Screening Of Nattokinase Producing Strains And Studies On Molecular Cloning And Expression Of Pro-nattokinase Gene

Thrombotic disease does harm to the human life gravely and bringsgreat loss to people. But the commonly used thrombolytic agent in clinicaltreatment have some defects, such as short of specificity, short half life, illeffect, inferior thrombolysis ability, need big dosage or difficult to produceand expensive. Natto is a traditional food in Japan, which is a kind offermented soybean by incubating Bacillus subtilis natto. During thefermentation Bacillus subtilis natto will produce nattokinase(NK), whichis an alkaline serine protease. NK is of strong fibrinolytic activity.Compared with traditional thrombilytic agent, NK has strongerthrombolysis activity and longer half life . NK can be taken orally orinjected into veins, besides, NK will not cause hemorrhage . All these virtuemake NK a promising second generation thrombolysis medicine.At the present time, Fermentation with Bacillus subtilis natto andpurification is still the only way of producing nattokinase(NK). But someproblems always arise from this course, for instance, strains grow slowly,low output of enzyme, etc. E.coli expression system has clear geneticbackground and high expression efficiency, it is easy to ferment to a highdensity. With the development of molecular biology of natto,it is of greatmeaning to construct genetic engineering strains for producing NK.In this reach, a high-yield NK producing strain NT2 is isolated andscreened from five kinds of Japanese natto by culture and fibrinolyticactivity assay. Chromosome DNA is isolated from screened subtilis natto.Pro-NK gene(encoding propeptide and mature peptide)is amplified byPCR. The gene is cloned into pGEM-T vector. The sequence analysisindicates pro-NK gene accord with NCBI reports. The recombinant clonevector and the prokaryotic expression vector pKK223-3 are digested by therestriction enzyme of EcoR I and Pst I. The recombinant expression vectorpkk-pro-NK is constructed and transformed into host strain E.coli JM105.Under 30℃and the induction of IPTG, Pro-NK is expressed and then partof which produces NK by an intermolecular self-processingmechanism.The expressed target protein amounts to18% of the total proteinof E.coli JM105 by thin-layer scan. The expressed NK amounts to 8%.SDS-PAGE analysis shows that pro-NK is accumulated mainly in depositand 50% of the expressed NK consists in supernatant after supersonictreatment and centrifugation. The fibrin plate assay indicats that thesupernatant has obvious fibrinolytic activity, while the deposit hasn't.According to the standard curve of urokinase activity, NK production is60U/ml. Subtilisin is an alkaline serine protease produced by various species ofBacillus subtilis. The nucleotide sequence and amino acid composition ofNK has great homogeneity with that of subtilisin. Compared with subtilisinE , subtilisin A , subtilisin Carlsberg , subtilisin BPN and subtilisin DY, NKshares homogeneity of 99.5% , 99.3%, 69%, 85%and 70%, respectively.Nk and subtilisin E has identical signal peptide and propeptide sequence, asfor mature peptide , only two amino acid is deferent. But NK is differentfrom other subtilisin in enzymological kinetics and specificity of substrate,The optimum substrate of NK is S2251 and NK shows strong fibrinolyticactivity. E.coli expression system is of high expression efficiency, while therecombinant protein is prone to form inclusion body, and can not...