Stuides On Protoplast Fusion Between Bacillus Subtilis And Aspergillus Niger By The He-Ne Laser

Bacillus subtilis has the better security, producing various types of enzyme, high proteinase activity, easy to be raised, demanding lower energy and high mycelium cell reproduction quantity, which is applied in many fields of production. However, its glucoamylase activity is lower, affecting the application scope and its effect. Aspergillus nige UV-11 is a high production strain of glucoamylase activity, but its enzyme system is unitary, and mold's nature is unable to be applied in many realms of production. This experiment, the fusion of Bacillus subtilis (abbreviated as MC7)and Aspergillus nige UV-11 (abbreviated as UV-11) protoplast through He-Ne laser-PEG compound induction, screen and achieve the new strain of high glucoamylase activity and the proteinase activity with Bacillus subtilis nature.Through orthogonal experiment, the best preparing conditions for MC7 and UV-11 protoplasts release and regeneration were determined as follows: MC7 protoplasts were cultured in 1mg/mL lysozyme for 1.5 hours at 37℃ and the best preparing conditions for UV-11 protoplasts were 1.5mg/ml lywallzyme, 2 hours and 34℃.Inducing the parents protoplast with single laser, the experimental result indicated that irradiating the MC7 protoplast for 4min, the rate of high positive mutation is 34.7% and irradiating the UV-11 protoplast for 4min, the rate of high positive mutation is 38.2%.The optimum conditions of inducing the parents protoplast by the polyethylene glycol (PEG) were: 40% PEG which induced protoplast for 4 minutes at 50℃, the fusion rate could achieve the highest which was 5.1 x10~-6 . By laser, the optimal irradiating time was 4 minutes, the fusion rate was the highest which was 8.5x10~-6. While by PEG and laser, the optimal irradiating time was 4min, the fusion could achieve the highest which was 10.2x 10~-6.The majority of the fusants's glucoamylase activity had been raised. Irradiated by laser for 4 minutes, the glucoamylase activity of 4-5# fusant could get 28.5U/mL, which was raised by 130.6% compared with parent MC7. In addition, its proteinase activity was 386.4U/mL, which was raised slightly.After analysed by the esterase isoenzyme patterns and compared with its parent strains, the results showed that the fusants' genie material was recombined, which led to the ability of producing glucoamylase improved remarkably and the ability of producing proteinase improved slightly. After subculture, the result showed that the hereditary stability of fusants was well.