title: Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum).
abstract: The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20 mM tris-CaCl2buffer (pH 8.2) with a flow rate of 0.5 mL min-1. The molecular weight and purity of ∼23 kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697 U mg-1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.
authors.affiliation: a Department of Biotechnology , Jamia Millia Islamia , New Delhi , India.
authors.forename: Tooba Naz
authors.initials: TN
authors.lastname: Shamsi
authors.affiliation: a Department of Biotechnology , Jamia Millia Islamia , New Delhi , India.
authors.forename: Romana
authors.initials: R
authors.lastname: Parveen
authors.affiliation: b Centre for Bioseparation Technology, VIT , Vellore , Tamil Nadu , India.
authors.forename: Priyankar
authors.initials: P
authors.lastname: Sen
authors.affiliation: a Department of Biotechnology , Jamia Millia Islamia , New Delhi , India.
authors.forename: Sadaf
authors.initials: S
authors.lastname: Fatima
mesh_terms: D000818:Animals; D029863:Cicer / Q000737:chemistry / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
chemical_list: C114880:trypsin-like serine protease; D012697:Serine Endopeptidases; D014357:Trypsin
keywords: BAPNA: Cajanus cajan trypsin inhibitor: CCTI; Cicer arientum; LBTI: lima bean trypsin inhibitor; Nα-benzoyl-DL-arginine-p-nitroanilide; SDS: sodium dodecyl sulfate; TEMED: tetramethylethylenediamine; chickpea trypsin: CpT; enzyme; food; ion-exchange chromatography; phenylmethyl sulfonyl fluoride: PMSF; purification; trypsin