Site directed mutagenesis in T83R and L287R positions on phytase enzyme from Yersinia intermedia

Phytate is the major phosphate storage compound in seeds of higher plants and forms complexes with multivalent metal ions such as iron, zinc, calcium and proteins, thereby showing anti nutritional effects. So phytase added mainly as an additive to the monogastric animal feed due to hydrolyze phytate and increase absorption of phosphorus and another elements.The purpose of this study was site directed mutagenesis in T83R and L287R positions on phytase enzyme from Yersinia intermedia to enhance thermostability. Mutation of amino acids in active site to arginine increases stability of enzyme.At first, the structure of this enzyme was prepared by SPDBD, Then the model was compared and superimposed to the used structure. Subsequently, all of the appropriate surface amino acids were identified for mutation. Then, by changing all these amino acids to arginine, the position of arginine was evaluated in the structure. Finally two of this positions were selected for this study.For this purpose, the primers containing the mutations were designed and PCR was performed with QuikChange™ site-directed mutagenesis method to make mutations. PCR product was transfered to DH5a competent cells. Accuracy of coloning was checked by colony PCR and sequencing. The results show that the specific selected amino acids were mutated to arginine correctly.

کلیدواژه ها:

Phytase ، Quikchange site directed mutagenesis ، Yersinia intermedia ،