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

IF 3.3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie Pub Date : 2025-02-01 DOI:10.1016/j.biochi.2024.09.014

Ankush Jain, Pradeep Kumar Anand, Jagdeep Kaur

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Abstract

Site-directed mutagenesis was employed to investigate the structure-function relationship of nattokinase (NK) and its effect on the enzymatic activity, thermostability, pH tolerance, and fibrinolytic properties of NK. Specific mutations (T270S, V271I, E262D, and A259T) were introduced within the nk gene, targeting regions predicted to be involved in substrate binding. The NK(E262D) mutant exhibited a significant increase in enzymatic activity (2-fold) and catalytic efficiency (2.2-fold) as assessed by N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) hydrolysis, compared to the wild type. In silico analysis supported these findings, demonstrating lower binding energy for the NK(E262D) mutant, suggesting stronger fibrin affinity. Thermostability assays revealed that NK(E262D) and NK(A259T) displayed exceptional stability, retaining enzyme activity at 60 °C. All mutants exhibited a broader pH tolerance range (pH 5.0–10.0) compared to the wild-type NK. The fibrinolytic activity assay revealed that the E262D mutant possessed the highest fibrinolytic activity (2414 U/mg), surpassing the wild-type. This study reported an NK variant with improved enzymatic activity, thermostability, and fibrinolytic properties.

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纳豆激酶的定点突变：揭示结构-功能关系以增强功能

为了研究纳豆激酶（NK）的结构-功能关系及其对 NK 的酶活性、热稳定性、pH 耐受性和纤维蛋白溶解特性的影响，我们采用了定点诱变技术。研究人员在 nk 基因中引入了特定的突变（T270S、V271I、E262D 和 A259T），这些突变针对的是预测与底物结合有关的区域。与野生型相比，NK(E262D)突变体的酶活性（2倍）和催化效率（2.2倍）均有显著提高。硅学分析证实了这些发现，证明 NK(E262D) 突变体的结合能更低，表明其纤维蛋白亲和力更强。热稳定性测定显示，NK(E262D)和NK(A259T)表现出了极高的稳定性，在60°C时仍能保持酶活性。与野生型 NK 相比，所有突变体的 pH 值耐受范围更广（pH 值为 5.0-10.0）。纤溶活性测定显示，E262D 突变体具有最高的纤溶活性（2414 U/mg ），超过了野生型。该研究报告了一种酶活性、热稳定性和纤维蛋白溶解特性均得到改善的 NK 变体。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biochimie 生物-生化与分子生物学

CiteScore

7.20

自引率

2.60%

发文量

219

审稿时长

40 days

期刊介绍： Biochimie publishes original research articles, short communications, review articles, graphical reviews, mini-reviews, and hypotheses in the broad areas of biology, including biochemistry, enzymology, molecular and cell biology, metabolic regulation, genetics, immunology, microbiology, structural biology, genomics, proteomics, and molecular mechanisms of disease. Biochimie publishes exclusively in English. Articles are subject to peer review, and must satisfy the requirements of originality, high scientific integrity and general interest to a broad range of readers. Submissions that are judged to be of sound scientific and technical quality but do not fully satisfy the requirements for publication in Biochimie may benefit from a transfer service to a more suitable journal within the same subject area.