Study On Structure And Zymological Characters Of The Mutants Of Hyperthermophilic Esterase APE1547

Three mutants of hyperthermophilic esterase APE1547 were constructed, using Glu instead of Asp34, using Gly instead of Arg287, and using Glu instead of Asp34, using Gly instead of Arg287 at the same time.And then we studied the structure and the zymological characters of the mutants of hyperthermophilic esterase APE1547. The study on the expression and purification of mutant and wild-type indicated that the total expression protein of mutants exceeded that of the wild-type, and the specific activity of the purified mutant enzyme performed a little higher than that of wile-type. The study on the mutant zymological characters the of hyperthermophilic esterase APE1547 shows that the optimum temperatures of wild-type is higher than mutants, both their optimum pHs are 8.0, and their substrate specificities are consistent. Mutants performed in a wide pH range compared to wild-type.The thermostability was investigated by half-lives of all the mutants.The results of thermostability indicated that the wild-type is more stable than the mutants. Fluorescence spectra and CD spectra were applied to detect the structures of APE1547 and its mutants,and the results indicated that the structures of the mutants D34E and R287G are almost consistent with the wide type,the structure of mutant D34E/R287G changes a little.In conclusion, these results give us a hint about the relationship between thermostability and structure which offers theory basis for its molecule reconstruction thereby extending its industry application.