The Thermostability Study Of Subtilisin Protein

The transformation of protein thermostablity is significant in both theory andindustry. On the one hand, as the protease is catalytic effectively and substratestereoselectivity, so it is increasingly used in chemical reactions. But the naturalproteases have low tolerance with salt, PH and temperature, which greatly limits theirapplication. So that designing a higher tolerance protease will facilitate its applicationin the industrial field; On the other hand, the structure and function of protein isrelative closely, researching the protein mechanism of heat resistance can in turnpromote the understanding of relationship between protein structure and function.Therefore, the studies on the mechanism of protein thermal stability and the rationaldesign methods for improving the protein thermal stability have become the hotspotsin the field of computational biology and protein engineering.This paper takes the subtilisin as the object of research, what we have done intwo areas is the identification of the thermostable critical areas in the subtilisin andthe analysis of protein stability factors. The spectific results are as follows:1. By the statistical coupling analysis of homologous sequence of subtilisin family,we have extracted the conserved residues and strong coupling residues; By themothed of molecular dynamics simulation, we have extracted the candidate mutationresidues on the surface of subtilisin. Integrate the rusults above, we put forward themothed of how to identify the thermostable critical areas on the surface of subtilisin,and using this method to determine10of thermostable critical areas on the surface ofsubtilisin. Compared the results with the existing experimental data of heat resistancemutations, we found that7of10forecast areas are within the experimental results.This results show that the method can be used to identify the thermostable criticalareas of protein.2. By the motheds of homologous modeling and molecular dynamics simulation, wehave studied the thermostable factors in subtilisin and its mutants. We selected fourfactors whice can affect the protein thermostability, these are hydrogen bond,electrostatic energy, van der Waals energy and salt bond. By the results of moleculardynamics simulation, we computed the above four factors of subtilisin and itscorresponding mutants. Comparing the results we found that the electrostatic energyand the numbers of salt bonds have larger variances between wild-type and its mutants, so the electrostatic energy and salt bonds are the major factor in affecting thethermostability of subtilisin; The number of hydrogen bonds and van der Waalsenergy have little variances between wild-type and its mutants, so the hydrogen bondsand van der Waals energy are the secondary factor in affecting the thermostability ofsubtilisin.The identification mothed of thermostable critical transformation area of surfaceon subtilisin can be used to identify the protein critical areas, it has guidingsignificance on protein thermostable transformation.