Abstract
Regulation of microbial urease activity plays a crucial role in improving the utilization efficiency of urea and reducing nitrogen emissions to the environment for ruminant animals. Dealing with the diversity of microbial urease and identifying highly active urease as the target is the key for future regulation. However, the identification of active urease in the rumen is currently limited due to large numbers of uncultured microorganisms. In the present study, we describe an activity- and enrichment-based metaproteomic analysis as an approach for the discovery of highly active urease from the rumen microbiota of cattle. We conducted an optimization method of protein extraction and purification to obtain higher urease activity protein. Cryomilling was the best choice among the six applied protein extraction methods (ultrasonication, bead beating, cryomilling, high-pressure press, freeze-thawing, and protein extraction kit) for obtaining protein with high urease activity. The extracted protein by cryomilling was further enriched through gel filtration chromatography to obtain the fraction with the highest urease activity. Then, by using SDS-PAGE, the gel band including urease was excised and analyzed using LC-MS/MS, searching against a metagenome-derived protein database. Finally, we identified six microbial active ureases from 2225 rumen proteins, and the identified ureases were homologous to those of Fibrobacter and Treponema. Moreover, by comparing the 3D protein structures of the identified ureases and known ureases, we found that the residues in the β-turn of flap regions were nonconserved, which might be crucial in influencing the flexibility of flap regions and urease activity. In conclusion, the active urease from rumen microbes was identified by the approach of activity- and enrichment-based metaproteomics, which provides the target for designing a novel efficient urease inhibitor to regulate rumen microbial urease activity.
Highlights
IntroductionUrease (urea amidohydrolase, EC 3.5.1.5), a nickel-dependent metalloenzyme, catalyzes the hydrolysis of urea into ammonia and carbon dioxide [1]
Urease, a nickel-dependent metalloenzyme, catalyzes the hydrolysis of urea into ammonia and carbon dioxide [1]
We focused on proteins with urease activity in the present study
Summary
Urease (urea amidohydrolase, EC 3.5.1.5), a nickel-dependent metalloenzyme, catalyzes the hydrolysis of urea into ammonia and carbon dioxide [1]. Urea is commonly used as a cost-efficient replacement for feed proteins to provide the sole nitrogen source for urease [2,3]. The high catalytic activity of microbial urease contributes to the fact that the rate of urea hydrolysis is fourfold more rapid than that of ammonia synthesis [5], which reduces the utilization efficiency of urea, and increases nitrogen emissions to the environment. The key to making full use of urea is to make ammonia synthesize microbial protein as much as possible by regulating rumen microbial urease activity
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