| TAP(thermostable alkaline phosphatase), coming from Thermus sp. FD3041, is composed of 501 amino acids, 40% of which is similar with bacterial alkaline phosphatase, and those residues related with active sites are more conserved which means the similar 3D structure and the same mechanism of catalysis between TAP and other alkaline phosphatases. TAP is a good moleculor model in the study of protein's thermostability due to its optimal reaction temperature above 70C.After the nonfunctional domain of TAP was deleted on the sites selected according to the rule of remaining the intactness of terminals' secondary structure predicted on the NPS@ server, TAPN34C25 with 34 amino acids deleted at N terminal and 25 at C terminal was obtained. Its optimal reaction temperature was 72 C, with 7C higher than TAPND27's, and its highest tolerable temperature 83C, with 16C lower than TAPND27's. In summary, we not only obtained a more exact TAP's functional domain than before, but also established a more efficient method, namely two terminals' secondary structure-determined method, for the thermostable enzymes' functional domain location in this study.The relationship among the substituted amino acids, the 3D structure simulated on PC through CPHmodels Serverand the thermostable performance of 4 thermostable alkaline phosphatase(TAP) mutants were analyzed to explore the mechanism of thermbstability change. These mutants are TAPA41V410-T),TAPP396S(P396^S),TAP2 (N100-S T320-I) and TAP4(N100-S P396-S, A410-V P490-S). The 3D structure neighboring the substituted amino acids was simulated by Swiss-PDBViewer to observe the relationship between the structure change and the thermostable performance of TAP and its mutants. The results displayed that all these amino acid substitutions except the T320-I mutant brought about only a little local change on TAP's 3D structure and very little effect on their optimal reaction temperature, but a significant decrease(nearly 10"C) on their highest tolerable temperature. However, the T320-I mutation due to close to TAP's active sites did bring about a significant descendents of the mutant in both the highest tolerable temperature and the optimal reaction temperature. Thus, it seems to be able to conclude that most of the amino acid substitutions, no matter where they locate and what structure change they may make, can cause TAP's highest tolerable temperature reduced significantly. What's more, if the mutation occurring near or in the active sites, it can also cause TAP's optimal reaction temperature reduced significantly at the same time.To explore the effects of metal ion, TAP and its mutants' activtity were checked in the condition of Mn2+, Zn2+, Mg2+ and Ca2+. The result revealed that Mn2+, Zn2+, Mg2+ were favored to their activity and thermostability, but Ca2+ wasn't.In order to analyse the relationship of complementary codons, CodonP, a statistical program of codon usage frequency, was developed in C# language, which could run on the .NET framework. Based on two localized databases of codon usage frequency and taxonomy, CodonP realized the statical analysis of correlation and parameters about codons.100 species selected randomly from every group of bacteria, archaebacteria and eukaryote were analyzed of the correlationship of usage frequency between codons and their complementary codons. The result showed that almost all the usage frequency of codon pairs was positively correlated which was more similar between archaebacteria and eukaryote than between bacteria and any one of them, and this kind of correlatinship lied on the symmetry of hydrogen bonds incodons. This study not only testified the correctness of codon bias, but also revealed the correlationship of codon pairs' usage frequency and its biological function. |
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