Disulfide bridges as essential elements for the thermostability of lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor.
Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in Tm, ranging from 2 to 9°C. Combining the positive mutations yielded additional improvements (up to 19°C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12°C increase in Tm and was able to retain and was able to retain no less than 60% of its activity after heat treatment.
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Magali
M
Tanghe
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Barbara
B
Danneels
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Matthias
M
Last
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Koen
K
Beerens
Industrial Catalysis and Adsorption Technology (INCAT), Faculty of Engineering and Architecture, Ghent University, Valentin Vaerwyckweg 1, B-9000 Ghent, Belgium.
Ingeborg
I
Stals
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Tom
T
Desmet
D001426:Bacterial Proteins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008958:Models, Molecular; D012984:Software; D048372:Streptomyces coelicolor / Q000201:enzymology*
D001426:Bacterial Proteins; D004220:Disulfides; D006899:Mixed Function Oxygenases
Streptomyces coelicolor LPMO10C; disulfide bridge; lytic polysaccharide monooxygenases; thermal stability