Study of the surface layer and parasporal body of Bacillus thuringiensis Israelensis) MH14) and prediction of Cry4Ba stabilization by point mutation method based on bioinformatics findings

Bacillus thuringiensis is the most important biological agent and by producing parasporal body it acts as a key role against agricultural pests. Furthermore, it produces Surface layer (S-layer) which is a protein or glycoprotein crystalline structure and has wide applications in nanobiotechnology. Accordingly it is significant to study more about this surface layer and toxin. In this study, purified parasporal body and mixed crystal/ spore were stained with coomasie blue G250 and were observed with light microscope. With a point mutation in CRY4Ba, protein stabilization was predicted and the location of protein cavities were predicted by Molegro software. Finally, the surface layer was extracted and its molecular weight and morphology were determined. To compare the surface layer and parasporal body, some features of them were estimated by the Protparam server. The results confirm the presence of polyhedral crystal proteins which were accumulated to form larger crystals after releasing spores. Also, it is predicted that in this research replacing the aspartic acid position- 451 with isoleucine, a more stable CRY4Ba pesticide protein is probably produced. This protein with three subunits contains 59 cavities and like the surface layer in this strain, it comprises low percent of methionine, histidine, cysteine and tryptophan.Discussion and In sporulation phase, Bacillus thuringiensis produces insecticidal crystals that integrate to form larger crystals. It is predicted that replacing the aspartic­ acid position- 451 with isoleucine would ameliorate the stability of CRY4Ba pesticide protein. This bacterium in vegetative phase produces a surface 100 KD protein which is similar to parasporal body in a shape and the percentage of some of amino acid.