra, Jagdeep Kaur
Small molecular weight Bacillus lipases are industrially attractive because of its alkaline optimum pH, broad substrate specificity and production in high yield by overexpression both in Escherichia coli and Bacillus subtilis. Its major limitation of being mesophilic in nature is constantly targeted by laboratory evolution studies. Herein metagenomically isolated Bacillus LipJ was randomly evolved by error prone PCR and library of variants were screened for enhanced thermostability. Point mutant Gln121Arg was extensively characterized and it showed dramatic shift of Temp...
July 2016: International Journal of Biological Macromolecules
Lada E Petrovskaya, Ksenia A Novototskaya-Vlasova, Elena V Spirina, Ekaterina V Durdenko, Galina Yu Lomakina, Maria G Zavialova, Evgeny N Nikolaev, Elizaveta M Rivkina
As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG)...
May 2016: FEMS Microbiology Ecology
Thaís Carvalho Maester, Mariana Rangel Pereira, E G Machado Sierra, Andrea Balan, Eliana Gertrudes de Macedo Lemos
Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase from Parvibaculum lavamentivorans [GenBank: WP012110575...
July 2016: Applied Microbiology and Biotechnology
Concetta De Santi, Bjørn Altermark, Marcin Miroslaw Pierechod, Luca Ambrosino, Donatella de Pascale, Nils-Peder Willassen
BACKGROUND: The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields. RESULTS: Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases...
January 19, 2016: BMC Biochemistry
Ximena Zottig, Fatma Meddeb-Mouelhi, Marc Beauregard
A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings...
March 1, 2016: Analytical Biochemistry
Olalla López-López, Kamila Knapik, Maria-Esperanza Cerdán, María-Isabel González-Siso
A fosmid library was constructed with the metagenomic DNA from the water of the Lobios hot spring (76°C, pH = 8.2) located in Ourense (Spain). Metagenomic sequencing of the fosmid library allowed the assembly of 9722 contigs ranging in size from 500 to 56,677 bp and spanning ~18 Mbp. 23,207 ORFs (Open Reading Frames) were predicted from the assembly. Biodiversity was explored by taxonomic classification and it revealed that bacteria were predominant, while the archaea were less abundant. The six most abundant bacterial phyla were Deinococcus-Thermus, Proteobacteria, Firmicutes, Acidobacteria, Aquificae, and Chloroflexi...
2015: Frontiers in Microbiology
Ana Popovic, Anatoly Tchigvintsev, Hai Tran, Tatyana N Chernikova, Olga V Golyshina, Michail M Yakimov, Peter N Golyshin, Alexander F Yakunin
This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions...
2015: Advances in Experimental Medicine and Biology
Amani D Alma'abadi, Takashi Gojobori, Katsuhiko Mineta
More than 99% of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel biocatalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities...
October 2015: Genomics, Proteomics & Bioinformatics
Carlina Peña-García, Mónica Martínez-Martínez, Dolores Reyes-Duarte, Manuel Ferrer
Nowadays, enzymes can be efficiently identified and screened from metagenomic resources or mutant libraries. A set of a few hundred new enzymes can be found using a simple substrate within few months. Hence, the establishment of collections of enzymes is no longer a big hurdle. However, a key problem is the relatively low rate of positive hits and that a timeline of several years from the identification of a gene to the development of a process is the reality rather than the exception. Major problems are related to the time-consuming and cost-intensive screening process that only very few enzymes finally pass...
2016: Combinatorial Chemistry & High Throughput Screening
Nam Hee Kim, Ji-Hye Park, Eunsook Chung, Hyun-Ah So, Myung Hwan Lee, Jin-Cheol Kim, Eul Chul Hwang, Seon-Woo Lee
A soil metagenome contains the genomes of all microbes included in a soil sample, including those that cannot be cultured. In this study, soil metagenome libraries were searched for microbial genes exhibiting lipolytic activity and those involved in potential lipid metabolism that could yield valuable products in microorganisms. One of the subclones derived from the original fosmid clone, pELP120, was selected for further analysis. A subclone spanning a 3.3 kb DNA fragment was found to encode for lipase/esterase and contained an additional partial open reading frame encoding a wax ester synthase (WES) motif...
February 2016: Journal of Microbiology and Biotechnology
Thorsten Masuch, Anna Kusnezowa, Sebastian Nilewski, José T Bautista, Robert Kourist, Lars I Leichert
The majority of protein sequence data published today is of metagenomic origin. However, our ability to assign functions to these sequences is often hampered by our general inability to cultivate the larger part of microbial species and the sheer amount of sequence data generated in these projects. Here we present a combination of bioinformatics, synthetic biology, and Escherichia coli genetics to discover biocatalysts in metagenomic datasets. We created a subset of the Global Ocean Sampling dataset, the largest metagenomic project published to date, by removing all proteins that matched Hidden Markov Models of known protein families from PFAM and TIGRFAM with high confidence (E-value > 10(-5))...
2015: Frontiers in Microbiology
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