Lactose Binding Alpha-1,2-Fucosyltransferase Enzyme Mutants
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Solution Overview
Problem
Current alpha-1,2-fucosyltransferases have low affinity for lactose, leading to inefficient production of 2′fucosyllactose and high undesired by-product formation of difucosyllactose, with a concentration ratio often exceeding 1:5.
Innovation Solution
Identification and utilization of newly discovered lactose binding alpha-1,2-fucosyltransferases with improved lactose binding and transferase properties, reducing side activity and by-product formation, resulting in a diFL to 2′FL concentration ratio of less than 1:10.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional alpha-1,2-fucosyltransferases are used for 2′FL production, then the synthesis process can be implemented, but the productivity is low due to low lactose affinity and high by-product formation
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of alpha-1,2-fucosyltransferase through site-directed mutagenesis. Specific amino acid residues are changed to improve lactose binding affinity and reduce difucosyllactose formation, thereby enhancing both productivity and manufacturing precision simultaneously
Solution Approach 2:
The patent creates improved enzyme variants by copying the wild-type alpha-1,2-fucosyltransferase structure and introducing specific mutations. Multiple mutant versions are generated and screened to find the optimal balance between lactose affinity and by-product reduction
2Productivity
If alpha-1,2-fucosyltransferases with high lactose affinity are used, then 2′FL production efficiency improves, but difucosyllactose by-product formation increases
Solution Approach 1:
The patent uses parameter changes by systematically varying amino acid sequences at specific positions in the enzyme. Mutations are introduced to decouple the correlation between lactose affinity and difucosyllactose formation, allowing high productivity with reduced harmful by-products
Solution Approach 2:
The patent employs a library of enzyme mutants where individual variants with suboptimal properties are discarded and replaced by improved versions. This approach allows rapid iteration to find enzymes that simultaneously achieve high productivity and low by-product formation
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enhances the production of 2′fucosyllactose with reduced unwanted by-products, achieving higher yields and purity levels, specifically with a diFL to 2′FL ratio lower than 1:10, thereby improving the efficiency and cost-effectiveness of the synthesis process.
Implementation Method 1
They catalyze the transfer of a fucose residue from a donor, generally guanosine-diphosphate fucose (GDP-fucose) to an acceptor
Data Source
AI summary
The present invention relates to methods for producing 2′ fucosyllactose (2′-FL), as well as newly identified fucosyltransferases, more specifically newly identified lactose binding α-1,2-fucosyltransferase polypeptides, and their applications. Furthermore, the present invention provides methods for producing 2-fucosyllactose (2′FL) using the newly identified α-1,2-fucosyltransferases.