Cooperation Of Osmolytes And Artificial Chaperone On Malate Dehydrogenase Refolding

Malate dehydrogenase (MDH) is an essential enzyme in TCA cycle. And it can be used in the separation of D-malic acid, which is an important chiral 4-carbon dicarboxylic acid in pharmaceutical industry. For its homo-dimer or tetrad structure, MDH is regarded as an ideal model for research on oligomeric protein structure and function. In this study, E.coli MDH (eMDH) IBs obtained by gene recombination was refolded in vitro, in order to define the refolding mechanism of oligomeric IBs protein in practical industrial production.First, eMDH was refolded with the assistance of osmolytes, which is present in most lives and in favor of protein protection. In this study, sucrose, trehalose and betaine was used to assist the refolding of eMDH. All of them could facilitate the refolding of eMDH with lower concentration, especially for sucrose and betaine. With their aidance, the specific activity of refolded eMDH was increased from 43U·mg^-1 to 73U·mg^-1 and 77U·mg^-1. But the results of fluorescence and CD indicated that their aidance mechanism were different. Sucrose could restrain the aggregation and inappropriate folding, and increased the content ofα-helix, which was regarded to be correlated with the activity of eMDH. Trehalose could protect the protein from aggregation, but didn't give any contribution on refolding. The effect of betaine was derived from its hydrophobic combine with protein.According to the conclusions above, the cooperation of osmolytes and artificial chaperone was studied. As the results showed, trehalose didn't have any influence to the assistance of artificial chaperone, but sucrose and betaine at lower concentration could facilitate it.. When 0.5mol·L^-1 sucrose and betaine were added in the stripping step withβ-CD, the specific activity of eMDH was increased to 133U·mg^-1 and 137U·mg^-1(with protein concentration of 0.1mg·ml^-1 and temperature of 4℃). With the aidance of osmolytes, the optimal ratio of artificial chaperone was reduced. Especially, the ratio of CTAB andβ-CD was decreased from 1:8 to 1:6, when 0.5mol·L^-1 betaine was added. With this effect of osmolytes, it is possible to increased the total concentration of artificial chaperone in practical production, to improve the refolding of eMDH.Then, the thermo-protection of sucrose and betaine, and their cooperation with artificial chaperone was applied to the refolding of eMDH with larger protein concentration and higher temperature. With the cooperation of betaine and artificial chaperone, the specific activity of refolded eMDH was increased to about 100U·mg^-1 under 15℃(with protein concentration of 0.5mg·ml^-1). Besides that, sucrose and betaine could protect refolded eMDH against higher temperature, maintain its activity. When protein concentration was increased to 1.0mg·ml^-1, the cooperation of sucrose/betaine and artificial chaperone was also observed, as a result, the activity of eMDH was advanced to about 65U·mg^-1.