Alpha-1,2-Fucosyltransferase Selectivity for LNFPI Production
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Solution Overview
Problem
Existing methods for producing lacto-N-fucopentaose I (LNFPI) using α1,2-fucosyltransferase result in the production of 2′FL as a by-product due to the enzyme's ability to react with lactose, necessitating the development of a protein with selective transferase activity that targets lacto-N-tetraose (LNT) over lactose.
Innovation Solution
The use of a microorganism-derived protein with a specific amino acid sequence, such as those from Neisseria or Francisella, exhibiting α1,2-fucosyltransferase activity to selectively transfer fucose to the non-reducing terminal galactose site of LNT, reducing by-product formation and enhancing LNFPI production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If α1,2-fucosyltransferase is used to produce LNFPI, then LNFPI production is achieved, but 2′FL is produced as a by-product due to the enzyme's broad substrate tolerance
Solution Approach 1:
The invention modifies the substrate specificity parameters of α1,2-fucosyltransferase through site-directed mutagenesis, specifically changing amino acid residues in the substrate binding region to reduce affinity for lactose while maintaining activity toward LNT, thereby eliminating 2′FL by-product formation
Solution Approach 2:
The invention introduces specific amino acid substitutions at particular positions (e.g., Ser174Phe, Ser174Ala) in the enzyme's substrate binding pocket to create localized changes in substrate recognition, enabling selective catalysis of LNT conversion to LNFPI without affecting lactose
2Object-generated harmful factors
If highly purified LNT is used as substrate to reduce by-products, then 2′FL formation is reduced, but substrate cost and process complexity increase
Solution Approach 1:
The invention enables the enzyme to selectively process LNT from crude milk oligosaccharide mixtures without requiring prior purification, as the mutated enzyme's enhanced substrate specificity allows it to distinguish LNT from other oligosaccharides including lactose, thereby eliminating the need for complex purification steps
3Loss of time
If enzyme reaction method is used instead of fermentation, then reaction time is reduced, but enzyme cost and purity requirements increase
Solution Approach 1:
The invention employs crude enzyme preparations or cell-free extracts containing the mutated α1,2-fucosyltransferase without requiring high purification, as the enzyme's inherent substrate specificity in the mutated form is sufficient to prevent by-product formation, thereby reducing enzyme preparation costs and simplifying the process
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
The described protein enables efficient production of LNFPI with reduced by-product formation, specifically targeting LNT and minimizing the production of 2′FL, thereby improving the overall yield and purity of LNFPI.
Implementation Method 1
a protein having an α1,2-fucosyltransferase activity... to selectively transfer fucose to the non-reducing terminal galactose site of LNT
Data Source
AI summary
An object is to provide a protein having an α1,2-fucosyltransferase activity and excellent productivity of LNFPI, and a method for producing LNFPI. A protein having a trans-fucosylation activity to lacto-N-tetraose (LNT) and consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, or 26, or a mutant protein or a homologous protein thereof.