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Prospecting for novel biocatalysts in a soil metagenome.

ABSTRACT: The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

SUBMITTER: Voget S

PROVIDER: S-EPMC201203 | biostudies-literature | 2003 Oct

REPOSITORIES: biostudies-literature

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Prospecting for novel biocatalysts in a soil metagenome.

Voget S S Leggewie C C Uesbeck A A Raasch C C Jaeger K-E KE Streit W R WR

Applied and environmental microbiology 20031001 10

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from t ...[more]

PMID: 14532085

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Project description:BackgroundCellulose, an abundant and renewable polysaccharides, constitutes the largest resource for bioconversion of biofuels. Plant polysaccharides hydrolysis is catalyzed by cellulases, which include endoglucanases, exoglucanases, and β-glucosidases. Converting cellulose and hemicellulose to short chains of oligosaccharides by endo-/exoglucanases is the key step for biofuel transformation. Intriguingly, β-glucanases with transglycosylation activity not only can relieve product inhibition of glucan hydrolysis but also has potential application as biocatalysts for functional materials.ResultsHere, a metagenomic fosmid library was constructed from a paddy soil for cellulase screening. One purified clone showing carboxymethylcellulase activity was isolated, and the complete β-glucanase gene (umcel9y-1) was cloned and overexpressed in Escherichia coli. Phylogenetic analysis indicated that β-glucanase Umcel9y-1 belonged to the theme C of glycoside hydrolase family 9. Amino acids sequence showed 58.4 % similarity between Umcel9y-1 and its closest characterized reference, cellulase Cel01. Biological characterization showed that Umcel9y-1 was an efficient endoglucanase and also exhibited high activities of exoglucanase and transglycosylation. The transglycosylation products of Umcel9y-1 including sophorose, laminaribiose, and gentiobiose, and transglycosylation was detected under all activated conditions. The order of catalytic efficiency for polysaccharides, cellooligosaccharides, and aryl-β-glycosides was p-nitrophenol-D-cellobioside, barley glucan, cellopentaose, cellotetraose, cellotriose, hydroxyethylcellulose, cellohexose, laminarin, and carboxymethylcellulose, respectively. The barley glucan was the optimal polysaccharides for Umcel9y-1 with K m and K cat/K m values of 13.700 mM and 239.152 s(-1) mM(-1), respectively.ConclusionBiological characterizations of recombinant Umcel9y-1 showed that the versatile β-glucanase had efficient endoglucanase activity to barley glucan and also exhibited high activities of exoglucanase and transglycosylation. The optimum conditions of recombinant Umcel9y-1 was pH 6.5-7.0 at 37 °C with predominant halotolerance and high-thermal stability. These results indicate that the novel metagenomic-derived β-glucanase may be a potent candidate for industrial applications.

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Prospecting for novel biocatalysts in a soil metagenome.

Publications

Prospecting for novel biocatalysts in a soil metagenome.

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