Study On Directed Evolution Of α-Amylase Gene In Vitro

Directed evolution is becoming increasingly important in biological research. It is used to probe the relationship between the structure and activity of proteins; to define the roles of particular proteins and protein assembles in the cell; to determine the logic and order of molecular events during differentiation and morphogenesis; to improve the activity of enzyme, antibiotics and so on.The routine method for inducing protein function evolution is imitating and speeding protein gene mutation. In this research α -Amylase gene was mutated in vitro by PCR with dNTP in which deoxythymidine triphosphate (dTTP)was partially replaced by 5-bromo-2'-deoxyuridine-5'-triphosphate(5-BdU). The higher the concentration is, the stronger its inducing ability is in a certain range. The experimental results showed that the suitable ratio for the 5-BdU and dTTP was about 1:1000. In this thesis the LBSP identification medium screening techniques was used. Only after one cycle, large quantities no-function recombination plamid and enzyme activity increasing 20 times recombination plasmid were obtained. In the second cycle the mutation type of enzyme activity increased 40 times was found.We sequenced three type mutation genes. One is no-function. The second amylase activity is 1/4 of the native amylase. The other amylase activity is 40 times of native amylase. The non-amylase activity gene has 9 base changes, resulting in 5 amino acid changes from the native enzyme. The low-enzyme activity gene has 2 base changes, resulting in short amino acid sequence with native enzyme. The high-enzyme activity has 2 base changes, resulting in long amino acid sequence with native amylase.This inducing method resolved the problem of non-effective induction as in base analogue induction. And the method we used provide a new measure for this kind of work.