The Change Of The Function And Structure Of The Molecular Mutant Of Thermostable Alkaline Phosphatse

Alkaline Phosphatse (short for AP EC3.1.3.1 ) is a Phosphomonoesterase which can be used for variety of research in molecular biology as the tools and isotope label. In resent years, scientists pay more attention to a kind of creature that can live in the place of high temperature, and researchers find that the enzymes produced by these creatures have specific structure and function, which is higher thermostability and the optimal reaction temperature; some enzymes even maintain the enzymatic activity in 110℃. Thermostablility and the optimal reaction temperature make the enzyme an excellent model in the research of the structure and function of the protein.By random mutation, there are more than 200 TAP gene mutants being obtained.Among which we select 8 to study in this experiment. We clone the sequence, express themutant TAP and purify the protein, then measure the activity, the optimal reactiontemperature and the thermostablility of the mutant. We submit the CDS sequence of the TAPmutant to the CPHmodels server of Technical University of Denmark to imitate the higherstructure of the protein. After obtaining the higher structure of the chain A of the mostcompatible mutant (D153hd330n) of the BAP which structure is known. We imitate thetertiary structure of the first 319 peptides of the mutant by means of the molecular imitationand predict the secondary structure of the TAP with SWISS-PDB Viewer. Through analyzingthe mechanism of the thermostablility, we find that:1) The optimal reaction temperature of TAP3,TAP6, TAP9 and TAP48 are decreased, while TAP5,TAP35 and TAP52 is unchanged on the whole while TAP25 increased a little. However the optimal temperature of all the mutants varied around the 70 ℃. Almost all the mutant appears the maximum enzyme activity; the amount of the protein produced by TAP25 and TAP35 are lower than the other mutant in the same condition of induction and...