| Aspergillus oryzae, an important microorganism in brewing manufacturing, plays a significant role in the production and flavor of food manufacturing industries, such as soy sauce, Chinese bean sauce, rice wine and so on. However, it often shows low production ability in factory practice, falling in the conditions of either fast-growing strains with low protease activity or high activity in slow-growing ones. Although protoplast fusion has been widely applied in breeding of filamentous fungus, the traditional method is comparatively complicated by marking the parental strains with auxotroph markers, which would affect some important characters of the strains by disturbing their normal metabolism. Hence, this research completes the fusion by using the inactivated protoplasts strain GIM3.13 and CICC2339 as the genetic donors. With their random integration, the fusant presents improved characters from the parent strains.The whole experiment can be divided into four periods. Firstly, the best collocation for protoplast isolation of Aspergillus oryzae, with the research on the species and concentration of lytic enzymes and other factors, is gained as: the mixed ratio of cellulase, lywallzyme and snailase as 5:3:1, fungus age as 15h, DDT addition in lytic reaction as 3mM, osmotic pressure stabilizer as 0.8MNaCl solution.Secondly, research on protoplast inactivation. GIM3.13 is heat inactivated, and best inactivation condition as 60℃ and 15min. CICC2339 is UV inactivated and best condition as 15W ultraviolet light 10cm away for 5min.Thirdly, research on the major factors to the fusion of theinactivated parent protoplasts. The best fusion condition collection is gained as 35%PEG6000 and 0.0 2MCaCl2in the solution with pH as 7.5, and inactivation time for 5min.Hence, the successful selection of the fusant shows that the fusion by using the inactivated protoplasts can be applied to the breeding of Aspergillus oryzae. And the primarily selected strains are of high protease activity and grow fast.
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