Random Mutagenesis And Screen Antithromboase Of Bacillus Subtilis And Studies On Biological Characters Of Mutant Enzyme

In our laboratory,constructed plasmid pUBH contains antithromboase(AT) gene from Bacillus subtil is N18. Plasmid pUBH in the protease-deficient Bacillus subtilis expresses successfully AT.AT not only can directly cleave cross-linked fibrin,but also can activate plasminogen to plasmin.So AT will be useful as a new type potent ideal thrombolytic agent.But there are some defects limiting the clinical application of AT,for example low specific activity.In order to overcome the above weaknesses of AT,we rebuilds AT by molecular breeding technology.However due to not know the relations of AT structure and AT function,it can't employ molecular design to transform the function of AT.At present the best measure of settling the difficult problem is protein molecular evolution engineering. The strategy of this engineering is to simulate the process of protein molecular natural evolution,to introduce the random mutation in the gene of target protein,and to select the excellent character of mutant.In this study,we use tow kinds of random mutagenesis method(chemical mutagen mutated immediately plasmid pUBH containing AT gene and base analogues PCR mutated the target AT gene) to obtain random mutant library;adopt tow-rank screening method(initially screening clones by the plate method and further sceening clones by the fermentation method) to gain the excellent character of mutant;study on the biological characters of purified mutant enzyme.There are various chemical mutagenes and mechanisms of chemical mutagenes are different.we select tow not noly different mechanisms but also extensive application kinds of chemical mutagenes,which are sodium nitrite of deamido mutagen and ethyl methane sulfonate(EMS) of alkylate mutagen,to mutate plasmid pUBH.The mutation principle of sodium nitrite is that sodium nitrite effects directly DNA,deaminates base and sequentially forms ketone group,changes hydrogen bond of electric potential,destroies base pairing rule,products base substitution.Used mutation method of sodium nitrite mutated plasmid pUBH,screen and acquire four mutant clones distinctly increase specific activity in comparison to wild type clone.According to quantity of transform and screen result.40mmol/L sodium nitrite ,37℃,1 hour is in favor of mutation the AT gene in plasmid plJBH.The mutation principle of EMS is that EMS reacts with water molecular to product carbon cation;carbon cation reacts with the amido of base;the hydrogen of changed amido and ketone group can't form hydrogen bond;base pairingrule is destroyed and the result causes mutation.Used mutation method of EMS mutated plasmid pUBH,screen and acquire five mutant clones distinctly increase specific activity in comparison to wild type clone.According to quantity of transform and screen result,0.7mol/L EMS,25℃ and 1hour is in favor of mutation AT gene in plasmid pUBH.The principle of base analogues PCR is that sodium nitrite of deamido mutagen reacts with the mixture of four kinds of dNTP that is natural substrate for PCR,deaminates base,and forms base analogues;used base analogues as substrate,Taq polymerase amplifies AT gene by PCR; base analogues can be introduced into AT gene during replication because Taq polymerase has no detectable 3'→5' proofreading exonuclease activity;due to base analogues presence,this leads to base mismatch and the result causes mutation.Mutation AT gene is obtains as inserted fragment by base analogues PCR;constructed shuttle plasmid pMA-AT is used as vector;they are digested by EcoRI and KpnI,then ligates in order that mutation AT gene substitutes natural AT gene;firstly the ligated DNA mixture is transformed into Escherichia coil;plasmid mixture is gained by extracted plasmid of all transforms;secondly plasmid mixture is transformed into Bacillus subtlis.Used shuttle plasmid and two time transform,obstacle that the efficiency of ligated DNA mixture transformed into Bacillus subtlis is low is conquered. Screened and acquired one mutant clone from mutant library distinctly increases specific activity in comparison to wild type clone.Mutant...