Abstract

A moderately thermophilic bacterium designated EP1001 isolated from an alkaline hot spring (conditions in the hot spring: temperature 53-54°C and pH 9.3) in Zimbabwe was investigated for protease production. Studies to determine the growth and protease enzyme production by the novel bacterium were carried out in ordinary batch, regulated batch and fed-batch modes of fermentation using a BioFlow III 3 L fermentor. Biomass production and protease production were dependent on fermentation mode. Higher biomass formation and protease production were obtained in fed-batch fermentations compared with that in ordinary and regulated batch cultivations. The protease enzyme was found to be a thermostable alkaline serine protease with optima activity at 75°C and pH 10. The enzyme had a half life of 45 min at 80°C and 12 h at 70°C. It was stable over the pH range of 5.0 to 11.0. The enzyme was inhibited by phenylmethane-sulfonyl fluoride and EDTA but not by N-Tosyl-L phenanylalanine chloromethyl, iodoacetamide and O-phenathroline. The ions Ca²⁺ and Fe²⁺ at 0.5 and 2.5 mM concentration were stimulatory, while Mg²⁺ and Mn²⁺ had little effect on the enzyme activity. The enzyme produced by bacterium (EP1001) was concluded to be an alkaline protease that requires calcium and iron ions for its activity.

Key words: Protease production, thermophilic bacterium, alkaline hot springs, alkaline protease.