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Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

ABSTRACT: The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

SUBMITTER: Findley SD

PROVIDER: S-EPMC3209001 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

Findley Seth D SD Mormile Melanie R MR Sommer-Hurley Andrea A Zhang Xue-Cheng XC Tipton Peter P Arnett Krista K Porter James H JH Kerley Monty M Stacey Gary G

Applied and environmental microbiology 20110923 22

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identifie ...[more]

PMID: 21948825

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Project description:The exploration and utilization of microbial salt-tolerant enzymatic and genetic resources are of great significance in the field of biotechnology and for the research of the adaptation of microorganisms to extreme environments. The presence of new salt-tolerant genes and enzymes in the microbial metagenomic library of the gastrointestinal tract has been confirmed through metagenomic technology. This paper aimed to identify and characterize enzymes that confer salt tolerance in the gastrointestinal tract microbe. By screening the fecal metagenomic library, 48 salt-tolerant clones were detected, of which 10 salt-tolerant clones exhibited stronger tolerance to 7% (wt/vol) NaCl and stability in different concentrations of NaCl [5%-9% (wt/vol)]. High-throughput sequencing and biological information analysis showed that 91 potential genes encoded proteins and enzymes that were widely involved in salt tolerance. Furthermore, two trehalose-6-phosphate hydrolase genes, namely, tre_P2 and tre_P3, were successfully cloned and expressed in Escherichia coli BL21 (DE3). By virtue of the substrate of p-nitrophenyl-α-D-glucopyranoside (pNPG) which can be specifically hydrolyzed by trehalose-6-phosphate hydrolase to produce glucose and p-nitrophenol, the two enzymes can act optimally at pH 7.5 and 30°C. Steady-state kinetics with pNPG showed that the K M and K cat values were 15.63 mM and 10.04 s-1 for rTRE_P2 and 12.51 mM and 10.71 s-1 for rTRE_P3, respectively. Characterization of enzymatic properties demonstrated that rTRE_P2 and rTRE_P3 were salt-tolerant. The enzymatic activity increased with increasing NaCl concentration, and the maximum activities of rTRE_P2 and rTRE_P3 were obtained at 4 and 3 M NaCl, respectively. The activities of rTRE_P2 increased by approximately 43-fold even after 24 h of incubation with 5 M NaCl. This study is the first to report the identification as well as molecular and biochemical characterization of salt-tolerant trehalose-6-phosphate hydrolase from the metagenomic library of the gastrointestinal tract. Results indicate the existence of numerous salt-tolerant genes and enzymes in gastrointestinal microbes and provide new insights into the salt-tolerant mechanisms in the gastrointestinal environment.

Dataset Information

Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

Publications

Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

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