Homologous Expression Of Ferulic Acid Esterase Gene In Aspergillus Niger

Ferulic acid esterase were plays an important role in plant cell wall degradation, and it hydrolyzed the easter bond between hemicellulose and cellulose in plant fiber materials, and released ferulic acid. Ferulic acid esterase mainly came from microorganisms, but its production was low, the expression level was not high, it can not meet the requirements of industrial production. It reported that ferulic acid esterase secretory expression in pichia expression system research, the expression was0.0679U/g, but the production level still had a gap distance for mass industrial production. Aspergillus niger was one of the world recognized safety production strains, it had excellent ability of protein secretion, efficient and accurate translation processing and with higher eukaryotic animal similar glycosylation modification system. These characteristics caused more and more attentions. Aspergillus niger was the main of industrialization production strains of ferulic acid esterase. By using of food-grade Aspergillus niger expression system expressed ferulic acid esterase was an efficient path which can increase production of ferulic acid esterase and reduce production cost.Ferulic acid esterase (faeA) coding region in Aspergillus niger was cloned by the method of PCR, constructed Aspergillus niger expression vector pSZHG-faeA and transformed Aspergillus niger in the use of agrobacterium-mediated method, screened out positive homologous transformants. The homologous recombination transformants of fermented supernatant on enzyme activity detection and SDS-PAGE analysis. The result indicated that this reserch realized homologous expression of ferulic acid esterase in Aspergillus niger. This research laid the foundation for mass industrial production of food-grade ferulic acid esterase.The main results in this study are as follows:1. Construction of Aspergillus niger expression vector pSZHGUsing the method of CTAB extraction of Aspergillus niger genomic DNA as a template, and the Aspergillus niger glucoamylase gene5’GLA fragment and3’GLA fragment were gained by using PCR amplification. Connected the two fragments with the vector pSZH, and constructed the Aspergillus niger expression vector.2. Construction of ferulic acid esterase (faeA) gene of Aspergillus niger expression vector pSZHG-faeAThe study take production glucoamylase’s Aspergillus niger CICC2462genomic DNA as a template, using gene specific primers, cloned Aspergillus niger homologous faeA gene by PCR. Respectively, using double enzyme(Apa Ⅰ/Cla I) digested the pMD-faeA plasmid and Aspergillus niger expression vector pSZHG, and connected them. Last, constructed the Aspergillus niger expression vector pSZHG-faeA.3. Agrobacterium-mediated transformation of Aspergillus niger, screening of transformants and homologous transformants identificationTaking of freeze-thaw method make Aspergillus niger expression vector pSZHG-faeA transformed into Agrobacterium AGL1. Transformed Aspergillus niger in the use of agrobacterium-mediated method. In the end,4positive homologous transformants were screened out by the method of PCR. Identification of4positive transformants with homologous recombination primers by PCR. As a result,4positive strains can amplify1701bp target fragments. It showed that they were homologous recombinants. The homologous recombinantion rate of transformation pSZHG-faeA was100%.4. Expression of ferulic acid esterase in Aspergillus nigerRespectively, using the method of DSWB and the method of ferulic acid methyl ester measured the ferulic acid esterase activity. The results showed that the ferulic acid esterase activity up to18.6mU/mL by the method of DSWB, and ferulic acid esterase activity up to22.12mU/mL by the method of ferulic acid methyl ester.By used of SDS-PAGE analysis of engineering strain culture supernatant, the result showed that all of4engineering strains had been secreted and expressed successfully, significantly higher than the starting strain. This research took the4recombinant strains protein bands to the company for protein spectrum analysis. The result showed that4recombinant strains proteins were really the ferulic acid esterase protein in Aspergillus niger. By the ALPHAIMAGER SYSTEMS software analysis, the protein expression quality was188-262μg/mL.