title: Expression, purification and identification of a thermolysin-like protease, neutral protease I, from Aspergillus oryzae with the Pichia pastoris expression system.
abstract: Neutral proteases are widely used in the textile, food and medical industries. This study was designed to obtain high expression levels of neutral protease I from Aspergillus oryzae 3.042 by using Pichia pastoris GS115 as the host strain for industrial purposes. The coding sequence of the target gene was modified, synthesized, and then cloned into the expression vector pHBM905BDM, which harbored the d1+2 × 201 AOX1 promoter and the MF4I leader sequence. The recombinant plasmid was transformed into Pichia pastoris GS115. The recombinant strain was used for high-density fermentation in a 4-L fermenter. The yield of the target protein reached 12.87 mg/mL, and the enzyme activity was approximately 49370 U/mL, which indicated that this enzyme was expressed in Pichia pastoris at a high level. The target protein was purified and characterized. Its optimum temperature and pH were 55 °C and 8.0, respectively. This enzyme was extremely sensitive to EDTA, which is consistent with the previous reports that it is a zinc-dependent metalloprotease. Our results indicated that low concentrations of zinc, calcium and magnesium ions stimulated the enzyme activity, whereas high concentrations inhibited its activity. In addition, calcium and magnesium ions increased the thermostability of the enzyme. All of the evidence indicated that this protease is a thermolysin-like peptidase.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China. Electronic address: 910062852@qq.com.
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Ma
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China. Electronic address: liuyunyun0823@126.com.
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China. Electronic address: 1343811610@qq.com.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Li
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China. Electronic address: 781793179@qq.com.
authors.forename: Lu
authors.initials: L
authors.lastname: Liu
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China. Electronic address: liyi@hubu.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Yi
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China. Electronic address: malixing@hubu.edu.cn.
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China. Electronic address: chaozhai2016@163.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhai
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases
keywords: Neutral protease I; Pichia pastoris; Thermolysin-like protease