Expression Of Kawachii Acid Protease In Aspergillus Niger

Acid protease has lower optimum p H(2~4) and under the condition of the low pH, enzymes which can hydrolyze protein effectively are amino acids and small peptides. Acid protease mainly come from the animal organs and the secretion of microorganism, lots of microorganism is capable of producing acid protease. It was first found in Aspergillus niger in the 50 s of this century, and in the early 80 s China has begun to product acid protease by Aspergillus niger solid or liquid fermentation. With subsequent application field expanding unceasingly, the demand for alcohol, liquor, beer, brewing, feed additive, leather processing and pharmaceutical industries is also gradually increasing.Acid protease which is from Zhaodong Richeng enzyme Ltd. has the characteristics of the liquid under the stable, avoiding the shortcomings of the most liquid acid protease of their short shelf life. However, compared with liquid fermentation has a high degree of mechanization, strict technical management, high enzyme production rate and stable quality, high product recovery rate, etc., solid-state fermentation methods of production limits its large-scale production and application. This experiment chooses saccharifying enzyme production fungus Aspergillus niger CICC2462 which was suitable for liquid fermentation as transformation receptor to express the acid protease, and explor ing new ways to create acid protease strains which has high yield under liquid stability that has important application value.The experimental research results are as follows The main experimental results are as follows:1. Identification of the Solid-state fermentation acid protease production strains and the productSolid-state fermentation acid protease production strains of ITS area was amplified by PCR. And the ITS sequence identification results showed that the strain belongs to Aspergillus. By SDS-PAGE and mass spectrometry, the results showed that the product is Aspergillopepsin I(EC.3.4.23.18) of Aspergillus saitoi acidic protease enzyme.2. The construction of acid protease gene cloning and expression vectorAccording to gene sequence pep1(GI:473517) from Aspergillopepsin I of Aspergillus saitoi acid protease, primers were designed, producing strain’s genomic DNA from solid-state fermentation acid protease as the template, and by the method of PCR, pep1 gene was cloned. Sequence analysis showed that Amplified fragment is 99 % similar to acid protease’s genome sequence of Aspergillus kawachii and encoding protein is 100 % similar to acid protease of Aspergillus kawachii, and it was named as pep B. Then the highly expressed genes of Aspergillus niger glucoamylase(gla A) site was as the targeted point, constructed the Aspergillus niger gene replacement expression vector p SZHG-pep B.3. Method of agrobacterium mediated transformation of Aspergillus niger and the screening of acid protease homologous recombinant transformation.Aspergillus niger recombinant expression vector p SZHG-pep B was transformed into agrobacterium AGL1 by agrobacterium-mediated method of freezing and thawing, then by the method of agrobacterium mediated to transformed the Aspergi llus niger mycelia for genetic transformation. The genome DNA of confrontational colonies was identified with homologous recombination premiers by the PCR technology and the results showed that the positive transformation p SZHG-pep B were all homologous recombinant strains and the homologous recombination rate was 100 %. After successive transfer culture of homologous recombination strains and screenin, we got successfully pSZHG-pep B pure source recombinant strains of the contract.4. Expression analysis of acid protease in the pure homologous recombination strainThe pure homologous recombination strain of p SZHG-pep B and the starting strains were cultured for shaking flask fermentation simultaneously, the acid protease enzyme activity was detected by the forint phenol method. the results showed that enzyme activity of p SZHG-pepB is up to 7614.307 U/m L, which is 209 times to the starting strain. SDS-PAGE detection results showed that pure contract source of recombinant strains fermented supernatant had obvious purpose stripe in 47 KDa.5. The acid protease enzyme analysis and stability analysisp SZHG-pepB purely contractual source of recombinant strains Characterization of the test results as follows: enzyme optimum temperature was 50 ℃, the optimum p H was 3.0.And the crude enzyme solution was set at 4 ℃ and 25 ℃, pH 3.0-4.0 were relatively stable when the p H is lower than 3.0 or higher than 4.0, activity has declined.