Biochemical and technological studies on alkaline protease produced by microorganisms

Production of extracellular alkaline protease, using an industrial strain of Bacillus licheniformis in different industrial media was optimized. Of different media used, the production medium composed of glucose and soybean meal as carbon and nitrogen sources, respectively, in addition inorganic phosphate, calcium chloride and magnesium chloride led to the maximal enzyme production after 96 h. The effect of glucose concentration on the cell growth and the enzyme production was studied in shaken flask culture, The maximal enzyme production was obtained on using glucose concentration of 60 g L. 1 in the industrial medium, Further optimization in the production process was achieved applying the optimum of aeration rate on small scale 16 L bioreactor. Whereas, the increase of airflow rate in the range from 0.5 to 2.0 v v. . 1min. 1 showed a negative effect on cell growth. The maximal enzyme production was obtained at airflow I 0 v v. -I min. 1 after 84 h at agitation rate of 500 rpm, When the process was scaled up to 500 L bioreactor and cultivated at the same airflow rate, the enzyme production reached the maximum after 80 h cultivation. Batch culture and fed-batch operations were used to enhance the production of the enzyme. Fed-batch cultivation was used for intermittent addition of glucose only after 48 h and intermittent addition of whole medium. Whole medium feeding led to highly enzyme production while feeding glucose only was not significant in enzyme production. Moreover, surfactant agents also, were used to improve the cell mass and enzyme production in shaken flasks with small scale, Tween 80 positively affected on the enzyme excretion and, production in a concentration of 0.75% (w/v) after 24 h. In small scale bioreactor, Tween 80 (0.75% w/v after 24 h) showed high enzyme productivity with reduced production time to 60 h. The apparent molecular weight enzyme was 33 kDa as determined using SDS electrophoresis, Enzyme purified was active at alkaline pH range from 8-11 and the highest activity was found at pH 8-10. The enzyme was also active over a broad range of temperature from 30- 90°C and it stable about (50%) at 50°C for 2 h and retained activity until 11 h. The effect of metal ions on protease activity showed that the cobalt chloride enhanced activity for about 140% while Ca. 2, Na. l, Zn. 2 or CU. 2 led to slight decreased in activity, Particularly, Fe. 2 decreased the activity to about 56.8%. Slight inhibition was exhibited by â -mercaptoethanol and EDT A. On the other hand, DMSO had no effect on the enzyme activity. Moreover, the enzyme was highly compatible with many commercial detergent.