Production and Purification of the Metalloprotease of Bacillus polymyxa

The nutritional and environmental factors relating to the production of an extracellular protease by Bacillus polymyxa were investigated. The enzyme was produced in all media that supported growth of the microorganism, irrespective of the carbon source used. Arabinose and hydrolyzed starch, however, gave highest yields. The nature of the peptone had a significant effect on the level of protease produced. Calcium and manganous ions exerted a beneficial effect on protease production. Highest enzyme levels were obtained when the initial pH of the medium was within the range 5.9 to 7.0. When the pH of the medium was not controlled during the fermentation, the accumulation of the enzyme paralleled the growth of the microorganism and reached a maximum towards the end of the exponential phase. With a fixed pH of 6.8, the level of protease was only one-fifteenth of that obtained when the culture was allowed to maintain its own pH. In addition, accumulation of the protease reached a maximum somewhat earlier, i.e., in the mid-log phase of growth. A 70-fold increase in the specific activity of the protease was obtained by ammonium sulfate and acetone fractionation followed by gel filtration on Sephadex G-100. The purified protease behaved as a homogenous entity when eluted by a sodium chloride gradient from CM-cellulose at pH 6.9. An overall enzyme recovery of 60% was obtained.