Engineered Carboxyesterase Amidation Activity

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Solution Overview

Problem

Current methods for amide synthesis, such as traditional chemical catalytic approaches, suffer from limitations including poor atom economy, significant waste generation, lack of enantioselectivity and chemoselectivity, and the use of toxic or explosive reagents.

Innovation Solution

Development of engineered carboxyesterase enzymes with significantly enhanced amidation activity, derived from the wild-type A. acidocaldarius Esterase 2, which can efficiently convert ester substrates to amides in the presence of amine substrates, thereby simplifying the synthesis process and reducing the need for stoichiometric activating agents.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Loss of substance

If traditional chemical catalytic approaches are used for amide synthesis, then amide bond formation can be achieved, but atom economy is poor and significant waste is generated

Engineering Contradiction:

Improvewaste generationVSAvoidamide synthesis efficiency

Core Design Contradiction:

Loss of substanceVSProductivity

Solution Approach 1:

The engineered carboxyesterase enzyme catalyzes the direct conversion of ester and amine substrates to amide products through a self-contained enzymatic mechanism, eliminating the need for external stoichiometric coupling reagents and minimizing waste generation while maintaining high productivity

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The patent applies directed evolution to engineer specific amino acid residues in the carboxyesterase enzyme, changing its catalytic parameters to achieve 785,000-fold improved amidation activity while maintaining high atom economy and reducing waste

Inventive Principle:

Principle #35Parameter changes

2Object-affected harmful factors

If traditional chemical catalytic approaches are used for amide synthesis, then amide bond formation can be achieved, but toxic or explosive reagents are required

Engineering Contradiction:

ImprovetoxicityVSAvoidamide synthesis efficiency

Core Design Contradiction:

Object-affected harmful factorsVSProductivity

Solution Approach 1:

The engineered carboxyesterase enzyme replaces expensive and hazardous stoichiometric coupling reagents with a biocatalyst that can be reused, eliminating toxicity concerns while maintaining high amide synthesis efficiency through enzymatic catalysis

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Solution Approach 2:

The patent replaces traditional chemical catalysis with enzymatic catalysis, substituting the enzymatic mechanism for chemical reagents and eliminating the need for toxic or explosive coupling agents while maintaining productive amide bond formation

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Manufacturing precision

If traditional chemical catalytic approaches are used for amide synthesis, then amide bond formation can be achieved, but enantioselectivity and chemoselectivity are lacking

Engineering Contradiction:

ImproveenantioselectivityVSAvoidamide synthesis efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The engineered carboxyesterase enzyme creates a specific chiral environment in its active site through precisely positioned amino acid residues, providing high enantioselectivity for amide bond formation while maintaining efficient catalysis through localized catalytic groups

Inventive Principle:

Principle #3Local quality

4Productivity

If wild-type carboxyesterase enzyme is used, then enzyme catalysis is achieved, but amidation activity is insufficient

Engineering Contradiction:

Improveamidation activityVSAvoidenzyme engineering complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent employs directed evolution to introduce specific amino acid changes at critical positions in the enzyme active site, achieving partial optimization steps that collectively result in 785,000-fold improved amidation activity while managing engineering complexity through systematic mutation approaches

Inventive Principle:

Principle #16Partial or excessive action

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 carboxyesterase enzymes exhibit a 785,000-fold improvement in amidation activity compared to the wild-type enzyme, allowing for direct synthesis of amides from simple ester and amine precursors with improved tolerance to water and alcohols, thus enhancing the efficiency and sustainability of amide synthesis.

Implementation Method 1

engineered carboxyesterase enzymes with significantly enhanced amidation activity, derived from the wild-type A. acidocaldarius Esterase 2, which can efficiently convert ester substrates to amides in the presence of amine substrates

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 2

Methodology Applied

Scientific EffectCatalysis: Catalysis

Data Source

PatentUS12331327B2Carboxyesterase biocatalysts

Publication Date: 2025.06.17 GLAXOSMITHKLINE INTPROP DEV LTD

AI summary

The present disclosure provides engineered carboxyesterase enzymes that have the ability to catalyze amide bond formation. Also provided are polynucleotides encoding the carboxyesterase enzymes, host cells capable of expressing the engineered carboxyesterase enzymes, and methods of using the engineered carboxyesterase enzymes to make commercially valuable amides. Also provided are amides that are made using the engineered carboxyesterase enzymes.