Study On The Construction Of Engineered Aspergillus Oryzae For Expressing Proteases And Their Effect On The Fermentation

Aspergillus oryzae has been widely used in production of Chinese soy sauce and in the fermentation of obtaining polypeptides.In this study five new recombinant engineered A.oryzae have been constructed with molecular biology techniques,and been used in fermentation of soybean meal to test the possibilities.The expressed protease were isolated and purified,and the properties of the purified two proteases were also studied.The research results could effect on exploring regulation mechanism of exogenous genes in A.oryzae and the breeding of excellent performance stain.Three alkaline protease genes and three acid protease genes were obtained from Bacillus licheniformis,Bacillus pumilus and Aspergillus niger by PCR amplified.Sequence analysis and amino acid sequence prediction of protease genes were carried out by bioinformatics.The expression frame of A.oryzae was constructed by using the binary expression vector pCAMBIA1304,and the amylase promoter and glycosidase terminator of A.oryzae were added into the vector to construct the reading frame.In the expression of the reading frame were linked to a single foreign protease gene to construct four expression vectors named paa-subC,paa-asp,paa-Ap,paa-pep.Self splicing peptide?T2A?is inserted between the two alkaline protease genes subC and asp as the connection,to construct alkaline protease tandem expression vector named paa-sTa,in order to study the expression of polycistron vector in A.oryzae.Filamentous fungal genetic transformation system is so complex that the efficiency of same transformation method different among different species,even among different subspecies.To provide a new way for the genetic transformation of A.oryzae,an efficient Agrobacterium mediated transformation system of A.oryzae have been establishand in this study.The method of Agrobacterium tumefaciens mediated transformation was used,and hygromycin used as the selection marker.To optimize the conditions for the transformation,influence factor such as spore suspension concentration,concentration of A.tumefaciens,temperature and time of co-culture have been studied.The induction should be added when pre-culture to improve the transformation rate.The results showed that when the OD₆₀₀00 of A.tumefaciens was 0.6,the spore concentration of A.oryzae was 10⁷/mL,and co-cultured for 3 days at 28?,the highest transformation rate could be obtained.Result of detection showed the target gene have been inserted into the A.oryzae genome.Thus,A.tumefaciens-mediated transformation?ATMT?may be a powerful tool for A.oryzae transformation.Engineered Aspergillus oryzae strins have been used in fermentation of soybean meal to test and compare the protease activity,polypeptide conversion rate and composition and antioxidant activity of fermented products.Remarkable improvements of protease activity were obtained in engineered Aspergillus oryzae after introduction of exogenous protease gene.The highest alkaline protease activity was 165 U/mL of A.oryzae asp,about 140%higher than that of the wild-type A.oryzae.The acid protease activity of A.oryzae pep was 143U/mL,about 242%higher than that of the wild-type A.oryzae.The hydrolysis efficiency of raw materials and the conversion rate of polypeptide products of engineered strains also improved.A major improvement in the polypeptide yield was achieved by these engineered A.oryzae.The polypeptide conversion rate of A.oryzae pep reached 35.89%,which was about twofold higher than that exhibited by wild-type A.oryzae.The result of amino acids contents analysis showed essential amino acids content and amino acids composition of the fermentation product were significantly improved when engineered A.oryzae strains were used.Fermentation with engineered A.oryzae,could obtain severe degradation of soybean meal,and higher degree of hydrolysis by SDS-PAGE electrophoretic map and scanning electron microscope?SEM?.Ultrafiltration membranes and gel filtration chromatography were used to separate the fermentation production by different A.oryzae,and the antioxidant activities of each component were compared.The results showed the ratio of small molecular peptide was larger of fermented products from A.oryzae pep.Compared with fermented products from wild-type A.oryzae,the antioxidant activity of different components from engineered strains were improved at different degrees,especially,fermentated product component I from A.oryzae pep had better antioxidant activity.To further improve the utilization to raw materials,A.oryzae pep with good fermentation performance was selected as strain to optimize.The fermentation condition of it to produce polypeptide from soybean meal was optimized by response surface test.The results showed that when fermentated by A.oryzae pep with inoculation amount of 8%for 108h with 31?,the polypeptide conversion rate reached 40.4%.The A.oryzae asp and A.oryzae pep were selected to isolation and purification due to the high protease activity.The salting out of ammonium sulfate,DEAE-Sepharose Fast Flow chromatography and gel chromatography were used to purify the alkaline protease ASP from A.oryzae asp and acid protease PEP from A.oryzae pep which have the high protease activity.The results of SDS-PAGE electrophoresis showed that the molecular weight of recombinant alkaline protease ASP was about 31kD,and the molecular weight of recombinant acid protease PEP was about 40kD.The purification of the two enzymes were up to 5.64 times and 3.85 times respectively.The properties of the purified two proteases were studied.The results have some reference value for the transformation,expression and regulation of using A oryzae as cell factory for exogenous gene expression.