title: Purification and characterization of a novel milk-clotting metalloproteinase from Paenibacillus spp. BD3526.
abstract: In this study, a milk-clotting enzyme (MCE) isolated from Paenibacillus spp. BD3526 was purified and characterized. The MCE was purified 8.9-fold with a 10.11% recovery using ammonium sulfate precipitation and anion-exchange chromatography and the specific milk-clotting activity (MCA) reached 6791.73 SU/mg. The enzyme was characterized as a 35kDa metalloproteinase, and the zymogen of which was encoded by a 1671 bp gene named zinc metalloproteinase precursor (zmp) with a predicted molecular weight of 59.6 kDa. The optimal temperature for MCA and proteolytic activity (PA) was 65°C and 60°C, respectively. The enzyme was stable over a pH range of 5.0-9.0 and at temperatures below 50°C. The MCA was completely inactivated when the enzyme was heated at 60°C for 30 min, and the PA was totally inactivated for 20 and 10 min when the enzyme was heated at 55°C and 60°C, respectively. The BD3526 enzyme was preferentially active towards κ-casein (κ-CN) and β-casein (β-CN), as determined by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), whereas the hydrolysis of αs-casein (αs-CN) was slow and comparable to that caused by chymosin and asparatic acid proteinase from Rhizomucor miehei. The cleavage site of the metalloproteinase in κ-CN was located at the Met106-Ala107 bond, as determined by mass spectrometry analysis.
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China; State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
authors.forename: Feng
authors.initials: F
authors.lastname: Hang
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
authors.forename: Qinbo
authors.initials: Q
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Hong
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
authors.forename: Peiyi
authors.initials: P
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
authors.forename: Zhengjun
authors.initials: Z
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
authors.forename: Zhenmin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China; Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, PR China. Electronic address: chenwei66@jiangnan.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry* / Q000302:isolation & purification*; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D056507:Paenibacillus / Q000201:enzymology*; D059748:Proteolysis; D013696:Temperature
chemical_list: D045726:Metalloproteases; D002118:Calcium
keywords: Metalloproteinase; Milk clotting activity; Paenibacillus; Proteolytic activity; Zymogen