Nattokinase Enzyme Engineering Service for Enhanced Activity and Stability

Nattokinase (NK), a potent fibrinolytic enzyme, is derived from the traditional Japanese food natto and is widely used in health supplements and drug development. Industrial production faces several challenges, including low yield of natto fermentation , insufficient enzyme activity for therapeutic use, and the enzyme's natural proneness to degradation during processing and storage.

CD Biosynsis offers a dedicated synthetic biology service focused on the robust Bacillus subtilis chassis. Our core strategy involves the modification of Bacillus subtilis promoters to significantly enhance the transcription and expression level of the NK gene. This is combined with the optimization of enzyme structural stability through directed evolution or rational design to protect the enzyme from heat, pH changes, and proteolysis. This integrated approach aims to establish a high-yield, high-activity, and stable NK production platform.

Pain Points

Scaling up high-quality Nattokinase production is challenged by these biological limitations:

• Low Fermentation Yield: Traditional natto fermentation is inefficient for industrial scaling, and even recombinant hosts may suffer from a low volumetric productivity of NK.
• Insufficient Enzyme Activity: The specific fibrinolytic activity of the produced NK may be too low for high-potency medical applications, requiring large doses.
• Proneness to Degradation: NK is a protease, but it is itself sensitive to thermal inactivation and degradation by other host proteases during fermentation and purification.
• Secretion System Bottlenecks: Efficient secretion of the target protein out of the Bacillus subtilis cell can be limited by the native signal peptide cleavage or saturation of the secretion machinery.

A cost-effective solution must maximize the enzyme's specific activity and its stability in the production environment.

Solutions

CD Biosynsis employs advanced host and enzyme engineering to enhance Nattokinase performance:

Modification of Bacillus subtilis Promoters

           

We utilize strong, inducible, or constitutive promoters and optimize their upstream activating sequences to achieve ultra-high transcription rates for the NK gene.

Optimization of Enzyme Structural Stability

We use rational design and/or directed evolution to introduce stabilizing mutations (e.g., disulfide bonds, surface charge modification ) that increase NK resistance to thermal and pH stress.

Host Protease Knockout

Key genes encoding extracellular proteases in B. subtilis are targeted for deletion to minimize the co-secretion of enzymes that can degrade the NK product.

Signal Peptide and Leader Sequence Tuning

We screen and optimize the NK signal peptide/pro-peptide sequence to enhance efficient translocation and correct folding , aiming for maximum functional enzyme secretion.

This systematic approach is focused on establishing a platform capable of high-titer production of a robust, highly active Nattokinase variant.

Advantages

Our Nattokinase engineering service is dedicated to pursuing the following production goals:

Enhanced Enzyme Activity

Structural optimization is focused on delivering a variant with a higher specific fibrinolytic activity (FU/mg), increasing therapeutic potential.

High Secretion Yield

Promoter and secretion system modifications aim to maximize the final enzyme output in the fermentation medium.

Increased Stability and Shelf Life

Structural stability optimization aims to make NK more resistant to thermal and pH degradation , improving product shelf life and processing ease.

Simplified Purification Potential

Protease knockout provides a cleaner fermentation broth, potentially reducing downstream processing steps and cost. [Image of Cost Reduction Icon]

Scalable and Controllable Production

Utilizing B. subtilis allows for a highly scalable and controlled fermentation process , replacing variable traditional methods.

We provide a biosynthetic platform aimed at maximizing the yield and quality of Nattokinase production.

Process

Our Nattokinase strain engineering service follows a standardized, iterative research workflow:

• Enzyme Characterization and Target Identification: Determine the native NK's specific activity and half-life (t}_{1/2) under stress conditions, establishing clear stability improvement targets.
• Promoter and Secretion Optimization: Screen a library of engineered promoters and signal peptides to maximize the transcription rate and efficient secretion of the NK gene.
• Structural Stability Engineering: Introduce targeted mutations or employ directed evolution to identify NK variants with enhanced thermal and pH stability .
• Host Protease Knockout: Systematically delete major extracellular protease genes in the B. subtilis host to prevent product degradation.
• Scale-Up and Fermentation Validation: Test the final engineered strain in fed-batch fermentation, measuring the final volumetric titer and product specific activity (FU/mL/h).
• Result Report Output: Compile a detailed Experimental Report including genetic modification data, enzyme stabilization data, and fermentation kinetics (titer, activity, and stability under stress) , supporting commercial assessment.

Technical communication is maintained throughout the process, focusing on timely feedback regarding enzyme activity and strain productivity.

Explore the potential for a high-performance, stable Nattokinase supply. CD Biosynsis provides customized strain and enzyme engineering solutions:

• Detailed Specific Activity and Stability Analysis Report , demonstrating the improvement in fibrinolytic units and stress tolerance.
• Consultation on fermentation and downstream purification strategies optimized for the engineered B. subtilis strain.
• Experimental reports include complete raw data on enzyme yield, t}_{1/2 at target temperature, and product purity , essential for formulation development.