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Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample

Paper ID Volume ID Publish Year Pages File Format Full-Text
22954 43402 2015 9 PDF Available
Title
Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample
Abstract

•We performed next generation metagenomic sequencing on soil samples.•Several genes for chitin and chitosan modifying enzymes were identified.•A novel sequence coding for chitinase was codon-optimized and synthesized.•Expression studies and dot assays revealed the chitinase to be active.•Metagenomic sequencing can be used as tool for novel enzymes.

Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378 bp amounting to a total sequence information of approximately 851Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500 bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5 mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value.

Keywords
Chitin/chitosan modifying enzymes; Chitinase; Soil; Next generation metagenomic sequencing; Dot assay
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Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 201, 10 May 2015, Pages 60–68
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us