Breeding Of High Producing Polyenzymes In Bacillus And Studies On Its Mechanism By N～+ Implantation

A mutated strain- ANO₂ with high yield of compound enzymes was obtained fromthe strain ANO₁ of Aspergillus niger by N⁺ implantation as well as UV irradiated, andscreened by using transparent zones of agar plate screening techniques. The results showedthat activities of acid-protease, Cellulase and Pectinase in Aspergillus niger were raisedfrom 71.6 IU.g^-1,141.7 IU.g^-1 and 264.8 IU.g^-1 to 996.5 IU.g^-1,940.4 IU.g^-1 and 906.5IU.g^-1 respectively. Characteristics of enzymes production in mutated strain Aspergillusniger ANO₂ were stabile by 5 times subculture. The optimum components mediums ofmutated strain Aspergillus niger ANO₂ was gotten by using single-factor research andorthogonal test. The optimal medium contained fresh bran 426.8g, bean cake 341.45g,corncob 341.45g, NH₄CL 17.07g, water 1000mL, inoculums concentration 10%.The initialpH was set at 5.5, and the culture temperature was controlled at 30℃ in 96h. The resultssuggested that it is a availability way to breed bacillus of enzyme by N⁺ implantation. The results of biological effects that the spores of Aspergillus niger were implantatedby N+ were as following: the treatment of vacuum hurted the vivid cell, The survival rateof spores in vacuum was decreased with the time delay, while the velocity of death wasslow. and the survival rate of spores was decreased also with increasing of implantation iondose ,but increased gradually when the dose of implantation was 20×2.6×10¹⁴ N⁺/cm²;while the survival rate of spores decreased when the implantation ion dose was 50×2.6×10¹⁴ N⁺/cm²; There was high positive mutation rate in the implantation ion dose of(20-60)×2.6×1014 N⁺/cm².And the lager the implantation dose was, the higher thespore mutation rate was. The ESR results showed that signal peaks of free radicals hadexisted in the spores and the control. Moreover, that the intensity of free radicals increasedwith the dose increasing. The SEM indicated that the control were integrity. While thesurface of spores was coarseness. Moreover, the damage of ionic etching and phenomenonof dilapidating cell wall could be seen. When implanted dose increased continually, thedamage to cells became more and more serious. Biological characteristics of the original strain ANO₁ and the mutant strain ANO₂were researched. Colonies appeared after ANO₂ had been cultured on standard medium for35h and ANO₁ for 25h. But the spores of the mutated strain ANO₂ appeared early andaround the clone there were transparent zones. The lamellas of ANO₂ appeared earlyerthan the original strain ANO₁. Biomass and enzyme activity of the mutant strain ANO2were increasing relativity. The biomass of the original strain ANO₁ was increasing before72h, while there was a stationary phase in 72-96h and then decreased. The enzyme activityof original strain ANO1 was not high, but the phase of producing was after the biomassgrowth phase.