Addition of proteinase K during the culture alter the physiology of Bacillus thuringiensis culture and the cry1Ac, nprX, nprA, and spo0A gene transcription.

Bacillus thuringiensis is the major bioinsecticide worldwide produced due to the Cry protein activity. Several studies have been done to improve the cost-productivity relation. The neutral protease A (NprA) is the major extracellular protein massively produced during the stationary phase by this bacterium, contributing to the Cry proteins' degradation. Also, the deletion of aprA and nprA genes enhanced the yield of Cry protein, stabilizing it. Therefore, to increase Cry production, one possibility is to degrade the NprA protease in the culture media. In the present study, proteinase K was used to hydrolyze the NprA to increase Cry production. Proteinase K was added during the exponential growth of B. thuringiensis culture. The bacilli and endospores were measured along all culture, while the Cry protein was measured at the end of the culture. The addition of PK affects the bacilli and spore kinetics positively but negatively to the Cry protein (there is no Cry protein detection). Therefore, the gene expression of the cry1Ac, nprX, nprA, and spo0A was measured. The expression of each gene was followed along all culture. Results demonstrated that PK alters both the transcriptional levels and the expression order of the genes.

High-energy ball milling treatment of soybean for Bacillus thuringiensis culture media.

Soybean meal has been intensively used as a substrate in culture media for several microorganisms. However, the fermentable sugar containing the soybean needs to be released from the solid matrix through different processes. Against this backdrop, the present study explores the use of high-energy ball milling as a one-step treatment method for expedited production of fermentable sugars of textured soybean. The best result is observed after only 5 min of milling, obtaining 34.1 times more fermentable sugars than untreated textured soybean, and 2.5 times more than commercially used soybean meal. Notably, the textured soybean ball-milled has been used as a substrate for Bacillus thuringiensis var. kurstaki HD-73 fermentation. The cell and spore production is also compared with a standard Rowe media. The maximum cell concentration obtained in the entire fermentation process using ball-milled textured soybean media is found to be higher than the concentration obtained using the standard Rowe media. In addition, it is observed that there is a direct correlation between maximum cell production and reducing sugar concentration generated by the high-energy ball milling treatment. No fermentation inhibitors or by-products are generated during the physical treatment.