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

The enzyme, as biocatalyst, has increasingly gained world-wide attentions during the past decades. Its development and industrialization has turned into a very important part in biotechnology in national economy. Although enzyme has broadly engineering application, its application under extreme condition is limited. Thermophilic and hyperthermophilic enzymes often have high thermostabilities and are resistant to organic solvent or denature. They are attractive in many biotechnology fields. Directed evolution has matured to a standard technology in the field of molecular enzyme engineering. After hyperthermophilic esterase APE1547 was firstly cloned by PCR, and then expressed in the E.coli. in the former works, our laboratory exploited the method of error-prone PCR and constructed a mutation library on the basis of APE1547. By application of a high-throughput screening method, a mutant with high activity and enantioselectivity was obtained. The E value (enantioseletivity factor) of mutant rises to 5.15 from 2.25 of wild-type by 2.26 times. DNA sequencing analysis indicated that there are five amino acid substitutions in the structure of mutant, which situated at medium and far away positions from the active site of the enzyme molecule. This paper focuses on the structural and mechanistic lessons which can be learned after researches on the structure and zymological characters. By comparing the structure of mutant with that of wild-type, it is found that they are almost identical only if the start position of the α-helix of N-terminal arm structure which archored on the C-terminal domain of of the mutant extend forward a length of two amino acid to a reverse direction compared to the wild-type. It has been confirmed that the archor structure of N, C-terminal region is the most important factor on protein stability during the thermal denaturation, it firstly deties from the aimed region. By structure analysis we found that the substitution of Arg11GLY played a key role in the viariation. It affects the electric charge interaction on the molecular surface, changes the potential of contracting solvent and ability to forming covalent bond. The study on the expression and purification of mutant and wild-type indicated that the total expression protein of mutant exceeded that of the wild-type by 50%, and the specific activity of the purified mutant enzyme performed a little higher than that of wile-type. It needed to be pointed that the operation that the purified method used is identical. The study on the mutant zymological characters the of hyperthermophilic esterase APE1547 shows that the optimum temperatures of wild-type and mutant are 90 C and 85 C, respectively, both their optimum pHs are 8.0, and their substrate specificities are consistent. Mutant performed in a narrow pH range compared to wild-type, and its activity decreased obviously in higher pH environment, while the wild-type can function well at alkalinous environment. Except that Zn2+ partially inhibited the activity of the mutant and Sr2+ partially activate the wild-type, absolutely most of the...