Cyclodextrin Glycosyltransferase Mutants for Organic Solvent Tolerance
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Solution Overview
Problem
Cyclodextrin glycosyltransferase enzymes exhibit reduced activity and stability in organic solvent environments, limiting their application in industrial processes involving hydrophobic substrates.
Innovation Solution
Mutating specific amino acids in the cyclodextrin glycosyltransferase, such as positions 146 and 539, to create mutants with enhanced solvent tolerance, including R146F, G539I, G539I/R146F, and G539I/R146F/D147N, which are expressed using recombinant vectors in host cells like E. coli or Bacillus subtilis, and optimized for use in organic solvents like DMSO, ethanol, and acetone.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If cyclodextrin glycosyltransferase is used in organic solvent environments, then the enzyme can process hydrophobic substrates, but the enzyme activity and stability are greatly reduced
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (positions 146, 147, and 539) in the cyclodextrin glycosyltransferase sequence to alter the enzyme's physical and chemical properties. This enables the enzyme to maintain stability and activity in organic solvent environments while retaining its ability to process hydrophobic substrates, thus resolving the contradiction between adaptability and reliability.
2Reliability
If molecular modification is performed to enhance catalytic activity and thermal stability, then enzyme performance improves, but solvent tolerance remains insufficient
Solution Approach 1:
The patent specifically modifies amino acid parameters at positions 146, 147, and 539 to enhance solvent tolerance. By changing the chemical properties of these residues through mutation, the enzyme gains improved tolerance to organic solvents while maintaining the catalytic activity and thermal stability achieved through previous molecular modification efforts.
3Productivity
If natural cyclodextrin glycosyltransferase is used, then the enzyme maintains good catalytic activity, but the enzyme stability in organic solvents is poor
Solution Approach 1:
The patent changes the amino acid composition parameters at critical positions (146, 147, and 539) of the enzyme sequence. This modification allows the enzyme to maintain its good catalytic activity characteristic of the natural enzyme while significantly improving its stability in organic solvent environments, thereby resolving the contradiction between productivity and stability.
Data Source
AI summary
The disclosure discloses a cyclodextrin glycosyltransferase with enhanced solvent tolerance and preparation thereof, belonging to the technical fields of enzyme engineering and genetic engineering. The disclosure constructs four cyclodextrin glycosyltransferase mutants with enhanced organic solvent tolerance. Among them, the mutant with the optimal tolerance to DMSO and methanol is G539I/R146F/D147N, which is 1.6 times and 1.7 times higher than that of WT, respectively; the mutant with the optimal tolerance to ethanol is R146F, which is 1.4 times higher than that of WT; the mutant with the optimal tolerance to acetone is G539I/R146F, which is 1.5 times higher than that of WT. The disclosure helps to expand the application of glycosyltransferases in organic reaction systems, improves the enzymatic efficiency of CGTase on natural hydrophobic substrates, and has great application prospects.