| Directed evolution of enzymes by changing the structure and the nature is an effective way to meet the need of industry. Now, hundreds of enzymes have been evolved by directed evolution technique to greatly improve their activities and efficiency, which have produced a great impact on the industry, agriculture, pharmaceutical etc. The success of directed evolution of enzymes depends on diversity of the constructed mutant library. Error-prone PCR is the representative of the random mutation. DNA shuffling technology is the representative of DNA recombination that is commonly used in directed evolution.To overcome the disadvantages of existing various methods of constructing mutant library, a new method to construct mutant library based on the assembly of protein secondary structure was established in this paper. The reassembly based on original protein secondary structures was performed. Compared with the existing methods, this method had a good diversity without destroy of protein functional structure, so it was easy to obtain a functional mutant. In order to facilitate the screening, the kanamycin resistance genes was used as the experimental object. Firstly, the secondary structure and the central area of catalytic activity were selected as the basic modules by analyzing the structure function information of low homologous kanamycin resistant protein. Two mutant library were constructed by the ligation of A-T and the ligation of overhangs caused by restriction endonuclease digestion, respectively. Finally, we got ten effective mutants by this method, and the highest resistance of kanamycin was 1920 ng/?L (which was 64 times of common kanamycin resistance).To sum up, we got a mutant, whose kanamycin resistance was significantly improved by this method. This method might obtain some targets for the prevention and treatment of a new discovery of the virus. This method could also get proteins or enzymes to meet the needs of the industrial, pharmaceutical, food, etc. |
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