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Site-directed mutagenesis of nattokinase: Unveiling structure-function relationship for enhanced functionality

Ankush Jain1, Pradeep Kumar Anand1, Jagdeep Kaur1

  • 1Department of Biotechnology, BMS Block-1, Panjab University, Sector 25, Chandigarh, India.

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|September 28, 2024
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Summary

Site-directed mutagenesis enhanced nattokinase (NK) properties. The E262D mutant showed improved enzymatic activity, thermostability, and fibrinolytic potential, offering a promising variant for therapeutic applications.

Keywords:
E262D mutationEnhanced NK variantNK-Fibrin interactionNattokinaseProtein engineeringThermostable NK

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Nattokinase (NK) is a serine protease with fibrinolytic activity.
  • Understanding the structure-function relationship of NK is crucial for improving its therapeutic potential.

Purpose of the Study:

  • To investigate the structure-function relationship of nattokinase (NK) using site-directed mutagenesis.
  • To enhance the enzymatic activity, thermostability, pH tolerance, and fibrinolytic properties of NK.

Main Methods:

  • Site-directed mutagenesis was used to introduce specific mutations (T270S, V271I, E262D, A259T) into the nk gene.
  • Enzymatic activity was assessed using N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) hydrolysis.
  • In silico analysis, thermostability assays, pH tolerance tests, and fibrinolytic activity assays were performed.

Main Results:

  • The NK(E262D) mutant showed a 2-fold increase in enzymatic activity and a 2.2-fold increase in catalytic efficiency.
  • In silico analysis indicated lower binding energy and stronger fibrin affinity for the NK(E262D) mutant.
  • NK(E262D) and NK(A259T) mutants exhibited exceptional thermostability, retaining activity at 60°C.
  • All mutants displayed a broader pH tolerance range (pH 5.0-10.0) compared to wild-type NK.
  • The E262D mutant demonstrated the highest fibrinolytic activity (2414 U/mg).

Conclusions:

  • Site-directed mutagenesis can effectively improve nattokinase properties.
  • The NK(E262D) variant presents enhanced enzymatic activity, thermostability, and fibrinolytic potential.
  • This study yields a nattokinase variant with superior characteristics for potential therapeutic applications.