Purification Of Tyrosinase With Distinct Properties And Cloning, Expression Of Mel Gene From Bacillus Thuringiensis

Tyrosinase, which is encoded by mel gene can catalyze tyrosine to form melanin via L-dopa. In the last few years, research interests in melanin has been growing because some important functions of melanin have been recognized, especially the ability of anti-irradiation of UV and removing free radicals. Bacillus thuringiensis is a biopesticide with most widely applications. However, its applications in the field have been limited for the decrease activity of its endotoxin proteins under irradiation of UV. In our previous work, we have found that Bacillus thuringiensis in almost all ubspecies can produce melanin at 42℃, which provides a new approach in improving the duration of B. thuringiensis preparations in the environment.In our study, "one step" method was employed to purify tyrosinase from B.thuringiensis. The purified tyrosinase showed distinct enzymatic properties, such as highly stability towards heat, highly substrate specifity, activated by EDTA in high concerntration. This enzyme provides a new model to study the relationship between protein structure and function.Furthermore, shotgun method was used to clone mel gene from B.thuringiensis. pUC18 was used as the cloned vector. A DNA fragment of 500bp was got and expressed in E.coli. The transformant producing melain has tyrosinase activity which indicated that this DNA fragment is mel gene. Then we sequenced the DNA fragment and analyzed it. It has the conserved region involoved in copper binding, which is likely the active center of tyrosinase. The results revealed that the secondary structure of tyrosinase from B.thuringiensis was different from those of other resources.Moreover, the expression of endotoxin genes in B.thuringiensis producing melaninwas detected. The results showed that whole expression of endotoxin genes was preincubation-dependent.In the end, we detected the effects of melanin on B.subtilis spores under stress environments. The results demonstrated that melanin enhance the resistance of spores to heat, cold and UV irradiation.