Cyclodextrin Glucosyltransferase Mutants for AA-2G Production
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Solution Overview
Problem
Current methods for producing 2-O-α-D-glucopyranosyl-ascorbic acid (AA-2G) using cyclodextrin glucosyltransferase suffer from low enzyme yield and conversion rates, hindering industrial production.
Innovation Solution
Development of cyclodextrin glucosyltransferase mutants with specific amino acid substitutions, such as K124E, G450S, I465F, I641T, K647E, and I631T, which are engineered using site-directed mutagenesis to enhance enzyme activity and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cyclodextrin glucosyltransferase is used for catalytic preparation of AA-2G, then the conversion rate is improved, but the enzyme yield is low
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence parameters of cyclodextrin glucosyltransferase through site-directed mutagenesis. Specific amino acid residues are mutated to alter the enzyme's catalytic properties, thereby improving both enzyme yield and conversion rate simultaneously. This resolves the contradiction by changing the fundamental parameters of the enzyme itself rather than adjusting external conditions.
2Productivity
If cyclodextrin glucosyltransferase is used for catalytic preparation of AA-2G, then the conversion rate is improved, but the purification process is complex
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at particular positions (such as K124E, G450S, I465F, I641T, K647E, I631T) to create localized changes in the enzyme's properties. These localized mutations confer specific characteristics that simplify purification while maintaining high conversion rate, allowing the enzyme to be differentiated and separated more easily from other proteins in the system.
3Productivity
If gene engineering means is used to increase enzyme yield, then the productivity is improved, but the manufacturing complexity is increased
Solution Approach 1:
The patent applies parameter changes through site-directed mutagenesis of the cyclodextrin glucosyltransferase gene, specifically mutating amino acid residues at positions 124, 450, 465, 641, 647, and 631. This targeted approach to changing gene parameters allows for increased enzyme yield through straightforward molecular biology techniques, avoiding the need for complex multi-step manufacturing processes while achieving high productivity.
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 exhibit significantly improved enzyme activity, with some showing up to 2.5 times the activity of the wild-type enzyme, facilitating high-yield industrial production and simplifying purification processes.
Implementation Method 1
the AA-2G is mainly generated through biological catalysis of glycosyltransferases among which cyclodextrin glucosyltransferase is the most commonly used catalytic enzyme
Data Source
AI summary
The present invention relates to preparation and application of a cyclodextrin glucosyltransferase mutant, belonging to the fields of gene engineering and enzyme engineering. By mutating amino acids of cyclodextrin glucosyltransferase, the enzyme activity of the obtained mutant can reach 2.5 times that of wild enzyme. In addition, the cyclodextrin glucosyltransferase mutant obtained in the present invention is simple in purification and suitable for industrial production.