Mutant Nitrile Hydratase Cal. t Nhase-A20V Thermal Stability
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Solution Overview
Problem
Current nitrile hydratase enzymes used in nicotinamide production suffer from decreased activity due to high temperatures and high concentrations of organic substrates and products, leading to low production efficiency and high energy consumption.
Innovation Solution
A mutant nitrile hydratase, Cal. t Nhase-A20V, is developed by mutating glycyl acid at position 20 of the α subunit to valine, which improves thermal stability and tolerance to substrates and products, allowing for higher enzyme activity and yield in nicotinamide production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If high temperature is used in the production process, then reaction rate increases, but enzyme activity decreases due to structural damage
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the nitrile hydratase enzyme structure. The mutation of glycine at position 20 to valine in the α subunit modifies the enzyme's thermal stability parameters, allowing it to maintain activity at higher temperatures that would otherwise denature the wild-type enzyme.
Solution Approach 2:
The patent applies local quality by introducing specific mutations at particular locations (positions 20, 150, 152, and 185) of the enzyme's α and β subunits. Each mutation locally modifies the enzyme structure to enhance thermal stability without compromising overall catalytic function, allowing the enzyme to withstand high-temperature conditions.
2Productivity
If high concentration of organic substrates and products is used, then production efficiency increases, but enzyme activity decreases due to structural destruction
Solution Approach 1:
The patent modifies the enzyme's tolerance parameters through amino acid mutations. The mutant enzymes exhibit changed interaction parameters with organic substrates and products, allowing them to maintain structural integrity and catalytic activity at high concentrations of 3-cyanopyridine and nicotinamide that would inactivate the wild-type enzyme.
Solution Approach 2:
The patent applies beforehand cushioning by pre-modifying the enzyme structure through mutations that create resistance to organic substrate and product damage. The mutant enzymes are prepared in advance with enhanced tolerance, cushioning them against the destructive effects of high-concentration organic compounds during the catalysis process.
3Productivity
If conventional nitrile hydratase is used for nicotinamide production, then production can be achieved, but energy consumption is high and production costs increase
Solution Approach 1:
The patent changes the operational parameters of the enzymatic process by using mutant nitrile hydratases with enhanced thermal stability. These mutants allow the reaction to proceed at lower temperatures while maintaining high activity, thereby reducing energy consumption for heating and cooling without compromising nicotinamide production efficiency.
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 nitrile hydratase, Cal. t Nhase-A20V, exhibits enhanced thermal stability and substrate/product tolerance, resulting in a significant increase in nicotinamide yield to 598 g/L and specific enzyme activity of 650 U/mg, compared to the wild enzyme.
Implementation Method 1
Nitrile hydratase (NHase) can be used to catalyze 3-cyanopyridine into nicotinamide with higher medicinal value
Implementation Method 2
The mutant nitrile hydratase, Cal. t Nhase-A20V, exhibits enhanced thermal stability and substrate/product tolerance
Implementation Method 3
the mutant also has better tolerance to a substrate and a product, and the final yield of nicotinamide produced by whole-cell catalysis reaches 598 g/L
Data Source
AI summary
The disclosure discloses a mutant of nitrile hydratase derived from Caldalkalibacillus thermarum, and belongs to the technical field of enzyme engineering. The nitrile hydratase mutant Cal. t Nhase-A20V provided by the disclosure has a half-life of about 10 min at 70° C., which does not change much compared with the thermal stability of the wild enzyme. The specific enzyme activity of the mutant Cal. t Nhase-A20V is 128% of that of the wild enzyme. At the same time, the mutant also has better tolerance to a substrate and a product, and the final yield of nicotinamide produced by whole-cell catalysis reaches 598 g/L. Therefore, the nitrile hydratase mutant Cal. t Nhase-A20V provided by the disclosure has good enzymatic properties and is beneficial to future industrial production.