2 research outputs found

    Data_Sheet_1_Analysis of lytic polysaccharide monooxygenase activity in thermophilic fungi by high-performance liquid chromatography–refractive index detector.docx

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    IntroductionMost current methods for analysing the activity of LPMO are based on the quantification of H2O2, a side product of LPMO; however, these methods cannot assay the LPMO activity of thermophilic fungi because of the low thermostability of H2O2. Therefore, we present a high-performance liquid chromatography–refractive index detector (HPLC-RID) method to assay the LPMO activity of the thermophilic fungus Thermoascus aurantiacus.ResultsAccording to the established method, the specific activities of nTaAA9A C1 and C4 oxidation were successfully analysed and were 0.646 and 0.574 U/mg, respectively. By using these methods, we analyzed the C1 and C4 oxidation activities of the recombinant TaAA9A (rTaAA9A) and mutated rTaAA9A (Y24A, F43A, and Y212A) expressed in Pichia pastoris. The specific activities of rTaAA9A C1 and C4 oxidation were 0.155 and 0.153 U/mg, respectively. The specific activities of Y24A, F43A, and Y212A C1 and C4 oxidation were 0.128 and 0.125 U/mg, 0.194 and 0.192 U/mg, and 0.097 and 0.146 U/mg, respectively.DiscussionIn conclusion, the method can assay the LPMO activity of thermophilic fungi and directly target C1 and C4 oxidation, which provides an effective activity assay method for LPMOs of thermophilic fungi.</p

    MOESM1 of Regioselectivity of oxidation by a polysaccharide monooxygenase from Chaetomium thermophilum

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    Additional file 1: Figure S1. SDS-PAGE of the purified Cu2+-CtPMO1 produced in Pichia pastoris. Figure S2. The N-terminal amino acid sequence analysis of CtPMO1 using LC-MS/MS. Figure S3. MALDI-TOF-MS/MS analysis of m/z 525 from MALDI-TOF-MS analysis. Figure S4. Types of fragmentation of CtPMO1 C4- and C6-oxidized products (m/z 525). Figure S5. 1H NMR spetra of CtPMO1 soluble reaction products with PASC as substrate in DMSO-d 6 . Figure S6. Sequence alignment of CtPMO1 and NCLPMO9C using ClastalW2. Figure S7. Homology model of the catalytic domain of CtPMO1 using SWISS-MODEL. Figure S8. Homology model of CtPMO1 binding with cellopentaose. Figure S9. Identification of the mutated CtPMO1 soluble reaction products oxidized by Br2 using with PASC as substrate MALDI-TOF-MS. Table S1. List of primers used for PCR of the CtPMO1 protein. Table S2. Fragmentation analysis of the peak of DP3-2 (m/z 525) according to Additional file 1: Figure S3, S4