title: Enzymatic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris.
abstract: A truncated neutral protease I (NpI) from  Aspergillus oryzae  3.042 was expressed in  Pichia pastoris  with a high enzyme yield of 43101 U/mL. Its optimum pH was about 8.0, and it was stable in the pH range of 5.0-9.0. Its optimum temperature was about 55 °C and retained >90% activity at 50 °C for 120 min. Recombinant NpI (rNpI) was inhibited by Cu(2+) and EDTA. Eight cleavage sites of rNpI in oxidized insulin B-chain were determined by mass spectrometry, and five of them had high hydrophobic amino acid affinity, which makes it efficient in producing antihypertensive peptide IPP from β-casein and a potential debittering agent. The high degree of hydrolysis (DH) of rNpI to soybean protein (8.8%) and peanut protein (11.1%) compared to papain and alcalase makes it a good candidate in the processing of oil industry byproducts. The mutagenesis of H(429), H(433), and E(453) in the deduced zinc-binding motif confirmed rNpI as a gluzincin. All of these results show the great potential of rNpI to be used in the protein hydrolysis industry.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu District, Guangzhou, Guangdong, People's Republic of China 510006.
authors.forename: Ye
authors.initials: Y
authors.lastname: Ke
authors.forename: Wei-Qian
authors.initials: WQ
authors.lastname: Huang
authors.forename: Jia-zhou
authors.initials: JZ
authors.lastname: Li
authors.forename: Ming-quan
authors.initials: MQ
authors.lastname: Xie
authors.forename: Xiao-chun
authors.initials: XC
authors.lastname: Luo
mesh_terms: D010367:Arachis / Q000737:chemistry; D001236:Aspergillus oryzae / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D030262:Soybean Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D007328:Insulin; D011994:Recombinant Proteins; D030262:Soybean Proteins; D008666:Metalloendopeptidases