Directed Evolution In Vitro Of α-Amylase Gene

This dissertation is studying on the evolution of alpha-amylase in vitro by mutational PCR (with 5-BrdUTP as substitute partly) technology. As known , alpha-amylase is one of the most important industrial enzymes,it has been widely used in many fields such as starch processing,fermentation trade,beer brewing ,textile industry and so on. Moreover, the study on alpha-amylase is one of the most active fields in enzyme industral fields.5-BrdUTP can partly replace dTTP when the process of replication was happened.As we know,Taq DNA polymerase has no the function of 3'- exonuclease, these characters may lead to the point mutagenesis.using this new method to mutate the alpha-amylase gene in vitro, and using the selective medium LBSP ,we screened five mutated genes with higher enzyme activity.after registered on Genebank five gene numbers were given: AY645667,AY645668,AY645559,AY645670,AY645671.The five new sequences were aligned with the the original gene by the biological software DNAman.Results show that: the mutant sites often happened from the NO.300-360 Amino acid(that is the NO. 4 conservative region).it means that the change of enzyme activity is mainly caused by the structure of the enzyme,not caused by the change of the promoter.further study show that :5-BrdUTP can not only lead to transition, but also lead to transversion.Mutational PCR (with 5-BrdUTP as substitute partly) technology is a new strategies for directed enzyme evolution in vitro . the study has proved the feasibility of this technology,and provide more material for further research of the evolution ofalpha-amylase.