Construction and characterization of a Myceliophthora thermophila lytic polysaccharide monooxygenase mutant S174C/A93C with improved thermostability

Xiao Guo; Luying Jiang; Yajing An; Fuping Lu; Fufeng Liu; Bo Wang

doi:10.1016/j.enzmictec.2023.110255

Construction and characterization of a Myceliophthora thermophila lytic polysaccharide monooxygenase mutant S174C/A93C with improved thermostability

Enzyme Microb Technol. 2023 Aug:168:110255. doi: 10.1016/j.enzmictec.2023.110255. Epub 2023 May 10.

Authors

Xiao Guo¹, Luying Jiang², Yajing An², Fuping Lu², Fufeng Liu³, Bo Wang⁴

Affiliations

¹ College of Chemical and Biological Engineering, Shandong University of Science and Technology, Qingdao 266590, PR China; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300450, PR China.
² Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300450, PR China.
³ Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300450, PR China. Electronic address: fufengliu@tust.edu.cn.
⁴ College of Chemical and Biological Engineering, Shandong University of Science and Technology, Qingdao 266590, PR China. Electronic address: wb@sdust.edu.cn.

PMID: 37178549
DOI: 10.1016/j.enzmictec.2023.110255

Abstract

Lytic polysaccharide monooxygenases (LPMOs) can oxidatively cleave the glycosidic bonds of crystalline polysaccharides, providing more accessible sites for polysaccharide hydrolases and promoting efficient conversion of biomass. In order to promote industrial applications of LPMOs, the stability of an LPMO of Myceliophthora thermophila C1 (MtC1LPMO) was improved by adding disulfide bonds in this study. Firstly, the structural changes of wild-type (WT) MtC1LPMO at different temperatures were explored using molecular dynamics simulations, and eight mutants were selected by combining the predicted results from Disulfide by Design (DBD), Multi agent stability prediction upon point mutations (Maestro) and Bridge disulfide (BridgeD) websites. Then, the enzymatic properties of the different mutants were determined after their expression and purification, and the mutant S174C/A93C with the highest thermal stability was obtained. The specific activities of unheated S174C/A93C and WT were 160.6 ± 1.7 U/g and 174.8 ± 7.5 U/g, respectively, while those of S174C/A93C and WT treated at 70 °C for 4 h were 77.7 ± 3.4 U/g and 46.1 ± 0.4 U/g, respectively. The transition midpoint temperature of S174C/A93C was 2.7 °C higher than that of WT. The conversion efficiency of S174C/A93C for both microcrystalline cellulose and corn straw was about 1.5 times higher than that of WT. Finally, molecular dynamics simulations revealed that the introduction of disulfide bonds increased the β-sheet content of the H1-E34 region, thus improving the rigidity of the protein. Therefore, the overall structural stability of S174C/A93C was improved, which in turn improved its thermal stability.

Keywords: Biomass conversion; Disulfide bond; Enzyme engineering; Lytic polysaccharide monooxygenase; Molecular mechanism; Thermostability.

MeSH terms

Disulfides
Mixed Function Oxygenases* / metabolism
Polysaccharides / metabolism
Sordariales* / genetics

Substances

Mixed Function Oxygenases
Polysaccharides
Disulfides

Supplementary concepts

Thermothelomyces thermophilus