Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091

• Published in: Food chemistry 2015-04, Vol.173, p.441-448
• Main Authors: Majumder, Rajib, Banik, Samudra Prosad, Khowala, Suman
• Format: Article
• Language: English
• Subjects: Animals; Aspartic Acid Endopeptidases; Casein; Caseins - metabolism; Cheese; Chymosin - metabolism; Edible mushroom; Enzymes; Fingerprinting; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Mass spectrometry; Metalloprotease; Metalloproteases - chemistry; Metalloproteases - isolation & purification; Metalloproteases - metabolism; Milk; Milk - metabolism; Milk clotting activity; Peptide mass fingerprinting; Peptides; Populus trichocarpa; Temperature; Termitomyces - enzymology; Termitomyces clypeatus; Two dimensional; Urea-PAGE
• ISSN: 0308-8146; 1873-7072
• DOI: 10.1016/j.foodchem.2014.10.027

•Edible mushroom T. clypeatus is a new source of milk-clotting protease (AcPs).•The milk-clotting protease was purified to homogeneity and characterised.•AcPs was characterised as a metalloprotease with high MCA/PA ratio.•AcPs hydrolysed milk casein in the order of κ-casein>α-caseins>β-casein.•T. clypeatus AcPs holds a potential use in biotechnological application. 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.