Modified Esterase for Stereoselective Cyhalothric Acid Hydrolysis
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Solution Overview
Problem
Existing methods for producing λ-cyhalothric acid, a key component of cyhalothrin with the highest insecticidal effect, face challenges in terms of yield and cost, and generate environmental waste due to chemical synthesis, necessitating an enzyme-based production method that can stereoselectively hydrolyze the (1R,3R) form of ester compounds efficiently.
Innovation Solution
A quintuple mutant modified esterase derived from Arthrobacter globiformis is engineered to enhance its reactivity towards (1R,3R)-methyl 3-(2-chloro-3,3,3-trifluoroprop-1-en-1-yl)-2,2-dimethylcyclopropane-1-carboxylate, achieved through specific amino acid substitutions and modifications, resulting in improved reactivity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis methods are used to produce CA isomers, then production capacity is achieved, but yield is low and cost is high
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (e.g., A102T, A109T, L151F, L151M, L151W, I153V, Y173F, Y197F, L200F, I211V, I211M, F217K, S220W, D222G, D227C, D227E, L229G, L242C, L242I, L242M, L242W, A245T, V257I, L298H, T301A, V326I, S329D, S329G, S329N, A350D, T371A) in the esterase enzyme to change its catalytic properties and substrate specificity, enabling high-yield production of λ-cyhalothric acid through enzymatic hydrolysis
2Productivity
If chemical synthesis methods are used to produce CA isomers, then production can proceed, but wastewater is generated causing environmental issues
Solution Approach 1:
The patent replaces chemical synthesis methods with enzymatic catalysis, substituting chemical reactions with biological catalysis. The modified esterase enzyme catalyzes the hydrolysis of methyl cyhalothrate to produce λ-cyhalothric acid, eliminating the need for chemical catalysts and reducing wastewater generation associated with chemical synthesis processes
3Adaptability or versatility
If existing esterases are used, then general ester hydrolysis occurs, but reactivity to (1R,3R)-MC is insufficient
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at defined positions in the esterase enzyme structure. These localized changes at specific residues (e.g., A102T, A109T, L151F) modify the enzyme's active site properties to enhance binding affinity and catalytic efficiency specifically for (1R,3R)-MC while preserving overall esterase functionality
Solution Approach 2:
The patent employs asymmetry by creating stereoselective enzyme variants that specifically recognize and hydrolyze the (1R,3R) enantiomer of methyl cyhalothrate. The amino acid substitutions introduce chiral recognition capabilities in the active site, enabling the enzyme to distinguish between enantiomers and preferentially catalyze hydrolysis of the desired (1R,3R) form
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 modified esterase effectively synthesizes (1R,3R)-cyhalothric acid with enhanced reactivity, overcoming yield and cost issues of chemical synthesis while reducing environmental impact.
Implementation Method 1
a modified esterase with improved reactivity to (1R,3R)-methyl 3-(2-chloro-3,3,3-trifluoroprop-1-en-1-yl)-2,2-dimethylcyclopropane-1-carboxylate
Implementation Method 2
stereoselectively hydrolyze only the (1R,3R) form of ester compound of λ-cyhalothric acid
Data Source
AI summary
The present invention provides any one of the following modified esterases (i) to (iii):(i) a modified esterase comprising an amino acid sequence in which at least one amino acid at a site selected from the group consisting of (1) to (24) below in the amino acid sequence of SEQ ID NO: 1 or 3 is substituted with another amino acid, (1) V64 (2) S65 (3) A102 (4) A109 (5) L151 (6) I153 (7) Y173 (8) Y197 (9) L200 (10) I211 (11) F217 (12) S220 (13) D222 (14) D227 (15) L229 (16) L242 (17) A245 (18) V257 (19) L298 (20) T301 (21) V326 (22) S329 (23) A350 (24) T371;(ii) a modified esterase in which one or more amino acids are further substituted (except for the amino acid site substituted in (i)), added, inserted, or deleted (except for the amino acid site substituted in (i)) in the modified esterase of (i), and the reactivity to (1R,3R)-methyl 3-(2-chloro-3,3,3-trifluoroprop-1-en-1-yl)-2,2-dimethylcyclopropane-1-carboxylate is improved compared to the reactivity of an esterase consisting of the amino acid sequence of SEQ ID NO: 1 or 3;(iii) a modified esterase in which an amino acid(s) other than the amino acid(s) substituted in (i) is further substituted with another amino acid(s), wherein the modified esterase has 70% or more identity with the modified esterase of (i) and has improved reactivity to (1R,3R)-methyl 3-(2-chloro-3,3,3-trifluoroprop-1-en-1-yl)-2,2-dimethylcyclopropane-1-carboxylate compared to the reactivity of the esterase consisting of the amino acid sequence of SEQ ID NO: 1 or 3.