title: Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption.
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.
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong, 30019, Republic of Korea.
authors.forename: Mi-Ji
authors.initials: MJ
authors.lastname: Yu
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong, 30019, Republic of Korea.
authors.forename: Sun-Hee
authors.initials: SH
authors.lastname: Yoon
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong, 30019, Republic of Korea. Electronic address: ywankim@korea.ac.kr.
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D002688:Chitinases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; D002688:Chitinases; D001205:Ascorbic Acid
keywords: Ascorbate; Bacillus subtilis; Enzyme assay; Expression; Lytic polysaccharide monooxygenase