Improvement Of Fibrinolytic Activity And Thermostability Of The Subtilisin Nattokinase By Site-directed Mutagenesis Expressed In Genetically Engineered Bacteria

Natto originated from ancient China,being introduced into Japan as a kind of food and becoming a symbol of food in Japanese food culture.In the fermentation process of natto food,natto bacteria can produce an alkaline serine protease,Nattokinase.Nattokinase can effectively dissolve fibrin which is the main component of thrombus in human body.As a new type of thrombolytic drug or health food,nattokinase has the advantages of low price and high safety,and has a broad market prospect compared with similar products like urokinase?In the nattokinase industrial production process will be heat loss and low enzyme activity.Through molecular biology methods to modify nattokinase to improve its thermal stability and enzyme activity,building the genetic engineering bacteria suitable for the production of Nattokinase will provide convenience and theoretical basis for large-scale industrial production of nattokinase.This study built pET-26b prokaryotic expression vector including Nattokinase mature peptide and propeptide+mature peptide then converted to Escherichia coli BL21.After induced by IPTG,the bacteria were centrifugally collected,and then the protein was expressed by SDS-PAGE.Enzyme activity detection of expression protein by fibrin plate method.Results show,both of Nattokinase mature peptide and propeptide+mature peptide could be expressed successfully in Escherichia coli;The supernatant and precipitation of mature peptide don't show fibrinolytic activity;The supernatant of propeptide+mature peptide shows fibrinolytic activity but not in precipitation.Through reading and bioinformatics prediction,166 glycine and xxx serine could influence the thermostability of nattokinase.Using overlap extension PCR technology,166 glycine was mutated to arginine,and xxx serine to proline.The prokaryotic expression vector pET-26b-NKt166 and pET-26b-NKtxxx were constructed and transferred into Escherichia coli BL21.After induced by IPTG,the bacteria were centrifugally collected,and then the protein was expressed by SDS-PAGE.Enzyme activity detection of expression protein by fibrin plate method.Results show,both of NKt166 and NKtxxx could be expressed successfully in Escherichia coli;The supernatant and precipitation of NKt166 don't show fibrinolytic activity;The supernatant of NKtxxx shows fibrinolytic activity but not in precipitation.After treatment at different temperatures,the heat loss rate of NKtxxx decreased by 40% compared with the wild type,and the total inactivation temperature increased from 63? to 65 ?;At the same time,its fibrinolysis activity increased by 18.10%.This study built pGAPZ?-a eukaryotic expression vector including Nattokinase propeptide+mature peptide then converted to pichia pastoris x-33.After induced by methanol,supernatant was collected and protein expression was detected by SDS-PAGE.Fibrin plate assay was used to detect the enzyme activity of the expressed protein.Results show,Nattokinase propeptide+mature peptide could be expressed successfully in pichia pastoris x-33;The supernatant of propeptide+mature peptide did not show fibrinolytic activity.