| Aspergillus oryzae has been broadly used in food industry, especially for producing soybean paste. Because of the demand for high quality and yiled, single strain of A. oryzae is adopted in soybean paste fermentation operation the manufacturing production of soybean paste. Nowdays A. oryzae 3.042 is the most popular strain. In this study, a wild A. oryzae originated from north-east region was induced by ultraviolet light, and a mutant Y29 with high genetic stability was finally separated from the induced strains. It was found that the amount of protease of Y29 is 1.2 times higher than A.3.042, and content of ammoniacal nitrogen is 0.74g/100ml, a little higher than A.3.042, the fermentation period is 2 days shorter than 3.042. Y29 and A.3.042 don't show significant differences in other parameters. The fermentation of soybean paste is dominated by the enzym, in A. oryzae. The research also proved that alkaline protease plays a key role in fermentation.Because alkaline protease in A. oryzae can not effectively catalyze substrate in the acid environment, it is imerative to make protease efficiency work at lower pH. This is accomplished by the directed evolution system with effective heterogeneous expressing. The alp gene in Y29 was amplified by RT-PCR. The alp gene was expressed heterogenously in P. pastoris GS115 with native signal peptide and withα-signal peptide. Two types of signal peptide were both identified and processed by P. pastoris. The yield of recombinant alkaline protease was 513mg/L and 336mg/L respectively. It was found that pro-region part of alp gene was important for its expression in P. pastoris.Recombinant alkaline protease from A. oryzae was purified by ion exchange and gel filtration for further characteristics. Recombinant and native alkaline protease from A. oryzae were not methylated. Their optimal temperature and pH were idenfical: 40℃and 9.0, and both are similar in pH stability and sensitivity to metallic ions and protease inhibitors as well. However, thermal stability of recombinant alkaline protease was inferior to native alkaline protease. The optimum inducement condition for recombinant alkaline protease was found to be few preferable 28.13℃, pH 6.56, methanol concentration 1.23%, the yield of recombinant alkaline protease from A. oryzae could be as high as 648mg/L.The directed evolution of alkaline protease from A. oryzae was carried out by over-lap PCR method. Three dimensional structure of alkaline protease was constructed homologously with the SWISS-MODEL system. Then based on dimensional structure and the results of three conservative amino acid sites of G78, A230 and G236 which are all near the active center, were chosen as mutant sites. The mutant genes were expressed in P. pastoris. The expressed mutant alkaline protease was purified for compare their characters. The results showed that the optimum pH of mutant enzyme G48R and A230E remain unchanged, whereas pH of mutant enzyme G78D and A230D reduced by 0.5 unit. Enzyme activity of G78D was 12% higher than wild alkaline protease enzyme activity of G236D was 40% lower than wild alkaline protease. At 40℃and pH 9.0, Km of G78D was 115.27% than wild enzyme, and Vmax and Kcat was 85.89% and 82.01% than wild enzyme respectively. Therefore, G78D was selected for further studies.To make functions in A. oryzae, two replacing target vectors were constructed. One vector, which includes two homologous arms and hygromycin B, was transformed into Y29 to get the mutant A. oryzae without alp gene Y30. The other vector, which includes mutant alp gene of G78D, was transformed into Y30 to get the mutant A. oryzae with alp gene (G78D) Y31. Compared with Y29, enzyme activity of acid and alkaline protease in Y31 was 14% and 20% respectively. Soybean paste was fermented with Y29, Y31 and A. oryzae 3.042. The ammoniacal nitrogen content was 0.74g/100mL, 0.80g/100mL and 0.70g/100mL, and fermentation period was 38d, 35d and 40d respectively. Soybean paste fermented with Y31 showed color darker. All testing parameters for Y31 met the national standard of soybean paste (SB/T 10309-1999).
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