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Novel lipolytic enzymes identified from metagenomic library of deep-sea sediment.

ABSTRACT: Metagenomic library was constructed from a deep-sea sediment sample and screened for lipolytic activity. Open-reading frames of six positive clones showed only 33-58% amino acid identities to the known proteins. One of them was assigned to a new group while others were grouped into Families I and V or EstD Family. By employing a combination of approaches such as removing the signal sequence, coexpression of chaperone genes, and low temperature induction, we obtained five soluble recombinant proteins in Escherichia coli. The purified enzymes had optimum temperatures of 30-35°C and the cold-activity property. Among them, one enzyme showed lipase activity by preferentially hydrolyzing p-nitrophenyl palmitate and p-nitrophenyl stearate and high salt resistance with up to 4?M NaCl. Our research demonstrates the feasibility of developing novel lipolytic enzymes from marine environments by the combination of functional metagenomic approach and protein expression technology.

SUBMITTER: Jeon JH

PROVIDER: S-EPMC3154406 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Novel lipolytic enzymes identified from metagenomic library of deep-sea sediment.

Jeon Jeong Ho JH Kim Jun Tae JT Lee Hyun Sook HS Kim Sang-Jin SJ Kang Sung Gyun SG Choi Sang Ho SH Lee Jung-Hyun JH

Evidence-based complementary and alternative medicine : eCAM 20110807

Metagenomic library was constructed from a deep-sea sediment sample and screened for lipolytic activity. Open-reading frames of six positive clones showed only 33-58% amino acid identities to the known proteins. One of them was assigned to a new group while others were grouped into Families I and V or EstD Family. By employing a combination of approaches such as removing the signal sequence, coexpression of chaperone genes, and low temperature induction, we obtained five soluble recombinant prot ...[more]

PMID: 21845199

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Project description:BACKGROUND:Enzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance. RESULTS:Here, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either pNP-?-d-galactopyranoside, pNP-?-d-xylopyranoside, pNP-?-l-arabinopyranoside or pNP-?-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the pNP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a ?-galactosidase (74%) and (2) a ?-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The ?-galactosidase- and ?-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the ?-xylosidase-like protein 2 showed activities with both pNP-Xyl and pNP-Ara in the temperature range 40-50 °C and pH range 8.0-10.0. Moreover, proteins 5 and 6 showed thermotolerant ?-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential "GGDEF" catalytic site, encoding ?-glucosidase activity, whereas that of protein 6 showed a "GDSL" site, encoding a 'new family' ?-glucosidase activity. CONCLUSION:Using a rational screening approach, we identified and characterized four thermo-alkaliphilic glycosyl hydrolases that have the potential to serve as constituents of enzyme cocktails that produce sugars from lignocellulosic plant remains.

Project description:Marine sediments constitute the world's most substantial long-term carbon repository. The microorganisms dwelling in these sediments mediate the transformation of fixed oceanic carbon, but their contribution to the carbon cycle is not fully understood. Previous culture-independent investigations into sedimentary microorganisms have underscored the significance of carbohydrates in the carbon cycle. In this study, we employ a metagenomic methodology to investigate the distribution and abundance of carbohydrate-active enzymes (CAZymes) in 37 marine sediments sites. These sediments exhibit varying oxygen availability and were isolated in diverse regions worldwide. Our comparative analysis is based on the metabolic potential for oxygen utilisation, derived from genes present in both oxic and anoxic environments. We found that extracellular CAZyme modules targeting the degradation of plant and algal detritus, necromass, and host glycans were abundant across all metagenomic samples. The analysis of these results indicates that the oxic/anoxic conditions not only influence the taxonomic composition of the microbial communities, but also affect the occurrence of CAZyme modules involved in the transformation of necromass, algae and plant detritus. To gain insight into the sediment microbial taxa, we reconstructed metagenome assembled genomes (MAG) and examined the presence of primary extracellular carbohydrate active enzyme (CAZyme) modules. Our findings reveal that the primary CAZyme modules and the CAZyme gene clusters discovered in our metagenomes were prevalent in the Bacteroidia, Gammaproteobacteria, and Alphaproteobacteria classes. We compared those MAGs to organisms from the same taxonomic classes found in soil, and we found that they were similar in its CAZyme repertoire, but the soil MAG contained a more abundant and diverse CAZyme content. Furthermore, the data indicate that abundant classes in our metagenomic samples, namely Alphaproteobacteria, Bacteroidia and Gammaproteobacteria, play a pivotal role in carbohydrate transformation within the initial few metres of the sediments.

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Novel lipolytic enzymes identified from metagenomic library of deep-sea sediment.

Publications

Novel lipolytic enzymes identified from metagenomic library of deep-sea sediment.

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