Engineered Transaminase Polypeptides for Chiral Amine Synthesis
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Solution Overview
Problem
Wild-type transaminases are not active enough, lack stability, or have insufficient stereoselectivity, making them unsuitable for industrial production of chiral amine compounds, which requires extreme conditions and generates waste, leading to high costs and pollution.
Innovation Solution
Engineered transaminase polypeptides with improved activity, stability, and stereoselectivity are developed through directed evolution, specifically designed to asymmetrically synthesize R-(+)-α-phenylethylamine with high efficiency and low inhibition by amino acid substitutions, insertions, or deletions, allowing for mild reaction conditions and higher yields.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If wild-type transaminase is used for chiral amine synthesis, then the reaction can proceed under mild conditions, but the enzyme exhibits low activity, poor stability, and insufficient stereoselectivity
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (e.g., V148L, T187I, L190M, S214C) to alter the enzyme's catalytic properties. These targeted mutations change the local structural parameters of the transaminase, thereby improving its activity, stability, and stereoselectivity while maintaining its ability to function under mild conditions
2Duration of action of moving object
If wild-type transaminase is used for chiral amine synthesis, then the reaction can proceed, but the enzyme exhibits poor stability
Solution Approach 1:
The patent uses parameter changes through specific amino acid substitutions (e.g., V148L, T187I, L190M, S214C) to enhance the structural stability of the transaminase enzyme. These mutations modify physical parameters of the enzyme structure, resulting in improved thermal stability and operational durability without sacrificing catalytic efficiency
3Manufacturing precision
If wild-type transaminase is used for chiral amine synthesis, then the reaction can proceed, but the enzyme exhibits insufficient stereoselectivity
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (e.g., V148L, T187I, L190M, S214C) that precisely tune the enzyme's stereoselectivity. These mutations alter the spatial and electronic parameters of the active site, enabling high enantioselectivity for producing R-(+)-α-phenylethylamine while preserving catalytic activity
4Manufacturing precision
If conventional chemical synthesis method is used for chiral amine preparation, then high chiral purity can be achieved, but extreme conditions and cumbersome resolution steps are required, generating large amount of waste
Solution Approach 1:
The patent replaces the conventional chemical synthesis mechanism with a biocatalytic mechanism using engineered transaminase. This substitution eliminates the need for extreme conditions, resolution steps, and hazardous chemicals, thereby achieving high chiral purity while significantly reducing environmental pollution and waste generation
Solution Approach 2:
The engineered transaminase exhibits self-service by inherently providing high stereoselectivity and catalytic activity without requiring external resolution steps or extreme conditions. The enzyme autonomously produces high chiral purity products under mild conditions, eliminating the need for additional purification processes that generate waste
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 engineered transaminase polypeptides achieve high stereoselectivity and stability, enabling efficient synthesis of chiral amine compounds with reduced waste and environmental impact, overcoming the limitations of wild-type transaminases.
Implementation Method 1
Transaminase is capable of catalyzing the transfer of an amino group from an amine donor to a ketone substrate to give a chiral amine product and a ketone by-product
Implementation Method 2
Transaminase has been favored by the industry for its high selectivity, high conversion rate and mild reaction conditions. Transaminase is capable of catalyzing the transfer of an amino group from an amine donor to a ketone substrate
Implementation Method 3
The reaction requires pyridoxal phosphate (PLP) as cofactor
Data Source
AI summary
Provided are amino acid sequences of engineered transaminase polynucleotide that are useful for asymmetrically synthesizing chiral amine compounds, and its preparation process and reaction process under industrial-relevant conditions. Also provided are polynucleotide sequences encoding engineered transaminase polypeptides, engineered host cells capable of expressing engineered transaminase polypeptides, and methods of producing chiral amine compounds using engineered transaminase polypeptides. Compared to other enzymes, the engineered transaminase polypeptides provided by this invention have better catalytic activity and stability, and are not inhibited by chiral amine products in the synthesis process. The use of the engineered polypeptides of the present invention for the preparation of chiral amine compound results in higher unit activity, and has good industrial application prospects.