Production, purification and properties of Bacillus thuringiensis neutral protease

Although the insect pathogenicity of Bacillus thuringiensis is reasonably well understood, relatively little is known about other facets of this microorganism's physiology. At the time of sporulation, the organism produces in addition to the spore and toxic paraspore (crystal) an extracellular proteolytic enzyme. This study concerns the conditions for production, the.purification and the properties of this enzyme.

It was found in studies relating to production of protease that B. thuringiensis var. kurstaki (HD-1) produced a considerable quantity of the enzyme in a protease production medium (PPM). This medium contained 7 x 10⁻³ M CaCl₂, 5 x 10⁻⁴ of M MnCl₂ and 1 x 10⁻³ M MgCl₂ in nutrient broth. Manganese was required for enzyme synthesis and calcium was required for enzyme stability.

Starting with a large volume of crude enzyme preparation obtained from the culture supernatant of B. thuringiensis grown in PPM, the enzyme was purified 97 x. The purification steps included Amicon ultrafiltration cell concentration, ammonium sulfate fractionation, and potato starch adsorption. Electrophoresis on SDS-polyacrylamide gels showed a single protein band at the last purification step.

The enzyme had a pH optimum around pH 6.5-7.0 and was sensitive to metal chelating agents such as EDTA and O-phenanthroline. The molecular weight of the neutral protease has been estimated to be about 37,000 by electrophoresis in SDS-polyacrylamide gels. In the presence of 0.1% calcium acetate, the enzyme is quite stable at 60 C after 10 minutes incubation. It lacks esterase activity when tested against acetyl-tyrosine ethyl ester and benzoylarginine ethyl ester. All the above properties indicate the similarity of the Bacillus thuringiensis neutral protease to those produced by other members of the genus Bacillus as well as to the other microbial neutral proteases.