Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design.

Xiaoli Zhou,Zhiqiang Xu,Yueqiu Li,Honghui Zhu,Jia He

doi:10.3389/fbioe.2021.815990

Abstract

Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T 50 15 , and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

Highlights

Lytic polysaccharide monooxygenases (LPMOs), the copper-dependent enzymes, catalyze the oxidative cleavage of the glycosidic bonds within crystalline polysaccharides such as cellulose and chitin and provide more binding sites for glycoside hydrolases, significantly improving the degradation efficiency of polysaccharides through cooperation with glycoside hydrolases (VaajeKolstad et al, 2010; Eibinger et al, 2014; Hemsworth et al, 2015)
We hypothesized that the introduction of disulfide bonds in the highly flexible area unrelated to the catalytic function will increase the rigidity of the LPMO molecule, improving its stability without destroying the activity of the enzyme
As a proof of concept, CjLPMO10A was engineered to improve its stability through the introduction of disulfide bonds

Summary

Introduction

Lytic polysaccharide monooxygenases (LPMOs), the copper-dependent enzymes, catalyze the oxidative cleavage of the glycosidic bonds within crystalline polysaccharides such as cellulose and chitin and provide more binding sites for glycoside hydrolases, significantly improving the degradation efficiency of polysaccharides through cooperation with glycoside hydrolases (VaajeKolstad et al, 2010; Eibinger et al, 2014; Hemsworth et al, 2015). Guo et al (2020) obtained an enzyme activity increased variant of a cellulose-active fungal LPMO, MtC1LPMO, by a site-directed mutation on the L2 loop, which provides an example of the activity improvement in LPMOs. In addition, enzyme stability is another limitation of using LPMOs in industries, where the enzyme proteins work under harsh conditions and are expected to last active. Enzyme stability is another limitation of using LPMOs in industries, where the enzyme proteins work under harsh conditions and are expected to last active To achieve this goal, Lo Leggio et al (2018) successfully obtained the stability increased AfAA9, an AA9 fungal LPMO from Aspergillus fumigatus, by substituting an aspartic acid to serine so changing the unfavorable charge interactions

Methods

Results

Conclusion