Extraction, purification and characterization of inhibitor of trypsin from Chenopodium quinoa seeds

Abstract

A novel trypsin inhibitor of protease (CqTI) was purified from Chenopodium quinoa seeds. The optimal extracting solvent was 0.1M NaCl pH 6.8 (p < 0.05). The extraction time of 5h and 90 °C was optimum for the recovery of the trypsin inhibitor from C. quinoa seeds. The purification occurred in gel-filtration and reverse phase chromatography. CqTI presented active against commercial bovine trypsin and chymotrypsin and had a specific activity of 5,033.00 (TIU/mg), which was purified to 333.5-fold. The extent of purification was determined by SDS-PAGE. CqTI had an apparent molecular weight of approximately 12KDa and two bands in reduced conditions as determined by Tricine-SDS-PAGE. MALDI-TOF showed two peaks in 4,246.5 and 7,908.18m/z. CqTI presented high levels of essential amino acids. N-terminal amino acid sequence of this protein did not show similarity to any known protease inhibitor. Its activity was stable over a pH range (2-12), temperatures range (20-100 °C) and reducing agents.Keywords: purification; characterization; inhibitor of trypsin; Chenopodium quinoa; seeds. Practical Application:The purification and characterization of this novel trypsin inhibitor allow its study for a diversity of applications such as antibacterial, anticancer, anti-inflammatory and biotechnology and its further use as drugs for human diseases.

New Protocol for Production of Reduced-Gluten Wheat Bread and Pasta and Clinical Effect in Patients with Irritable Bowel Syndrome: A randomised, Double-Blind, Cross-Over Study

“…The lyophilized was dissolved in 10 mmol/L Tris-HCl, pH 8.0 and protein concentration was determined using Coomassie blue staining by Bradford method [36]. The albumin composition was analyzed by Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel (SDS-PAGE) according to the procedure of Schägger and von Jagow [37] with some modifications [38]. The albumin ‘solutions were mixed with a sample buffer (0.05M Tris-HCl at pH 6.8, 4% ( w / v ) SDS, 12% ( v / v ) glycerol, 0.01% ( w / v ) bromophenol blue, and 2% ( v / v ) 2-ME) and heated for 3 min in a boiling water bath, then centrifuged at 10,000 rpm for 3min.…”

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