Expression, purification and identification of a thermolysin-like protease, neutral protease I, from Aspergillus oryzae with the Pichia pastoris expression system

• Published in: Protein expression and purification 2016-12, Vol.128, p.52-59
• Main Authors: Ma, Xiaojian, Liu, Yunyun, Li, Qingqing, Liu, Lu, Yi, Li, Ma, Lixin, Zhai, Chao
• Format: Article
• Language: English
• Subjects: Aspergillus oryzae - enzymology; Aspergillus oryzae - genetics; Fungal Proteins - biosynthesis; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Gene Expression; Metalloendopeptidases - biosynthesis; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metalloendopeptidases - isolation & purification; Neutral protease I; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Thermolysin-like protease
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2016.08.008

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. •The recombiant neutral protease was expressed in Pichia pastoris at a high level under high-density fermentation.•The gene of recombiant neutral protease was modified based on the codon usage bias of P. Pastoris.•Low concentrations of zinc, calcium and magnesium ions stimulated the recombiant neutral protease enzyme activity.•All of the evidence indicated that the recombiant neutral protease is a thermolysin-like peptidase.