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


Xiaojian Ma;


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 dl+2x201 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 a4-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 were55℃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.




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