Aims:  To clone and characterize genes encoding novel cellulases from metagenomes of buffalo rumens. Methods and Results:  A ruminal metagenomic library was constructed and functionally screened for cellulase activities and 61 independent clones expressing cellulase activities were isolated. Subcloning and sequencing of 13 positive clones expressing endoglucanase and MUCase activities identified 14 cellulase genes. Two clones carried two gene clusters that may be involved in the degradation of polysaccharide nutrients. Thirteen recombinant cellulases were partially characterized. They showed diverse optimal pH from 4 to 7. Seven cellulases were most active under acidic conditions with optimal pH of 5·5 or lower. Furthermore, one novel cellulase gene, C67‐1, was overexpressed in Escherichia coli, and the purified recombinant enzyme showed optimal activity at pH 4·5 and stability in a broad pH range from pH 3·5 to 10·5. Its enzyme activity was stimulated by dl‐dithiothreitol. Conclusions:  The cellulases cloned in this work may play important roles in the degradation of celluloses in the variable and low pH environment in buffalo rumen. Significance and Impact of the Study:  This study provided evidence for the diversity and function of cellulases in the rumen. The cloned cellulases may at one point of time offer potential industrial applications.

No abstract

Background: Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.

A novel prephenate dehydrogenase gene designated pdhE-1 was cloned by sequence-based screening of a plasmid metagenomic library from uncultured alkaline-polluted microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis indicated that PdhE-1 and other putative prephenate dehydrogenases were closely related. The putative prephenate dehydrogenase gene was subcloned into pETBlue-2 vector and overexpressed in Escherichia coli BL21(DE3) pLacI. The recombinant protein was purified to homogeneity. The maximum activity of the PdhE-1 protein occurred at pH 8.0 and 45 °C using prephenic acid as the substrate. The prephenate dehydrogenase had an apparent K m value of 0.87 mM, a V max value of 41.5 U/mg, a k cat value of 604.8/min and a k cat/K m value of 1.16 × 10(4)/mol/s. L-Tyrosine did not obviously inhibit the recombinant PdhE-1 protein. The identification of a metagnome-derived prephenate dehydrogenase provides novel material for studies and application of proteins involved in tyrosine biosynthesis.