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Catalytically Perfect Enzymes

Catalytically Perfect Enzymes

The theory of catalytically perfect enzymes was first proposed by W.J. Albery and J. R. Knowles in 1976. These enzymes catalyze biochemical reactions at high-speed. Their catalytic efficiency values range from 108-109 M-1s-1. These enzymes are also called 'diffusion-controlled' as the only rate-limiting step in the catalysis is that of the substrate diffusion into the active site. Examples include triose phosphate isomerase, fumarase, and superoxide dismutase.

Most enzymes...

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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Published on: March 25, 2020

In Vitro Enzyme Self-Selection Using Molecular Programs.

Adèle Dramé-Maigné¹, Rocío Espada¹, Giselle McCallum¹

¹Gulliver UMR CNRS 7083, ESPCI Paris, Université PSL, 75005 Paris, France.

ACS Synthetic Biology

|January 11, 2024

Summary

This summary is machine-generated.

We developed a new in vitro self-selection method for enzyme engineering, linking enzyme function to gene replication. This approach enables high-throughput screening of millions of variants for improved enzyme activity and stability.

Keywords:

biocatalysis chemical reaction networks directed evolution enzymes in vitro microfluidics molecular programing ultrahigh-throughput selection

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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

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Published on: August 8, 2016

Area of Science:

Synthetic Biology
Enzyme Engineering
Biotechnology

Background:

Directed evolution is key for in vitro enzyme engineering.
Current high-throughput screening methods (microfluidics) are complex.
In vitro self-selection offers higher throughput but is limited to few activities.

Purpose of the Study:

To generalize in vitro compartmentalized self-selection using synthetic molecular networks.
To create a programmable circuit linking enzymatic activity to gene replication.
To enable screening-free enrichment of enzyme variants with desired phenotypes.

Main Methods:

Developed a programmable circuit architecture for autonomous selection.
Encapsulated bacterial expression libraries with the selection circuit.
Applied autonomous selection for thermostability and catalytic efficiency of Nt.BstNBI (NBI).
Utilized nanopore sequencing for mutational activity landscape analysis.

Main Results:

Achieved single-step, screening-free enrichment of enzyme variants.
Successfully manipulated up to 10^7 microcompartments and 5x10^5 variants.
Identified mutations enhancing NBI activity (20x) and thermostability (3°C).
Revealed mutational landscapes suggesting electrostatic interactions with DNA are crucial.

Conclusions:

The synthetic molecular network approach generalizes in vitro self-selection.
This method is modular, requires no complex instrumentation, and is broadly applicable.
It significantly enhances enzyme engineering efficiency and discovery.