Comparison Of Basic Culture Characteristics Of Different Aspergillus Oryzae Strains And Heterology Expression Of Lipase

Aspergillus oryzae is used as a common strain of traditional brewed foods because of the ability to secrete large amounts of hydrolases.The hydrolase secreted by A.oryzae mainly includes proteolytic enzymes and saccharide hydrolases.These two types of enzymes decompose the amino acid,polypeptide,oligosaccharide,monosaccharide and other small molecules produced by the substrate,which is the main source of the flavor of traditional fermented food.A.oryzae with excellent enzyme production performance is of great significance to the fermentation industry.In recent years,A.oryzae has received increasing attention due to its strong ability to express heterologous proteins.A.oryzae has a stronger post-translational modification function than Escherichia coli and Pichia pastoris;compared with plants and insects,A.oryzae can lives in more demaning growth conditions and has shorter growth cycle.So,A.oryzae is considered to be an ideal host for the expression of heterologous proteins.In this paper,several strains of A.oryzae preserved in our laboratory were compared.The main research contents are as follows:1.Comparative analysis of culture characteristics of different Aspergillus oryzae strainsThe sporulation of eight strains of A.oryzae was compared by plate culture,and each plate of the strain was able to collect spores of 10⁷.Among them,H4 had the highest sporulation,with an average of 7.9×10⁷/plate,which was 4.72 times of the average value of B strain with the lowest sporulation.Under the condition of liquid fermentation,the pH value of several A.oryzae fermentation broths decreased first and then increased.The wet weight of hypha increased at the beginning of fermentation,and remained stable after the third day.Some strains'weight were increased slightly at the fifth day,and concentrated around 3.5 g finally.A simple analysis of the enzyme production in liquid fermentation revealed that AS11 had the highest?-1,4-Glucan glucohydrolace activity?13.04 U/mL?.The?-amylase activity of H4 and H5 were the highest,were 19.02 U/mL and 15.71 U/mL,respectively.The maximum?-1,4-Glucan glucohydrolace activity was second only to AS11,which was about 8.5 U/mL.B has the lowest protease activity and is basically stable at 0.1 U/mL throughout the fermentation process,but its glucoamylase?7.0 U/mL?and?-amylase?8.5 U/mL?vigor are in the eight strains medium level.2.Construction of pUC18-hygB-LIP recombinant expression vectorIn order to compare the ability of several strains of A.oryzae to express heterologous proteins,an expression vector was constructed based on pUC18-LIP.When selecting resistance markers,we explored the sensitivity of strains to multiple antibiotics.The results showed that 0.1?g/mL pyridine thiamine,2?g/mL benomyl and 0.1 mM chlorpromazine+150?g/mL hygromycin B could completely inhibit the growth of A.oryzae strains.It means the ptrA,benA,and hygB genes can be used as a selection marker for the expression vector.Based on various factors,the hygB gene was selected as a resistance marker and the vector pUC18-hygB-LIP was successfully constructed.3.Comparative analysis of the ability of different Aspergillus oryzae strains to express heterologous proteinsThe conditions for the preparation of protoplasts were investigated.It was found that when the fresh spores were inoculated,the culture time was controlled at about 14hours.When the inoculated spores were in the frozen state of glycerol,the culture time could be extended to about 16 hours;The optimal enzymatic hydrolysis time of strains AS11,A and H4 was 3.5 h,and the regeneration rates were 34%,28%,and 33%,respectively.The optimal enzymatic hydrolysis time of strains B,C,and H1 was 3h,and the regeneration rates were 30%,27%,and 26%,respectively.The optimal enzymatic hydrolysis time of strains H5 and H6 was 2.5 h,and the regeneration rates were 28%and 26%,respectively.The Rhizopus chinensis lipase gene was transformed into A.oryzae with PEG-CaCl₂,and the expression of lipase was compared with A.oryzae strains as host under liquid fermentation conditions.The results showed that H4had the best expression ability in several strains of A.oryzae,and the lipase activity was 10.16 U/mL higher than that of the host strain.The maximum difference of enzyme activity before and after transformation was from large to small:A?8.21U/mL?,H6?8.17 U/mL?,B?7.88 U/mL?,H1?6.27 U/mL?,H5?5.75 U/mL?,C?4.44 U/mL?.