In Vitro Directed Evolution Of Nattokinase From Bacillus Natto

Thrombotic diseases, especially acute myocardial infarction (AMI), imperil the lives and health of humanity in modern life. Compared with widely used thrombolytic agents, such as tissue plasminogen activator (t-PA) and urokinase, several cheaper and safer resources have been extensively investigated over the years. Among them, nattokinase (NK), which was extracted from a traditional Japanese fermented natto, has attracted interest for its use in thrombolytic therapy. The molecular mass and isoelectric point of NK are about28kDa and8.6respectively. NK has sufficient stability of pH and temperature to be stable in the gastrointestinal tract. NK directly cleaves cross-linked fibrin in vitro, catalyzes the conversion of plasminogen to plasmin or inactivates the fibrinolysis inhibitor(PAI-1). Until recently, most studies of NK have focused on its thrombolytic effects and mechanism, heterologous expression and purification. Currently, whether nattokinase may become a widely used thrombolytic agent mainly depends on the enhancement of its properties, e.g., prolonging the half-life with oral administration and improving the stability and catalytic efficiency. The complete nucleotide sequence of the subtilisin NAT aprN has been obtained using shotgun cloning, and the amino acid sequence has been deduced from the DNA sequence. In vitro molecular evolution strategies are the most efficient methods for creating proteins with improved or novel properties. In the current study, we investigated how to improve the fibrinolytic activity of NK using directed evolution to broaden its medical or commercial applications. The ideal screening method for the mutant library is that the expression products is active and secreted into the extracellular. During this study, we firstly constructed three secretion expression system E.coli BL21(DE3)plysS-pETSN, Bacillus subtilis WB600-pSGNK and pichia pastoris GS115-pPINK. Then a high-throughput screening method was established by combining the nattokinase activity assay method and plate screeing strategy. The mutant library were transferred to the skim milk plate, clear zone could be seen around the colonies with NK activity. This method is simple, low cost and effective for screening the variants with improved fibrinolytic activity.We generated a library of NK variants by the family shuffling of genes encoding NK, subtilisin BPN’ and subtilisin Carlsberg. The three encoding genes were recombined and shuffled to establish chimeric gene libraries. To screen large libraries, the NK variants were expressed in E. coli. BL21(DE3)pLysS using a prokaryotic signal peptide, PelB, for efficient secretion. NK variants were selected based on zone-forming activity on agar plates with skim milk for initial screening and with fibrin for second screening. A mutant NK (MN) with increased fibrinolytic activity compared to the wild-type NK from Bacillus natto was obtained.The amino acid sequence alignment of the three parents and MN revealed that the catalytic triad (D32, H64, S221) and the substrate-binding site (S125, L126, G127) were conserved. There were nine amino acid substitutions that were derived from SB, and the rest were derived from SB or SC. No new mutations were introduced into the mutant enzyme sequence.To understand the functions of the amino acid substitutions, the identified mutations in the selected MN was distributed throughout the model of the MN structure based on the3D model of NK that was previously constructed by our lab. The three-dimensional structure showed that the strictly conserved residues of the catalytic centre (D32, H64, S221) and the substrate-binding sites (S125, L126, G127) were positioned in the pocket, which was comprised of two a-helices and seven P-strands. However, in the current study, none of the mutations were located in those strictly conserved regions throughout the MN. Most of the mutations were located in the surface regions and far away from the pocket with the exception of the substitutions A150V and T224S, which were very close to the Ser221in the catalytic centre of the enzyme. This change may not be involved in hydrogen bonding with other residues. However, the combination of this change with other substitutions may result in the formation of a larger active-site pocket to improve the catalytic efficiency. These results indicate that the hydrophobic pocket plays an important role in the substrate association with the enzyme.We also studied and compared the enzymatic properties of NK and MN. We found that NK and MN can be both expressed actively in E. coli, and28kDa protein were detected by SDS-PAGE and Western blotting analysis. All of them were purified to homogeneity. The fibrinolytic activity for fibrin and catalytic efficiency for succinyl-Ala-Ala-Pro-Phe-p-nitroanilide of MN were increased2.1-fold and2.13-fold respectively compared with that of NK. In additon, the antioxidant capacity of MN was stronger than that of NK. The optimum pH and pH stability of NK and MN were of little difference, in the range of pH5-11, the enzyme activity were stable. The optimum temperature and thermal stability of NK and MN were also of no significant difference, only the thermal stability of MN is slightly higher than that of NK. The results of our work have demonstrated that it is feasible to generate a mutant library of nattokinase using the DNA family shuffling method. With better enzymatic properties, the mutant may become a desirable and economical source for thrombolytic therapy.