Studies On Enhanced Thermal And PH Stability Of Nattokinase

Thrombotic diseases have increasingly threatened the human health,so developing novel thrombolytic drugs has become a hot research field.Nattokinase?NK?is an alkaline serine protease with strong thrombolytic activity produced during the fermentation process of natto.NK has several advantages,such as no immunogenicity,low cost and oral administration,which is considered to be the most promising thrombolytic drug.However,the low stability of wild-type NK severely limits its production and application.In order to overcome the defects of natural NK,this study comprehensively utilized the rational design and semi-rational design strategies to functionally evolve the stability of NK.Firstly,the thermostability and acidic stability of NK were enhanced by site-directed mutagenesis.On this basis,we designed combinatorial mutations to improve the NK comprehensive properties.Moreover,based on molecular dynamics simulation,the acid-sensitive regions in NK were found.Through screening and modification,the effect of amino acid sites or amino acid types on the acidic stability of NK was studied.Finally,we obtained the information on the structure and acid stability of NK.The main results are summarized as follows:?1?Asn and Gln located on the surface were mutated to Asp and Glu,respectively,which improved the activity and thermal stability of NK.The activity of Q59E increased to 1.54times of WT,and the half-life at 55?of N218D was increased from 11.9 min to 28 min.The thermal stability of the double mutant Q59E-N218D was further enhanced,and its half-life(t_1/2-32 min)was 2.72 times that of the wild type NK.?2?Rational design strategy was adopted to improve the acid resistance of NK.Mutants S78T and Y217K with remarkably improved acidic stability were chosen to construct the double mutant S78T-Y217K.Its residual activity remained above 90%after incubation for 4 h at pH 4 while WT only retained 25%under the same condition.?3?To further improve the above two characteristics of nattokinase,we constructed mutants:Q59E-S78T-N218D?Q59E-Y217K-N218D and Q59E-S78T-Y217K-N218D.The acid resistance of mutants has been all increased to different levels.Among these,Q59E-S78T-N218D exhibited a characteristic thermalstability,of which the half-life value was approximately 2.8-fold of WT.And the activity of Q59E-Y217K-N218D was 30%higher than that of WT.?4?The molecular dynamics simulation method was used to simulate the states of wild-type NK at different pH values,and four regions R1-R4 sensitive to acid were obtained.Through modification,it was identified that the site P40 and S78 have an important influence on the acid stability of NK.After analysis,we found that improving the rigidity of loop linked to the active center is beneficial to enhance the stability of NK under acid conditions.