Helicobacter Pylori Fucosyltransferase Mutants for Complex Substrate Labeling
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Solution Overview
Problem
Existing mutants of Helicobacter pylori α-1,3-fucosyltransferase struggle to recognize and efficiently catalyze complex substrates, such as large molecule donor substrates and complex receptor substrates, leading to low enzyme activity and difficulty in expressing the enzyme in a soluble form in E. coli, which hampers the development of engineered cells for therapeutic applications.
Innovation Solution
Mutants of Helicobacter pylori α-1,3-fucosyltransferase are generated through error-prone PCR, specifically altering amino acids at positions 108 and/or 139, resulting in mutants A108V and D139E, which are expressed in E. coli BL21 (DE3) and demonstrate improved catalytic efficiency on complex substrates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing mutants of Helicobacter pylori α-1,3-fucosyltransferase are used, then enzyme activity on simple substrates is maintained, but enzyme activity on complex substrates (large molecule donor substrates and complex receptor substrates) is very low
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues (particularly in the substrate binding pocket) to alter the enzyme's substrate recognition properties. This allows the enzyme to accommodate larger molecular weight substrates while maintaining catalytic activity, resolving the contradiction between reliability on simple substrates and adaptability to complex substrates.
2Productivity
If fucosyltransferase is used to transfer macromolecular proteins to cell membrane surfaces, then therapeutic effectiveness of cell therapies is enhanced, but enzyme activity is reduced by nearly a thousand times compared to small molecule substrates
Solution Approach 1:
The patent modifies the enzyme's catalytic parameters through site-directed mutagenesis, specifically optimizing the transition state stabilization and substrate binding affinity. This enables the enzyme to maintain high productivity with macromolecular substrates by changing the kinetic parameters (Km and kcat) to accommodate larger substrates, thereby resolving the contradiction between therapeutic effectiveness and enzyme activity reliability.
Solution Approach 2:
The patent applies local quality changes by modifying specific regions of the enzyme (particularly the substrate binding pocket and catalytic domain) while leaving other regions unchanged. This localized optimization allows the enzyme to handle macromolecular substrates effectively without compromising overall enzyme stability and function, thus resolving the productivity-reliability contradiction.
3Reliability
If substitution mutations are introduced to enhance enzyme activity, then enzyme activity may be improved, but expression in E. coli becomes difficult with formation of inclusion bodies
Solution Approach 1:
The patent uses parameter changes to optimize the balance between enzyme activity and solubility by systematically varying amino acid substitutions and evaluating their dual effects on catalytic activity and expression solubility. This allows identification of mutations that enhance activity while maintaining proper folding and soluble expression in E. coli.
Solution Approach 2:
The patent employs chaperone proteins or co-expression systems as intermediaries to facilitate the proper folding of mutated enzymes in E. coli, preventing inclusion body formation while preserving the enhanced enzymatic activity. This mediator approach resolves the contradiction between improved enzyme activity and ease of manufacture.
4Adaptability or versatility
If Km of enzyme is low for complex substrates, then enzyme can recognize complex substrates, but commercial detection kits cannot detect enzyme activity
Solution Approach 1:
The patent develops alternative detection methods that copy or mimic the natural complex substrates, allowing enzyme activity to be measured using simplified assay systems. This enables detection of enzymes with low Km for complex substrates by using surrogate substrates that retain the essential recognition features while being amenable to standard detection techniques.
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 mutants A108V and D139E exhibit 1.75-1.82 times higher catalytic efficiency in coupling N803 onto cell membranes compared to the wild type, with D139E showing three times higher enzyme activity and increased enzyme loading, enhancing the potential for therapeutic applications by extending the half-life of drugs and reducing infusion frequency.
Implementation Method 1
Mutants of Helicobacter pylori α-1,3-fucosyltransferase are generated through error-prone PCR, specifically altering amino acids at positions 108 and/or 139
Implementation Method 2
Fucosyltransferase is an enzyme that transfers L-fucose from GDP-fucose (guanosine diphosphate fucose) to polysaccharides, participating in the synthesis of terminal polysaccharide structures. The α -1,3-fucosyltransferase of Helicobacter pylori catalyzes branched fucosylation
Data Source
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AI summary
Disclosed in the present invention is a mutant of Helicobacter pylori α-1,3-fucosyltransferase, a polynucleotide encoding the mutant, and an expression vector and a host cell comprising the polynucleotide. Further disclosed in the present invention are a method for labeling a target molecule on a target cell or a target protein by using the mutant of Helicobacter pylori α-1,3-fucosyltransferase, a cell or protein labeled according to the method, and a use of the cell or protein in the preparation of a disease treatment drug. Compared with the wild type, the mutant of Helicobacter pylori α-1,3-fucosyltransferase provided by the present invention can label a target molecule on the surface of a cell membrane with better efficiency, and a method for quantitative detection of enzyme activity and an application prospect of a labeled cell or protein in the preparation of a disease treatment drug are provided.