Expressing Of The Protease Gene From The A.oryaze

Aspergillus oryzae is an important industrial micro-organisms. It is rich in enzymes which have high stability, high temperature capability protease and carbohydrase. So far, it have not found the fungus Aspergillus toxins. So it is widely used in food and feed industries because of its high security pharmaceutical. It is the duty to improve their activity to the biological workers in production.In this paper, total RNA was successfully extracted from Aspergillus oryzae fermentation product. The alkaline and neutral protease genes were amplified by RT-PCR. Also the protease genes are expressed in Escherichia coli and Pichia pastoris respectively.The main content are illustrated as following:1. The total RNA extracted successfully from the solid-state fermentation of Aspergillus oryzae, cloned the neutral and alkaline protease gene;2. The genes were inserted into a expression vector 6HisT-pRX2 to get the recombinant plasmid 6HisT-pRX2-NIP and 6HisT-pRX2-ALP. The recombinant plasmid transformed into E.coli BL21 and induce respectively by lactose and IPTG;3 The recombinant neutral protease with 6His-Tag was purify by Ni-NTA His.Bind Resin; The recombinant neutral protease's activity is 304U/g by Fu Linfen method;4 The genes were inserted into a expression vector pPIC9K to get the recombinant plasmid pPIC9K-NIPJT, pPIC9K-ALPZT, the recombinant plasmid transformed into yeast GS115 and induce by methanol. Compare the different signal peptides impaction of the own signal peptide Aspergillus oryzae and the yeast's own signal peptide...