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

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Title Cupincin: A Unique Protease Purified from
Rice (Oryza sativa L.) Bran Is a New Member
of the Cupin Superfamily
 
Creator Roopesh, Sreedhar
Purnima Kaul, Tiku
 
Subject 05 Enzymes
07 Waste utilization
01 Rice
 
Description 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.
 
Date 2016
 
Type Article
PeerReviewed
 
Format application/pdf
 
Language en
 
Rights —
 
Identifier http://ir.cftri.com/12240/1/PLosOne_2016.pdf
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.