title: Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit.
abstract: Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The Kmand Vmaxvalues of the enzyme for casein were obtained to be 1.29mg/ml and 0.035μmol Tyr/min, respectively. Optimal temperature and pH were 65°C and 5.5, respectively. After incubation of enzyme at 65°C for 1h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl2reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses.
authors.affiliation: Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Salehi
authors.affiliation: Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran. Electronic address: aghamaali@guilan.ac.ir.
authors.forename: Mahmoud Reza
authors.initials: MR
authors.lastname: Aghamaali
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Reza H
authors.initials: RH
authors.lastname: Sajedi
authors.affiliation: Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran.
authors.forename: S Mohsen
authors.initials: SM
authors.lastname: Asghari
authors.affiliation: Department of Biology, Faculty of Science and Engineering, Gonbad kavous University, Gonbad Kavous, Iran.
authors.forename: Eisa
authors.initials: E
authors.lastname: Jorjani
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000302:isolation & purification*; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D004795:Enzyme Stability; D005638:Fruit / Q000201:enzymology; D006868:Hydrolysis; D008892:Milk / Q000737:chemistry; D008970:Molecular Weight; D032341:Withania / Q000201:enzymology
chemical_list: D002364:Caseins; D057055:Aspartic Acid Proteases; D016282:Aspartic Acid Endopeptidases; C022517:rennin-like enzyme (Aspergillus ochraceus)
keywords: Aspartic protease; Milk-clotting activity; Withania coagulans