Microbiology Research Journal International

Bacillus thuringiensis has shown a great biotechnological potential due to its insecticidal Cry-proteins production. Also, this bacterium produces different extracellular enzymes. The study aimed to evaluate the amylolytic system of B. thuringiensis strain Bt LBIT 418 cultured in a liquid medium, with starch as a carbon source, where the bacteria secreted the maximum amount of endoamylase after 10h of incubation, in the middle of the exponential phase of growth. Its’ cleaves action-released dextrins composed for five of more glucose residues. An exoamylase able to convert these dextrins in maltotriose, maltose and glucose appeared at once, given rise to a new increment of the bacterial growth. An α-glucosidase was also secreted, but covering the 40h of the incubation period. Other related hydrolases as glucoamylase and maltase were not found, but proteases whose synthesis profile correlated with the endoamylase were detected, although both activities presented independent synthesis regulations.

In contrast, the exoamylase could be purified 98 times with a recovering of 14%, by fractionating lyophilised supernatants from 13h of cultures, in columns of CM-Sephadex G-50 and G-100. The enzyme showed a MW of 83.2 kDa and maximum activity at pH 7-8 and 50°C, while the enzyme stability was higher at pH 6-7.5 and 55°C. The enzyme was inactivated with 0.01 M EDTA and reactivated with 0.01 M CaCl₂, showed that this amylase was a metalloenzyme. Furthermore, its’ thiol-dependent characteristic was unclear, because the enzyme was inhibited by 0.01M HgCl₂, but did not by 0.01 M pHMB. The results suggest that Bt LBIT 418 produced at least three different amylases and it is the first report concerning about characterisation of the amylolytic system of B. thuringiensis. This supports the hypothesis that it is possible to obtain in a sole fermentation process, the insecticidal Bt microcrystals, as well as extracellular enzymes with potential biotechnological applications as the amylases.