Cloning and Expression of Acidic Protease of Aspergillus kawachii in non-spore Aspergillus niger SH-2 and Analysis of Enzymatic Properties
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Abstract:
Acid protease of Aspergillus kawachii, which has the functions of dissolving the particles of the fermentation raw material , promoting microbial propagation, degrading yeast cell protein and other functions to improve liquor flavor, has been widely used in liquor production in the process of fermentation. Aspergillus niger SH-2, which had the property of low endogenous protein expression after genetic engineering transformation, was used in this study to express acid protease gene pepB from Aspergillus kawachii.. The gene pepB and expression elements glucoamylase promoter PglaA, glucoamylase terminator TglaA, orotic acid nucleoside-5-phosphate decarboxylase marker gene pyrG were obtained by PCR., and the pepB expression vector was constructed on the basis of pMD18-T vector. The recombinant strains were fermented for 240 h in the fermentor and the enzyme activity of the fermented crude enzyme was 9722 U/g, which was 8.5 times as much as that of the reported original enzyme strain, and SDS-PAGE pattern showed that the molecular weight of the expressed product was about 47 ku. The results of enzyme properties analysis showed that the optimum reaction temperature of the acidic protease was 35 oC and the optimum pH was 4.0. Mn2+ and Cu2+ had significant activation effect on the enzyme activity. Finally, the changes in the enzyme activity of recombinant strains under different fermentation pH were investigated. The results showed that in the range of pH 4.5-6.5, the initial pH of the fermentation was properly increased, and the activity of acid protease increased. Consequently, this study has successfully constructed a strain that can express the acid protease of Aspergillus kawachii.
