Quorum Sensing

Chapter 24

Directed Evolution of Quorum-Quenching Enzymes:

A Method for the Construction of a Directed Evolution

Platform and Characterization of a Quorum-Quenching

Lactonase fromGeobacillus kaustophilus

Maybelle Kho Go, Jeng Yeong Chow, and Wen Shan Yew

Abstract

A thermostable quorum-quenching lactonase fromGeobacillus kaustophilus(GKL) was used as a template
for in vitro directed evolution experiments. Here we describe the overexpression and purification of wild-
type GKL, the construction of a quorum-quenching directed evolution platform using bioluminescence as
a reporter, and the in vitro kinetic assay for the determination of kinetic parameters of wild-type GKL and its
mutants.

Key wordsN-Acyl-homoserine lactones, N-Acyl-homoserine lactonases, Directed evolution,

Bioluminescence

1 Introduction

The amidohydrolase superfamily of enzymes consists of members

bearing a conserved mononuclear or binuclear metal center within

a(β/α) 8 -barrel structural scaffold [1]. This superfamily was first

reported by Holm and Sander in 1997 [2] and has since expanded

to cover more than 30 reactions involving a diverse range of sub-

strates [3, 4], including quorum-sensingN-acyl-homoserine lac-

tones (AHLs) [5].

Quorum sensing is an integral part of microbial interaction. It

is responsible for the virulence and pathogenicity of disease-causing

bacteria [6]. Current studies show that modulation and perturba-

tion of a quorum-sensing pathway is an effective anti-microbial

strategy [7]. The disruption of pathways does not present the

selective pressure that always results in the development of resis-

tance in microbes. This suggests the possible use of quorum-

quenching enzymes, such as lactonases, as attractive therapeutic

biomolecules.

Livia Leoni and Giordano Rampioni (eds.),Quorum Sensing: Methods and Protocols, Methods in Molecular Biology,
vol. 1673,https://doi.org/10.1007/978-1-4939-7309-5_24,©Springer Science+Business Media LLC 2018

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