Production of a protease activity from Bacillus thuringiensis rt in a complex media

Abstract

Bacillus thuringiensis is a Gram-positive, facultative anaerobic bacterium that produces crystal proteins with insecticidal properties. In addition, it is an excellent producer of proteases and other lytic enzymes that can increase the insecticidal effect by acting in synergy with the crystal protein. In this work, the production of proteases was carried out by submerged fermentation using a complex media for possible use in different industries such as food, chemical, textile and bioinsecticides. The strain Bacillus thuringiensis RT (Genebank: EF638795, 16S partial sequence) was used. The production was carried out in a Labfors 3 fermenter (Infors HT, Bottminguen, Switzerland) in 3000 ml at 30°C and free pH. A complex culture media called M5 developed from economical substrates was used; it contains (g/L): cerelosa 2, whey 10, skim milk powder 5, vinasse 5, sucrose 2, starch 2 and soybean meal 7. Protease activity was determined in triplicate in culture supernatant every 24 h during 5 days. One hundred and twenty μl of supernatant was mixed with 480 μl of azocasein (1% w/v) in phosphate buffered saline. One unit of activity is defined as the amount of enzyme that produces an increase in 0.01 OD420 in 30 min at 30°C. The values of protease activity during fermentation were (U/L): 0.13 ± 0.02 x 106 (24 h), 0.26 ± 0.02 x106 (48 h), 0.35 x106 ± 0.01 x106 (72 h), 0.98 ± 0.07 x106 (96 h) and 1.36 ± 0.09 x106 (120 h). Thus, the maximum value of protease activity was obtained at 120 h (α = 0.05) displaying a volumetric productivity of 11421.97 U/Lxh. In addition, the final pH value of the culture was 9.51. In conclusion, this lab-scale study gives useful information to continue with the scaling-up of the process.