Research Of Pepa Gene From Aspergillus Usamii Expression In Aspergillus Niger

Acid protease is one kind of aspartic protease with the optimal pH of2.5-5.0. Fungal acid protease can hydrolyze proteins effectively at low pH, so it is widely used in wine-making, food handing, feed additives, leather processing and so on, is one important product of the industry of enzymatic preparation with good commercial development value. Aspergillus usamii is the traditional seed lot in producting acid protease by fermentation. The output of traditional fermentation using in the product of acid protease is not ideal, so it is effective way to obtian high-yield strains of the acid protease through genetic engineering technique.Aspergillus niger is one kind of industrial microorganism widely used on the account of its abundant enzyme systems and metabolites. As the recipient bacterium of genetic engineering, Aspergillus niger has special advantages which are not exist in bacterium and yeast, such as high ability of secreting proteins in high lever(Some glucoamylase producing strains in ideal culture conditions secret glucoamylase up to20g/L from Fermentation liquid), correct processing after translation, the similarity with higher eukaryotic animals, accepted safety in production and mature fermentation and post-treatment procedure. Aspergillus usamii and Aspergillus niger both belong to Aspergillus, with the same mechanism of gene expression regulation. Therefore, the expression of Aspergillus usamii acidic protease gene under the regulation of Aspergillus niger glucoamylase gene promoter is expected to establish Aspergillus niger engineering bacteria with high efficient expression of acidic protease. It is important to increase the output of acid protease and simplify the fermentation conditions.In this study, A-1347bp pepA gene from Aspergillus usamii and glucoamylase homologous arms5’GLA and3’GLA are spliced by overlap extension PCR, made pepA regulated by the promoter of glucoamylase.Through this method, the expression vector of Aspergillus niger was constructured. Then the mycelium of Aspergillus niger was transformed via Agrobacterium mediation and the expresssion activity of acid protease was analyzed in order to lay the foundations for industrial production and application. The main results of the study is as follows:1. Construction of the expression vector of Aspergillus nigerThe gene of acid protease (pepA) in aspergillus usamii, the upstream and downstream genetic fragments of the gene of glucoamylase in Aspergillus niger(GLA5and GLA3) were obtained by PCR ampliation. Using overlap extension PCR, the homologous recombination expression fragment (GLA5-pepA-GLA3) was obtained by splicing the GLA5, GLA3and pepA,The expression vector of Aspergillus niger (pSZH-pepA) was constructured via Agrobacterium mediation, T-DNA of which consequently connected two homologous recombination expression fragments of the genes of acid protease and hygromycin.2. Establishment and optimization of genetic transformation system of the mycelium of Aspergillus niger via Agrobacterium mediationThe genetic transformation system of the mycelium of Aspergillus niger via Agrobacterium mediation was investigated by using the strain screening of hygromycin B and cefotaxime sodium, the concentration of acetosyringone, the choice of culture medium, the cell age of Aspergillus niger, the time and temperature of co-culture. The optimum transformation condition was when the PDA as co-culture medium, the concentration of acetosyringone was200μmol/L, the cell age was5d, the time and temperature of co-culture was48h and28℃.3. Expression of acid protease in Aspergillus nigerThe resulting plasmid named with pSZH-pepA was transformed into Agrobacterium tumefaciens AGL1using a freeze-thaw method, and then was transferred into Aspergillus niger mycelium, which is a high glucoamylase expression recipitent strain, through the mediation of Agrobacterium Tumefaciens. Aspergillus niger transformats were identified by PCR amplification, there are37positive strains and3homologous recombination transformants in69stable genetic strains. Pick the stable transformants into fermentative medium with300r/min grown at34℃for3d. Take the culture supernatant for SDS-PAGE after TCA concentration.The result show that the acidic protease of transformants with40Kda bands contrast original strain’s, so the gene of acid protease was expressed in Aspergillus niger under the glucoamylase production conditions. Using Forint-phenol method to determine the activity of zymotic fluid supernatant of recombinant strain, the result showed that the gene of acid protease expressed in recombinant and its greatest activity reached45.56U/mL, which was11.16U/mL in initial strain, so pepA expressed under the regulation of the promoter of glucoamylase gene.4. Research of fermentationRecombinant strain was cultured by shaking culture in different pH, culture temperatures and time in order to investigate the influence of these factors and determine the appropriate fermentation condition. The result showed that when the primary pH was5.0, the culture temperature was34℃and the culture time was72h, the output of acid protease was relatively ideal and the activity was45.56U/mL.