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.

Acetone↗

PubMed1973

Physiochemical properties of the native, zinc- and manganese-prepared metalloprotease of Bacillus polymyxa.

The neutral protease of Bacillus polymyxa had a broad pH optimum (6.0 to 7.2) for activity at 37 C. The enzyme was most stable at pH 5.6 to 5.8. The protease had an optimum temperature of 37 C and was quite thermostable up to 35 C, but at higher temperatures the stability decreased rapidly. The substrate specificity of the protease was similar to that of the neutral proteases of other members of the genus Bacillus. The enzyme was shown to be a zinc metalloprotease. However, manganous ions had a greater activating and stabilizing influence on the activity of this enzyme than zinc. Replacement of zinc in the native enzyme by manganese resulted in a 50% increase in activity. In addition, the prepared manganese metalloprotease had higher temperature and more alkaline pH optima than the native enzyme.

Apoproteins↗

PubMed1972