title: Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091.
abstract: Milk-clotting enzymes are valued as chymosin-like protease substitutes for cheese making industries. An extracellular metalloprotease (AcPs) with high milk-clotting activity was purified from edible mushroom Termitomyces clypeatus and characterised. AcPs was preferentially active towards κ-casein, analysed by Urea-PAGE and LC-ESI-MS, whereas the degradation of α and β-casein components by AcPs proceeded slowly justifying its suitability for cheese making. RP-HPLC peptide profiling revealed that the AcPs activity on milk casein was similar to that of a commercial milk coagulant. The enzyme exhibited pH and temperature optima at 5.0 and 45 °C, respectively and showed a pI value of 4.6. One- and two dimensional zymographies revealed a single polypeptide band with proteolytic signal. The MALDI-TOF/MS followed by peptide mass fingerprinting revealed homology with a predicted protein of Populus trichocarpa. To our knowledge, this is the first report on a metalloprotease from T. clypeatus, and the results indicate that this enzyme can be considered as a potential substitute for chymosin in cheese manufacturing.
authors.affiliation: Drug Development Diagnostics and Biotechnology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, India.
authors.forename: Rajib
authors.initials: R
authors.lastname: Majumder
authors.affiliation: Maulana Azad College, Department of Microbiology, 8, Rafi Ahmed Kidwai Road, Kolkata 700013, India.
authors.forename: Samudra Prosad
authors.initials: SP
authors.lastname: Banik
authors.affiliation: Drug Development Diagnostics and Biotechnology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, India. Electronic address: sumankhowala@iicb.res.in.
authors.forename: Suman
authors.initials: S
authors.lastname: Khowala
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002364:Caseins / Q000378:metabolism*; D002611:Cheese; D012085:Chymosin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008892:Milk / Q000378:metabolism*; D013696:Temperature; D055434:Termitomyces / Q000201:enzymology*
chemical_list: D002364:Caseins; D045726:Metalloproteases; D016282:Aspartic Acid Endopeptidases; C022517:rennin-like enzyme (Aspergillus ochraceus); D012085:Chymosin
keywords: Edible mushroom; Metalloprotease; Milk clotting activity; Peptide mass fingerprinting; Termitomyces clypeatus; Urea-PAGE