Amino Acid Dehydrogenase Cofactor Regeneration via Ketoreductase
Solution Overview
Problem
Current processes for biosynthesis of chiral amino acids using amino acid dehydrogenases are limited by inefficiencies in biocatalytic reactions, requiring enhancements to improve yield and specificity.
Innovation Solution
A coupled system involving L- or D-amino acid dehydrogenase (AADH) coupled with a cofactor regenerating system comprising a ketoreductase (KRED) is used to convert 2-oxo acids into chiral amino acids, utilizing a reaction medium with an ammonium ion donor, NAD+/NADH or NADP+/NADPH, and a secondary alcohol to achieve enantiomeric excess.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If amino acid dehydrogenase is used for biosynthesis of chiral amino acids, then enantiomeric excess is achieved, but reaction efficiency and yield are limited
Solution Approach 1:
The patent combines amino acid dehydrogenase with a cofactor regenerating system comprising ketoreductase and secondary alcohol in a coupled system. This merging of enzymatic functions allows continuous regeneration of NADH/NADPH cofactors, thereby maintaining high reaction efficiency while preserving the enantiomeric excess produced by the dehydrogenase.
Solution Approach 2:
The patent introduces parameter changes by adding a cofactor regenerating system that modifies the redox state parameters through ketoreductase-catalyzed reduction of NAD+ to NADH using secondary alcohol. This parameter change enables sustained high-yield production while maintaining the stereochemical precision of the dehydrogenase reaction.
2Productivity
If cofactor regenerating system with ketoreductase is added, then reaction efficiency is enhanced, but system complexity increases
Solution Approach 1:
The ketoreductase enzyme performs multiple functions: it regenerates NADH/NADPH cofactors for the dehydrogenase reaction and simultaneously produces ketone byproducts that can be removed to drive reaction equilibrium. This multi-functionality enhances reaction efficiency without proportionally increasing system complexity.
Solution Approach 2:
The cofactor regenerating system is designed to be self-sustaining, where the secondary alcohol substrate automatically replenishes NADH/NADPH levels as they are consumed by the dehydrogenase. This self-service mechanism maintains high productivity without requiring external cofactor addition or complex control systems.
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
This approach significantly enhances the efficiency and specificity of chiral amino acid biosynthesis, allowing for high yields of chiral amino acids with improved enantiomeric excess, leveraging engineered ketoreductases for increased activity and stability.
Implementation Method 1
Amino acid dehydrogenases comprise a group of coenzyme-dependent enzymes that catalyze the reversible oxidative deamination of an amino acid to its keto acid and ammonia with the concomitant reduction of either cofactor NAD+, NADP+ or FAD
Implementation Method 2
a cofactor regenerating system comprising a ketoreductase and a lower secondary alcohol, under conditions where the compound of formula I is converted to the chiral amino acid of formula IIa and the lower secondary alcohol is converted to a ketone
Data Source
AI summary
The present disclosure relates to the use of an amino acid dehydrogenase in combination with a cofactor regenerating system comprising a ketoreductase. In particular embodiments, the process can be used to prepare L-tert-leucine using a leucine dehydrogenase.