Abstract
Rice bran is an underutilized agricultural by-product with economic importance. The unique phytochemicals and fatty acid compositions of bran have been targeted for nutraceutical development. The endogenous lipases and hydrolases are responsible for the rapid deterioration of rice bran. Hence, we attempted to provide the first comprehensive profiling of active serine hydrolases (SHs) present in rice bran proteome by activity-based protein profiling (ABPP) strategy. The active site-directed fluorophosphonate probe (rhodamine and biotin-conjugated) was used for the detection and identification of active SHs. ABPP revealed 55 uncharacterized active-SHs and are representing five different known enzyme families. Based on motif and domain analyses, one of the uncharacterized and miss annotated SHs (Os12Ssp, storage protein) was selected for biochemical characterization by overexpressing in yeast. The purified recombinant protein authenticated the serine protease activity in time and protein-dependent studies. Os12Ssp exhibited the maximum activity at a pH between 7.0 and 8.0. The protease activity was inhibited by the covalent serine protease inhibitor, which suggests that the ABPP approach is indeed reliable than the sequence-based annotations. Collectively, the comprehensive knowledge generated from this study would be useful in expanding the current understanding of rice bran SHs and paves the way for better utilization/stabilization of rice bran.
Highlights
Rice bran is an underutilized agricultural by-product with economic importance
These esterase inhibitors had no effect on the labeling of both 50 and 27 kDa proteins, which further confirmed that these two proteins could be serine proteases
activity-based protein profiling (ABPP) assay indicated that there were many putative serine hydrolases active during the assay conditions, and these were sensitive to the tested inhibitors
Summary
Rice bran is an underutilized agricultural by-product with economic importance. The unique phytochemicals and fatty acid compositions of bran have been targeted for nutraceutical development. We attempted to provide the first comprehensive profiling of active serine hydrolases (SHs) present in rice bran proteome by activity-based protein profiling (ABPP) strategy. There are no reports or attempts made to provide comprehensive profiling of active SH enzymes present in rice bran. With the advent of Activity-based protein profiling (ABPP), the functional annotation is possible in the native biological s ystems[18, 19]. Serine hydrolases (SHs) play a significant role in virtually all physiological processes It consists of a wide range of enzymes that carry an activated serine residue in their catalytic site. We attempted to provide the first comprehensive profiling of active serine hydrolases present in O. sativa bran proteome by the ABPP approach. The biochemical characterization of the 12S storage protein was demonstrated as a serine protease in this study
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