Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption |
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| Affiliation: | 1. Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain;2. IN2UB, Institute of Nanoscience and Nanotechnology, University of Barcelona, Joan XXIII, s/n, 08028 Barcelona, Spain;1. Department of Geoscience and Natural Resources, University of Copenhagen, Rolighedsvej 23, 1958 Frederiksberg C, Denmark;2. Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark;3. Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway;4. Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21250;5. Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, P. O. Box 4950, Nydalen, N-0424 Oslo, Norway;6. Department of Microbiology, Clinic for Diagnostics and Intervention, Oslo University Hospital, Rikshospitalet, P. O. Box 4950, Nydalen, N-0424 Oslo, Norway;1. NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, N-7491, Trondheim, Norway;2. Department of Biotechnology and Nanomedicine, SINTEF Materials and Chemistry, N-7034, Trondheim, Norway;1. Department of Chemistry, Biotechnology, and Food Science, The Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432 Ås, Norway;2. INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France;1. Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100, Copenhagen, Denmark;2. Novozymes Inc, 1445 Drew Ave, CA, 95618, Davis, United States;3. Novozymes A/S, Smørmosevej 25, 2880, Bagsværd, Denmark;4. Novo Nordisk, Diabetes Structural Biology, Novo Nordisk Park, 2760, Måløv, Denmark |
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| Abstract: | Lytic polysaccharide monooxygenases (LPMOs) are copper ion-containing enzymes that degrade crystalline polysaccharides, such as cellulose or chitin, through an oxidative mechanism. To the best of our knowledge, there are no assay methods for the direct characterization of LPMOs that degrade substrates without coupled enzymes. As such, in this study, a coupled enzyme-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs. To establish this new assay method, a chitin-active LPMO from Bacillus atrophaeus (BatLPMO10) was cloned as a model enzyme. An expression system using B. subtilis as the host cell yielded a simple purification process without complicated periplasmic fractionation, as well as improved productivity by 3.7-fold higher than that of Escherichia coli BL21(DE3). At the optimum pH determined using a newly developed assay, BatLPMO10 showed the highest activity in terms of promoting chitin degradation by a chitinase. In addition, the assay method indicated that BatLPMO10 was inhibited by sodium ions, and BatLPMO10 and a chitinase mutually enhanced each other’s activities upon degrading chitin as the substrate. In conclusion, this hydrolase-free ascorbate assay allows quantitative analysis of BatLPMO10 without a coupled enzyme. |
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| Keywords: | Lytic polysaccharide monooxygenase Expression Ascorbate Enzyme assay |
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