Formate Dehydrogenase Mutant Disulfide Bond Stability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Wild-type formate dehydrogenase (CboFDH) from Candida boidinii has low specific enzyme activity and poor operational stability, limiting its efficiency in regenerating NADH for industrial production of chiral compounds.
Innovation Solution
A formate dehydrogenase mutant with an amino acid sequence mutation at the 10th site from alanine to cysteine, encoded by a specific nucleotide sequence, is expressed using a recombinant expression vector in genetically engineered E. coli bacteria, enhancing enzyme activity and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type CboFDH is used for NADH regeneration, then the enzyme can catalyze formic acid to generate CO2 and NADH, but the specific enzyme activity is low and operational stability is poor
Solution Approach 1:
The patent applies site-specific mutagenesis to change the amino acid at position 10 from alanine to cysteine in the CboFDH enzyme. This parameter change in the enzyme's primary structure results in improved operational stability (6.8 times increase in half-life at 60°C) while maintaining or enhancing specific enzyme activity (1.3 times increase), thereby resolving the contradiction between reliability and productivity
Solution Approach 2:
The patent introduces a localized modification at position 10 of the enzyme sequence, creating a cysteine residue that forms an intramolecular disulfide bond with cysteine at position 30. This local structural change specifically enhances the enzyme's resistance to copper ion inactivation and thermal stability without affecting the overall catalytic function, thus improving reliability while preserving productivity
2Ease of manufacture
If wild-type CboFDH is used, then the enzyme structure is simple and easy to obtain, but the enzyme shows poor tolerance to copper ions and low acid resistance
Solution Approach 1:
The patent modifies the enzyme's amino acid sequence parameter at position 10, introducing cysteine to form a disulfide bond that specifically enhances copper ion tolerance (30 times improvement) and acid resistance (2.0 times improvement at pH 4), while maintaining the simplicity of production through recombinant expression in E. coli
Solution Approach 2:
The introduced cysteine at position 10 forms an intramolecular disulfide bond with cysteine at position 30, creating a protective structural feature beforehand that cushions the enzyme against copper ion inactivation and acid denaturation, preventing damage before it occurs
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 mutant enzyme shows improved specific activity by 1.3 times, increased half-life by 6.8 times at 60°C, enhanced copper ion tolerance by 30 times, and improved acid resistance and affinity to NAD+, facilitating more efficient NADH regeneration for bioconversion production.
Implementation Method 1
Formate dehydrogenase (FDH, EC 1.2.1.2) catalyzes formic acid to generate carbon dioxide and generates NADH along with the reduction of NAD+
Implementation Method 2
generates NADH along with the reduction of NAD+
Implementation Method 3
the cysteine obtained by mutating the amino acid residue at 10th site can form a correct disulfide bond with the original cysteine residue at 30th site
Implementation Method 4
so that the oxidation of the free cysteine is prevented from improving the resistance of the mutant enzyme to copper ions
Data Source
AI summary
The present invention discloses a formate dehydrogenase mutant with improved enzyme activity and stability and a construction method thereof, which belongs to the technical field of genetic engineering. The mutant of the present invention is obtained by mutating alanine at a 10th site to cysteine based on the amino acid shown in SEQ ID NO. 2. The specific enzyme activity of the mutant enzyme obtained by the present invention is improved by 1.3 times compared with that before the mutation, a half-life period (t1/2) at 60° C. is increased by 6.8 times compared with that in the mutation period, the copper ion tolerance is increased by 30 times compared with that before the mutation, and when pH is 4, the stability is improved by 2.0 times, and the catalytic efficiency is increased by 1.4 times. The present invention shows that an amino acid residue at a 10th site is mutated to the cysteine which forms a correct disulfide bond with a cysteine residue at a 30th site of the natural formate dehydrogenase, so that the stability and the catalytic efficiency of the enzyme are improved, and the industrial application potential of the enzyme is improved.