Constrution Of β-Mannanase Gene And Bovine Prochymosin Gene Engineering Strains Of Aspergillus Niger

β-mannanase (endo-1,4-β-D-mannanase;mannohydrolase;EC3.2.1.78) are a kind ofsemi-cellulose enzymes which be able to hydrolyze mannose polysaccharides of β-1,4--D-mannosidase endo bond.β-mannanases are produced by various organisms, such asanimal, plant and microorganism. Now β-mannanase has been widely used in the food, feed, papermaking, washing,spinning, oil exploration and other aspects of biological research techniques.TheUnited States ChemGen developed β-mannanase product "beauty enzyme, its price up to600,000yuan/t.Therefore, we want to break the monopoly of foreign companies on the domestic market price,to achieve large-scale application of β-mannanase, the application of construction of geneticengineering bacteria mannanase is an effective way to solve this problem.Chymosin is the most essential to the industrial production of cheese since it can cause proteincohesion and lead milk to condense. The traditional method is to slaughter the newborn calves toextracte chymosin from the abomasum, due to the the cheese constantly increasing demand, resultingin worldwide shortage of calves and making chymosin sources are not stable. In order to resolve thiscontradiction, the genetic engineering of chymosin and good economic benefits in abroad.In this study, we took Aspergillus niger CICC2462as the receptor material to construct the genereplacement vector pSZH-MAN and pSZH-GCYM directing towards high expression ofglucoamylase gene loci,and transformed the gene using Agrobacterium-mediatedTransformation(ATMT).In order to achieve the efficient expression of target gene, thereby laying thefoundation for constructing a safe and efficient engineered strains of Aspergillus niger. This researchprovides new ways for simplifying the β-mannanase and chymosin fermentation production process,improving production β-mannanase and chymosin.The main results are as follows:1.Expression of β-mannanase gene in Aspergillus nigerThe sequence of β-mannanase gene MAN (1275bp)was amplified from Aspergillus nigerCICC2462,by overlap extension PCR make MAN gene fragmen, the glucoamylase gene5’homologyarm, the3’ homologous arms for splicing homologous recombination expression cassette GMG. Therecombination expression cassette was inserted into expressional vector pSZH-CYM to form theexpressional vector pSZH-MAN of Aspergillus niger. The pSZH-MAN was transformed into Aspergillus niger CICC2462by ATMT. Four positive transgenic strains were detected by hygromycinselection and identification of PCR. One homologous recombinant strains were obtained whichreplaced glucoamylase gene locus. Then through continuous passage, screening and PCR to theobtaine homologous recombinant strains of homozygous.By fermentation cultivation,enzyme activitydetection and SDS-PAGE analysis of the recombinant strains,showing that the saccharifying enzymeshake flask fermentation conditions, recombinant strains culture solution supernatant of β-mannanaseactivity as high as12467.2U/mL,which was72times that of the starting strain and12-15times that ofthe level of production. The results of this study provides a new way to simplify the process offermentation of β-mannanase, improve enzyme production.2.Expression of bovine chymosin gene in Aspergillus nigerThe pSZH-CYM expressional vector which has been constructed in our lab was transformed intoAspergillus niger CICC2462by ATMT. Three positive transgenic strains were detected byhygromycin selection and identification of PCR. One homologous recombinant strains were obtainedwhich replaced glucoamylase gene locus. Then through continuous passage, screening and PCR to theobtaine homologous recombinant strains of homozygous. By fermentation cultivation,enzyme activitydetection recombinant strains, showing that the saccharifying enzyme shake flask fermentationconditions, recombinant strains culture solution supernatant of chymosin activity as high as960u/mL.Iorder to further improve the expresstion of CYM gene in the Aspergillus nigerCICC2462,making the CYM genes fused with glucoamylase gene which has high expression inAspergillus niger CICC2462for the purpers of fusion expression. Constructing fusion expressionvector pSZH-GCYM. The expressional vector pSZH-GCYM was transformed into Aspergillus nigerCICC2462by ATMT.Four positive transgenic strains were detected by hygromycin selection andidentification of PCR. Two homologous recombinant strains were obtained which replacedglucoamylase gene locus,,but has not yet been detected enzyme activity.