Study On Recombinant Expression And Enhanced Thermostability Of Nattokinase From Bacillus Natto

The heart and cerebrovascular disease caused by thrombus is one of the diseases withhighest incidence and mortality rates. Researches on thrombolytic drugs and functional foodwith thrombolytic activity have developed very rapidly in recent decades. Nattokinasebecomes one of the hot research topics due to its high thrombolytic effect. Nattokinase is aserine protease identified from Bacillus natto during the process of soybean fermentement,and has several advantages, such as safe, oral administration, and prolonged effects.In this study, recombinant nattokinase was expressed in Escherichia coli and Bacillussubtilis, and the enzymatic properties were analyzed after purification; two amino acids wereidentified which were critial for fibrinolytic activity and thermostability of nattokinase. Ourmain results are listed as follows:(1) The gene pro-nk(encoding propeptide and mature peptide) were amplified from B.natto chromosome by PCR. The vector pET-24a-pro-nk was constructed and was furthertransformed into E. coli BL21(DE3) to express recombinant nattokinase. The expressionproduct showed a strong fibrinolytic activity.(2) The recombinant nattokinase was expressed in B. subtilis, which is recognized as aGRAS microorganism (generally regarded as safe) by the FDA. Three signal peptides (wapA,yncM, pre) mediated nattokinase expression in B. subtilis were studied. The recombinantnattokinase with wapA signal peptide was expressed at the highest level and exhibited thestrongest fibrinolytic activity. The specific activity of purified nattokinase using Hitrap SP HPcolumn was1052FU·mg-1. Enzymatic properties showed that the optimum temperature was50℃and the optimum pH was8.0, and the purified nattokinase was stable at pH5.0-11.0andless than60℃.(3) Based on analysis of sequence aligments and crystal structures, two key amino acidsin nattokinase for thermostability were found out, and two nattokinase mutants (P14L andN76D) were achieved by side-directed mutagenesis. The half-life of mutants P14L and N76Dat65℃were improved from20min (wild-type nattokinase) to30min and50minrespectively.