Studies On Characters Of Heat-shock Protein GroEL From A. Ferrooxidans And Attachment Of EPS To Sulfide Ores

As a crucial bacteria in bioleaching,the environment conditions ,such as temperature,pH and so on,affect the activation of Acidithiobacillus ferrooxidans. Generally,the rate of decomposition is sufficdiently fast at 40℃or 50℃for many sufides. And in bioleaching,the temperature exceeds 35℃universally.But the best growth temperature for Acidithiobacillus ferrooxidans is about 30℃.The elevated temperature restrains the activation of Acidithiobacillus ferrooxidans. So the thermotolerance of Acidithiobacillus ferrooxidans is important.In this study,the gene of GroEL and GroES from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and successfully expressed in Escherichia coli,finaly purified by one-step affinity chromatography. The molecular mass was 57kDa and 10 kDa measured by SDS-PAGE respectively.The ATPase activity and chaperonin activity were tested. The maximum activety was observed at about 50℃. The ATPase activity of GroEL is temperature dependent.The highest rate of ATP hydrolysis by GroEL is observed in the range of heat-shock temperatures. ATPase activity of GroEL is significantly inhibited by the co-chaperonin GroES. GroES suppress the hydrolysis of ATP by GroEL to a very similar extent in the temperature range of 20-50℃.Interestingly,at 55℃or higher temperatures ,GroES,didn't inhibit the ATPase activity of GroEL.It is very interesting that GroEL could facilitate the refolding of the denatured rusticyanin from Acidithiobacillus ferrooxidons ATCC 23270 at elevated temperature. The sequences alignment and the molecular modeling of the GroEL suggest that Leu31 is important for ATP binding,and Y199,S201,Y203,F204 L234,L237,L259,V263,V264 are important for polypeptides binding. The results of microcalorimetric show that Ni²⁺ enhances the positive cooperativity of GroEL.