A Research On Screening New Strain Of Aspergillus Oryzae With Protoplast Fusion

Aspergillus oryzae is the important microorganism in the factory of brewage. It excreted many kinds of enzyme during its process of growth. Among them, the most important enzyme is protease which can decompose the legume protein into multi-peptide and amino acids. So Aspergillus oryzae 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, the strains of Aspergillus oryzae often shows low production ability in factory practice, falling in the conditions of either fast-growing strain with low protease activity or high activity in slow-growing ones. This phenomenon dropped the ratio of producing, and difficult to form a product line. At present, the means of screening Aspergillus oryzae mostly adopted mutation in our country, but it's difficult to gain an ideal strain. Hence, our experiment is screening Aspergillus oryzae with protoplast fusion. Protoplast fusion is a process of gain a new fusant from the regeneration cell. Firstly, we decompose the parents' microorganism cell respectively by the enzyme which can set free the cell wall, and make them become protoplast. Then join the physics or chemistry or biological condition to help fusion, make parents strains' protoplast collected together, and make gene commutation and reorganization through cytoplasm or nucleus fusion. Then can reborn the cell wall under the feasible condition, and we can screen them through reasonable process. Hence, we can acquire new strains.This experiment select Aspergillus oryzae strain AS3.951 and CICC2339 as the parents strain. Firstly, we choose cellulase, lywallzyme and snailase as lytic enzymes, and the mixed ratio of them is 7:3:1. We gain protoplast after strains' cell wall decomposed by lytic enzymes, and the ratio of protoplast formation is about 10~6/ml. Secondly, we research on protoplast inactivation. The methods of inactivation are ultraviolet-inactivate, microwave-inactivate and ultrasonic-heat inactivate. After research the best condition for inactivation by every different method, we gain the result that AS3.951 inactivated by ultraviolet and CICC2339 inactivated by ultrasonic-heat. On the base of inactivation, we research on the electro fusion. The better condition of electro fusion is 1MHz for pulse frequency, 40V for pressure of being string, 400V for fusion pulse pressure, 80us for pulse width, 6 for number of pulse. Through electro fusion, we gain some new strains and compared with the strains which fused by PEG At last, we screened three fused strains which named EF102, EF113 and EF222. Both of them represented well characteristics. The protease activity reached 6540U/g, 7680U/g and 6500U/g respectively, the activity of EF113 much higher than parent strain AS3.951.