Chitooligosaccharide (COS) Bioproduction Engineering Service

Chitooligosaccharides (COS) are low-molecular-weight chitosan derivatives known for their excellent biological activities, including anti-tumor, immune-enhancing, and antimicrobial properties, making them valuable in medicine and agriculture. Commercial production primarily relies on the enzymatic hydrolysis of chitosan using chitosanase. Key challenges include the low efficiency in chitosanase hydrolysis , which requires high enzyme loading and long reaction times, and the resulting uneven oligosaccharide polymerization degree (DP) , which compromises the functional consistency and purity of the final product.

CD Biosynsis offers a synthetic biology service focused on optimizing both the enzyme and the process for high-quality COS production. Our core strategy involves directed evolution of chitosanase modification to enhance its specific activity, substrate affinity, and ability to generate specific DP products. This is coupled with the coupling of fermentation and enzymatic hydrolysis processes to enable continuous substrate supply or simultaneous product removal, improving overall conversion rate and reducing product inhibition. This integrated approach aims to deliver a high-efficiency, high-purity, and industrially scalable bioproduction route for specific COS fractions.

Pain Points

Maximizing the efficiency and quality of COS production through enzymatic hydrolysis faces these critical limitations:

• Low Chitosanase Hydrolysis Efficiency: Chitosan is a highly crystalline polymer, making it difficult for the enzyme to access cleavage sites. This results in low conversion rates and high enzyme usage .
• Uneven Oligosaccharide Polymerization Degree: Native chitosanases often produce a broad spectrum of DP products (DP}2 \text{ to } \text{DP}6$), making downstream separation challenging and leading to functional inconsistencies.
• Product Inhibition: The final product (COS) can inhibit the activity of chitosanase , preventing complete hydrolysis and lowering the final product titer.
• Enzyme Stability: Chitosanase is often used under harsh conditions (low pH or high temperature) required for substrate solubility, leading to rapid enzyme denaturation and loss of activity.

A cost-effective solution must improve enzyme performance and control product distribution.

Solutions

CD Biosynsis utilizes advanced enzyme and process engineering to optimize COS production:

Directed Evolution of Chitosanase Modification

           

We employ rational design and directed evolution to enhance the enzyme's substrate affinity and specific activity and modify the active site for selective cleavage (e.g., favoring DP2 or DP3 production).

Coupling of Fermentation and Enzymatic Hydrolysis Processes

We develop integrated process strategies, such as membrane reactor coupling or immobilized enzyme systems, to continuously remove COS products and maintain high enzyme activity.

Enhanced Enzyme Stability and Tolerance

We introduce mutations to improve the thermal stability, pH tolerance , and resistance to product inhibition of the engineered chitosanase, increasing its effective lifespan.

Heterologous Expression System Optimization

We optimize the expression host (e.g., Pichia pastoris or E. coli ) and fermentation conditions for high-titer production and easy purification of the engineered chitosanase itself.

This systematic approach is focused on improving enzyme performance and achieving tight control over the final COS product profile.

Advantages

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

High Specificity and Uniform DP

Engineered enzymes deliver a narrow and specific distribution of DP products , enhancing functional consistency and therapeutic value.

Reduced Enzyme Consumption

Enhanced specific activity and stability mean a lower enzyme dosage is required to achieve high substrate conversion, cutting operational costs.

Simplified Purification

A narrow DP distribution eliminates complex chromatographic separation of different oligomers, streamlining downstream processing. [Image of Cost Reduction Icon]

Increased COS Titer

Coupled processes and reduced product inhibition allow for higher final product concentration in the reaction mixture.

Controlled Biological Functionality

Specific DP fractions (e.g., DP3) are often associated with enhanced bioactivity, allowing for tailored product design .

We provide a specialized platform aimed at maximizing the quality and minimizing the purification cost of COS production.

Process

Our COS engineering service follows a rigorous, multi-stage research workflow:

• Chitosanase Gene Source and Cloning: Isolate high-potential chitosanase genes and optimize their sequence for heterologous expression .
• Directed Evolution/Rational Design: Perform multiple rounds of mutation and screening to select variants with higher specific activity and altered substrate binding pockets for specific DP production.
• Enzyme Expression and Purification: Optimize the host fermentation protocol for the high-titer production and efficient recovery of the engineered enzyme.
• Enzymatic Hydrolysis Process Optimization: Optimize reaction parameters (pH, temperature, substrate concentration) and test continuous flow systems to maximize conversion and control product inhibition.
• Product Profile Validation: Analyze the final COS product profile using HPLC or MALDI-TOF to confirm the uniformity of the DP distribution and target yield.
• Result Report Output: Compile a detailed Experimental Report including enzyme engineering data, reaction kinetics, and final COS yield and DP profile , supporting commercialization.

Technical communication is maintained throughout the process, focusing on timely feedback regarding enzyme performance and COS quality.

Explore the potential for a high-quality, uniform COS supply. CD Biosynsis provides customized enzyme and process solutions:

• Detailed Enzyme Kinetic and Stability Report , illustrating improved performance under industrial conditions.
• Consultation on continuous processing or recycling strategies for the engineered chitosanase.
• Experimental reports include complete raw data on substrate conversion rate and product DP uniformity , essential for high-end applications.