Study On Characteristics And Functions Of M23/37Family Proteases From Bacillus Thuringiensis

Bacillus thuringiensis （Bt），most widely used biological pesticide in the world, hasbeen a concern since it was found. But its application in the field is still faced with greatdifficulties, and the biggest one is that insecticidal crystal is susceptible to a variety ofenvironment factors, which would lead to the degradation. With the development of science,more and more new technologies and new ideas are presented and applied to the study ofthe crystal protection. Among the means, constructing engineering bacteria which caninhibit cell lysis is an ideal way. The main component of the cell wall is peptidoglycan, so itmay become possible to protect the crystals by finding a key peptidoglycan hydrolase geneand constructing the mutant. M23/37family proteases can hydrolyze peptidoglycan, so theyshow great potential and prospects in this regard.The genome sequence of Bt_HD73has been published, and some genes involved inmother cell lysis have also been studied, but the structure and function of M23/37familyproteases in Bt is still unknown. Under this background, this paper pays attention to thestructure and function of two M23/37family proteases produced by Bt.Firstly the bioinformatics analysis of two M23/37family proteases （HD73₀859,HD73₁661） from Bt_HD73was done, such as the secondary structure, tertiary structure,catalytic domain, active sites, etc. According to the analysis, both the two secrete proteaseswere predicted as peptidoglycan hydrolase. Then HD73₀859, HD73₁661genes werecloned and a corresponding series of proteins were expressed. But all these proteins showno enzyme activity to cell wall, Cry1Ac crystal or BSA in subsequent experiments,although they could combine with cell wall. Reason for this phenomenon might be thepeculiarity of M23/37family proteases that they have a very complex mechanism to maturethe pre-pro-enzyme which is the original inactive form of protein. So, the proteases in thisfamily were usually inactive when they expressed in E. coli.To solve the above problem, we changed experimental methods and pay attention tostudying the proteases in their original host Bt_HD73. On the one hand, we constructedHD73₁661-GFP fusion protein and located HD73₁661protein by Laser ScanningConfocal Microscope. It showed that the protein was bonded with cell wall and the wall ofspores. On the other hand, we constructed a mutant HD （Δ1661） and carried out preliminaryexperiments to analyze its traits. Comparing with the wild-type strain, it has significantdifferences in the presence of cell division and cell separation. In this study, a preliminary analysis of M23/37family proteases of Bt have been done.It provides theoretical and experimental basis for further study of secreted protease in Bt,and at the same time it efficiently provides ideas and clues to build a new generation Btengineered bacteria which have strong resistance, long duration and high-efficiency.