Cupincin: A Unique Protease Purified from Rice (Oryza sativa L.) Bran Is a New Member of the Cupin Superfamily

Roopesh, Sreedhar and Purnima Kaul, Tiku (2016) Cupincin: A Unique Protease Purified from Rice (Oryza sativa L.) Bran Is a New Member of the Cupin Superfamily. PLoS ONE, 11 (4). pp. 1-20.

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Abstract

Cupin superfamily is one of the most diverse super families. This study reports the purification and characterization of a novel cupin domain containing protease from rice bran for the first time. Hypothetical protein OsI_13867 was identified and named as cupincin. Cupincin was purified to 4.4 folds with a recovery of 4.9%. Cupincin had an optimum pH and temperature of pH 4.0 and 60°C respectively. Cupincin was found to be a homotrimer, consisting of three distinct subunits with apparent molecular masses of 33.45 kDa, 22.35 kDa and 16.67 kDa as determined by MALDI-TOF, whereas it eluted as a single unit with an apparent molecular mass of 135.33 ± 3.52 kDa in analytical gel filtration and migrated as a single band in native page, suggesting its homogeneity. Sequence identity of cupincin was deduced by determining the amino-terminal sequence of the polypeptide chains and by and de novo sequencing. For understanding the hydrolysing mechanism of cupincin, its threedimensional model was developed. Structural analysis indicated that cupincin contains His313, His326 and Glu318 with zinc ion as the putative active site residues, inhibition of enzyme activity by 1,10-phenanthroline and atomic absorption spectroscopy confirmed the presence of zinc ion. The cleavage specificity of cupincin towards oxidized B-chain of insulin was highly specific; cleaving at the Leu15-Tyr16 position, the specificity was also determined using neurotensin as a substrate, where it cleaved only at the Glu1-Tyr2 position. Limited proteolysis of the protease suggests a specific function for cupincin. These results demonstrated cupincin as a completely new protease.

Item Type:	Article
Uncontrolled Keywords:	cupin,protease, rice bran
Subjects:	600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes 600 Technology > 08 Food technology > 16 Nutritive value > 07 Waste utilization 600 Technology > 08 Food technology > 21 Cereals > 01 Rice
Divisions:	Protein Chemistry and Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	10 Aug 2016 11:10
Last Modified:	10 Aug 2016 11:10
URI:	http://ir.cftri.res.in/id/eprint/12240

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