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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Published on: November 7, 2012

Highly active enzymes produced by directed evolution with stability-based selection.

Ryo Kurahashi¹, Shun-Ichi Tanaka¹, Kazufumi Takano¹

¹Department of Biomolecular Chemistry, Kyoto Prefectural University, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan.

Enzyme and Microbial Technology

|September 11, 2020

Summary

Directed evolution can stall when proteins reach peak activity. This study introduces a stability index to guide proteins to new fitness peaks, enabling the creation of highly active variants through a two-step selection process.

Keywords:

Esterase Fitness landscape Sequence space Sulfolobus tokodaii

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Published on: April 1, 2016

Area of Science:

Protein Engineering
Biotechnology
Enzyme Evolution

Background:

Directed evolution aims to improve protein function but can reach a plateau where no further improvements are possible.
Reaching a local maximum in the fitness landscape necessitates a strategy to descend and find higher peaks for enhanced protein activity.
Protein stability is a crucial factor that can guide protein evolution towards improved functionality.

Purpose of the Study:

To develop a novel strategy for overcoming plateaus in directed evolution by utilizing protein stability.
To identify a method for selecting intermediate protein variants with high stability but potentially lower activity.
To demonstrate the efficacy of a two-step selection process combining stability and activity screening for enhanced enzyme evolution.

Main Methods:

Initial selection of stability-keeping variants through five rounds of random mutagenesis and halo formation assay at 70°C after heat treatment.
Subsequent activity-based selection of pooled stable variants using cell-free extracts and p-nitrophenyl butyrate assay at 75°C.
Two rounds of random mutagenesis applied to the stable variants to identify significantly improved enzymes.

Main Results:

Stability-keeping variants were successfully isolated, likely representing diverse starting points in the protein fitness landscape.
A variant with a 9-fold increase in activity compared to the wild-type esterase was obtained after the two-step selection process.
The proposed two-step selection strategy proved effective in generating significantly improved enzyme variants.

Conclusions:

A two-step selection approach, prioritizing stability followed by activity, facilitates the discovery of highly active protein variants.
The stability index offers a valuable guideline for navigating the fitness landscape in directed evolution.
This method enhances the efficiency of directed evolution for creating enzymes with markedly improved activity.