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In-vitro Mutagenesis

In-vitro Mutagenesis

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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

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Structure-Guided Iterative Mutagenesis Drives Dual-Functional Evolution of α-1,3-Fucosyltransferase.

Mengli Li¹, Ruoyu Jia¹, Jiawei Yao¹

¹State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu 214122, China.

Journal of Agricultural and Food Chemistry

|September 3, 2025

Summary

This summary is machine-generated.

Researchers enhanced alpha-1,3-Fucosyltransferase (α-1,3-FucT) enzyme activity and stability using computational design and combinatorial mutations. This improves its industrial application for synthesizing human milk oligosaccharide 3-fucosyllactose (3-FL).

Keywords:

catalytic activity molecular modification rational design thermostability α-1,3-fucosyltransferase

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Published on: February 5, 2019

Area of Science:

Enzyme Engineering
Biocatalysis
Glycobiology

Background:

Alpha-1,3-Fucosyltransferase (α-1,3-FucT) is crucial for synthesizing 3-fucosyllactose (3-FL), a human milk oligosaccharide.
Industrial application of α-1,3-FucT is limited by low activity and poor thermostability.

Purpose of the Study:

To enhance the catalytic performance and thermostability of α-1,3-FucT.
To develop an efficient strategy for enzyme rational design and industrial enzyme engineering.

Main Methods:

Computer-aided design and greedy combinatorial strategy were employed for enzyme optimization.
Four computational tools identified potential mutation sites, followed by library screening.
Stepwise combinatorial approach generated beneficial single and quadruple mutants.

Main Results:

Six single mutants showed 28%-84% higher specific activity and improved thermostability.
A quadruple mutant (K132I-E134M-N199F-K301P) exhibited a 116% increase in enzymatic activity and an extended half-life.
Mutations optimized substrate binding and conformational stability by remodeling hydrophobic clusters and hydrogen-bond networks.

Conclusions:

The study presents an efficient strategy for rational enzyme design using combinatorial mutations.
Optimized α-1,3-FucT demonstrates significant improvements, paving the way for industrial applications.
Enzyme engineering via combinatorial mutations is critical for industrial biocatalysis.