Studies On The Radioactive Breeding For High ALDC Activity And Optimization Of Fermentation Conditions

A Bacillus Lichenifornis strain BL4 with a-acetolacetate decarboxylase (ALDC) activity as the initial strain was induced by UV radioactive breeding.Through optimizing microbial culture conditions and studying extract condition, the enzyme yield was increased.The research results are showed as following:l.By studying extract condition of ALDC,the optimum disrupting cell condition has been gotten: ultrasonic frequency 25KHz, ultrasonic power 500W, cell density 2%, radiate 2 second each tune, total radiate 4.6 seconds, 2 second interval.With that condition ,we can obtained the highest ALDC activity.Adopting 80% ammonium sulfate to salting-out ALDC and 30% ammonium sulfate to get rid of others. The ALDC activity will reduce with the deposits time increasing.2. Adopting ultraviolet radiation with wavelength 260nm, power 500W, to radiate at a distance of 30cm, a strain with the stable ALDC activity which was 30% higher than the initial one was obtained.3.The optimum fermentation medium of strain BL4 consisted of 0.3% beef extract, 2% pancreas peptone, 1% glucose, 0.5% citric acid, 0.5% K2HPO4-3H2O , 0.005% ZnSO4.7H2O. The ALDC activity can reach 7.08U/mL under such optimum condition: pH 6.0, temperature 33 C,start culture medium 100mL,seed volume 10%,after 18 hours.4.The release of ALDC was also carried out in this paper.It was found that the increaseof ALDC yield would reach the toppest: 7.37U/mL,while 0.02% Glyine and 3.2 ten thousand units /mL sodium penicillin were added to the shaking flask culture of strain BL4 at lag phase and log phase respectively. Certain amount of Glyine and sodium penicillin could contribute to the release and the increase of ALDC yield.5.According to studies on semifinished enzyme, the most suitable reaction temperature is about 35~45癈 and the optimum reaction pH is 5.5.ALDC has good stability below 45 C,ranging between pH5.0 and pH6.5. Cu2+, Ca2+, Ba2+, Mn2+can restrain the enzyme activity to some different extent,while other metals ion and EDTA have no influence on the enzyme activity.