Thermostble Module Enzyme By Rational Design And Its' Module Reconstruction

Protein rational design is a technique of protein engineering arisen decade years ago. Based on the understanding of protein structure, protein refining is realized by protein engineering techniques, and new function is obtained. Compared with directed evolution based on non-rational design, this method can obtain the expected protein more rapid。Our lab in my previous job, have carried out respectively on from Aeropyrum thermophilic ancient fungus pernix K1 thermophilic esterase was the ape1547 and Sulfolobus strain from Tokodaii (JCM545) 7T (dibenzyloxyphosphoiyl peptide release) st0779 molecular evolution mechanism was studied. Through analysis of sequence and structure, we find that after ape1547 has a similar catalytic structure with st0779, but the difference of the propeller structure domain is larger, and their substrate specificity gap is amazing. So we don't produce a idea, will ape1547 thrusters structure domain st0779 transfer to the catalytic structure domain, expectation can produce interesting phenomenon. Therefore we first through computer aided method confirmed our design reorganization about the stability of the enzyme. Under the environment of high temperature in the 70 degrees of restructuring enzyme AS executive molecular dynamics simulation 500ps compared with starting conformation after Lord Thermal stability also happen a drastic change. Compared with ST, no matter in peptide enzyme and substrate esterase was, they optimal substrate activity have improved greatly increased, the specificity. To pTNPC3, for example, is AS and ST optimal lipid substrates, and AS pNPC3 catalytic vigor for improved 1.58 times compared to ST. But more than ST high thermal stability was 10 degrees. The value of main chain of RMSD is less than 2, visible in the actual water enzyme AS reorganization of environmental its thermal stability is very high. And experimental proof, reorganizing the enzyme AS in enzymology character on the show before with us, not only in the idea of enzymatic activity AS happened on the huge change, but also in the substrate specificity and enzymes enzymes。The significance of the work could be summaried as follows:peptides with different functions indeed can be used as "building blocks" to "transplant" between the proteins with low homology and similar structure. This may be one of the evolution mechanisms for protein molecular: inserting different peptides into the frame of primitive enzymes with promiscuity will introduce different functions, and then a protein superfamily with different function has formed initiatorily. By the following "subtle regulation" among the members of super family through mutagenesis in primary sequence, divergent evolution for function and sequence occurred among them. Second, enzymes with "promiscuity" can be used as the primitive enzymes for molecular evolution. With the primitive enzymes for protein engineering selected, guided by the rational design spirit, the combination of directed-evolution based on site mutagenesis and protein elements recombination technique will speed up the process of protein engineering, and then increase the resource of enzyme/protein with excellent characters significantly.