Structure-guided engineering of a Thermobifida fusca cutinase for enhanced hydrolysis on natural polyester substrate

Qilei Dong,Lingqia Su,Qiaoling Zhao,Jiahai Zhou,Shuguang Yuan,Jing Wu,Weixue Huang,Lian Wu

doi:10.1186/s40643-020-00324-8

Abstract

Cutinases could degrade insoluble polyester, including natural cutin and synthetic plastic. However, their turnover efficiency for polyester remains too low for industrial application. Herein, we report the 1.54-Å resolution X-ray crystal structure of a cutinase from Thermobifida fusca and modeling structure in complex with a cutin mimic oligo-polyester C24H42O8. These efforts subsequently guided our design of cutinase variants with less bulky residues in the vicinity of the substrate binding site. The L90A and I213A variants exhibit increased hydrolysis activity (5- and 2.4-fold, respectively) toward cutin and also showed enhanced cotton scouring efficiency compared with the wild-type enzyme.

Highlights

Cutin, a natural polyester, forms a three-dimensional network structure of protective cuticle in higher plants, in which waxy material and pectin are embedded (Fig. 1a) (Kolattukudy 2001)
The parental T. fusca cutinase plasmid was removed by digesting with DpnI (Biolabs, UK), and PCR products were transformed to DMT chemical competent cells, amplified to E. coli DH5α cells and BL21 (DE3) for expression
Structural elucidation of T. fusca cutinase variants with enhanced activity To explain the enhanced performances of variants L90A and I213A, we modeled the complex structure of the two variants with the substrate mimic C24H42O8, using an identical molecular dynamics (MD) simulation protocol as that for WT T. fusca cutinase

Summary

Introduction

A natural polyester, forms a three-dimensional network structure of protective cuticle in higher plants, in which waxy material and pectin are embedded (Fig. 1a) (Kolattukudy 2001). Scouring is carried out in the hot NaOH aqueous solution. Such non-specific alkaline scouring generates environmental pollutants and decreases fabric tension. Cutinases belong to the serine hydrolase superfamily, and catalyze the hydrolysis of ester bond. These enzymes have broad substrate ranges, including soluble fatty acids, triglycerides, and insoluble polyesters like cutin, polyethylene terephthalate (PET), highlighting their potentials for use in industrial applications such as bioscouring and the degradation and/or synthesis of plastics (Nyyssola 2015; Liu et al 2009)

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Results

Conclusion