Fructooligosaccharide (FOS) Engineering Service for Conversion Rate and Yield

Fructooligosaccharides (FOS) are non-digestible carbohydrates widely used as prebiotics, dietary fibers, and low-calorie sweeteners in the food and health supplement industries. Current production methods face two major challenges: natural plant extraction yields a low amount of FOS , which is often complex and costly, while the dominant enzymatic synthesis method (using fructosyltransferase, FTase) often results in a low conversion rate from sucrose, limiting final yield and purity.

CD Biosynsis offers a synthetic biology service focused on enhancing the enzymatic synthesis route. Our core strategy involves the directed evolution of fructosyltransferase (FTase) to maximize its transfructosylation activity relative to its hydrolysis activity, thereby increasing the conversion of sucrose to FOS. This is coupled with the optimization of the Aspergillus niger fermentation system to achieve high, stable yields of the engineered FTase enzyme itself, ensuring a cost-effective biocatalyst supply. This integrated approach aims to deliver an efficient, high-purity, and industrially scalable FOS production process.

Pain Points

Improving FOS production efficiency requires addressing these limitations in the enzymatic route:

• Low Conversion Rate: FTase naturally possesses both transfructosylation (desired) and hydrolysis (undesired) activities . High hydrolysis activity breaks down the sucrose substrate and the FOS product, severely limiting the maximum conversion rate.
• Enzyme Production Cost: Relying on microbial fermentation (often A. niger ) to produce the FTase biocatalyst can be expensive if the enzyme yield or specific activity is low .
• Byproduct Formation: Low conversion efficiency leads to a final product mixture containing high levels of unconverted sucrose and glucose/fructose monomers , requiring complex and costly purification steps.
• Enzyme Stability: The FTase enzyme may suffer from poor thermal stability or pH tolerance during high-substrate concentration reactions, restricting reactor conditions.

A cost-effective solution must improve the enzyme's selectivity while ensuring a cheap, high-yield supply of the biocatalyst.

Solutions

CD Biosynsis utilizes enzyme and microbial engineering to enhance FOS synthesis efficiency:

Directed Evolution of Fructosyltransferase (FTase)

           

We employ high-throughput screening to evolve FTase variants with a significantly higher ratio of transfructosylation to hydrolysis activity , maximizing FOS yield and purity.

Optimization of Aspergillus niger Fermentation System

We modify the A. niger host strain's genome to increase the secretion and stability of the target FTase enzyme, reducing biocatalyst production costs.

Enzyme Immobilization Strategies

We develop methods for immobilizing the engineered FTase onto carriers, enabling continuous reuse of the enzyme and further reducing overall operational costs.

Substrate and Product Tolerance Improvement

We engineer the FTase for better tolerance to high sucrose concentration and high temperature , allowing for improved reaction kinetics.

This systematic approach is focused on creating a superior biocatalyst and an optimized production platform for cost-effective FOS synthesis.

Advantages

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

High Conversion Efficiency

Engineered FTase aims to significantly increase the final yield of FOS from the sucrose substrate.

Simplified Purification Process

Higher conversion rates result in lower levels of unconverted sugar byproducts , simplifying and reducing the cost of downstream separation.

Low Biocatalyst Cost

Optimized A. niger fermentation provides a high-titer, cost-effective source of the FTase enzyme for industrial use. [Image of Cost Reduction Icon]

Increased Enzyme Reusability

Enzyme immobilization techniques are designed to allow for multiple reaction cycles , reducing the enzyme dosage required over time.

Controlled FOS Composition

Enzyme engineering can potentially control the degree of polymerization (DP) of the FOS product, tailoring it for specific functional properties.

We provide a biosynthetic platform aimed at maximizing the quality and cost-effectiveness of FOS production.

Process

Our FOS engineering service follows a standardized, iterative research workflow:

• FTase Library Construction: Generate a focused FTase mutant library based on rational design or random mutagenesis, targeting residues near the active site that influence water access .
• High-Throughput Screening: Implement a screening assay that rapidly identifies variants with the highest transfructosylation-to-hydrolysis ratio at high substrate concentrations.
• A. niger Host Optimization: Modify the A. niger host strain, potentially removing competing proteases and optimizing promoter sequences for stable, high-level FTase secretion.
• Immobilization Protocol Development: Develop and validate a robust method for covalent or physical immobilization of the final engineered FTase variant onto an industrial-scale carrier.
• Biocatalysis Validation: Perform batch or continuous enzymatic synthesis using the immobilized enzyme, measuring final FOS yield, purity, and operational stability .
• Result Report Output: Compile a detailed Experimental Report including FTase mutation data, enzyme kinetics (kcat/KM for transfer vs. hydrolysis), and final FOS yield/purity metrics , supporting commercial implementation.

Technical communication is maintained throughout the process, focusing on timely feedback regarding FTase selectivity and enzyme stability.

Explore the potential for a high-efficiency FOS synthesis route. CD Biosynsis provides customized enzyme and fermentation system solutions:

• Detailed Kinetic and Stability Analysis Report , demonstrating the improvement in FTase selectivity and lifetime.
• Consultation on continuous reactor design optimized for the immobilized FTase system.
• Experimental reports include complete raw data on sucrose conversion rate, FOS purity ($\% \text{w/w), and byproduct formation , essential for product formulation.