ed to hydrolyse five with the substrate over two h, with inhibitor and 0.4 mM substrate (diluted from one hundred mM in DMSO) in water. Inhibitor concentrations from 0 to 50 M or 0 to 25 M were monitored for fluorescence continuously for up to 2 h. To test enzyme recognition specificity, inhibition was measured with glucosyl-(1,four)-cyclophellitol (GGcyc) [36] or xylosyl(1,4)-xylocyclophellitol (XXcyc) [35]. To test the impact from the distinct linker chemistries, inhibition kinetics have been also measured working with Biotin-ABP-Xyn [35] and Biotin-ABP-Cel [36].Abbreviations ABP: Activitybased probe; ABPP: Activitybased protein profiling; BCA: Bicin choninic acid; bMLG: Barley mixedlinkage glucan; CAZyme: Carbohydrate active enzyme; cGM: Carob galactomannan; CMC: Carboxymethyl cellulose; DMSO: Dimethylsulfoxide; DTT: Dithiothreitol; GH: Glycoside hydrolases; IAA: Iodoacetamide; LPMO: Lytic polysaccharide monooxygenase; MW: Molecular weight; PVPP: Polyvinylpolypyrrolidone; SDSPAGE: Sodium dodecyl sulphate polyacrylamide gel electrophoresis; TEAB: Tetraethylammonium bicarbonate; TMT: Tandem mass tag; wAX: Wheat arabinoxylan.Polysaccharide hydrolysis was measured via the detection of decreasing ends using the BCA assay. Briefly, enzyme ( 10 g/mL) was mixed with substrate in 50 mM pH 4.0 NaOAc buffer with one hundred mM NaCl and incubated at 30 for 15 min. The reaction was stopped by the addition of freshly mixed BCA reagent (250 mM Na2CO3, 140 mM NaHCO3, 2.5 mM bicinchoninic acid, 1.25 mM CuSO4, two.five mM l-serine); then colour was created by incubation at 80 for 10 min ahead of measuring A563. Minimizing ends were determined relative to a glucose calibration series from 10 to 200 M. A substrate blank was measured and subtracted from every single sample measurement. Minor activities have been quantified by the same process using 50 g/mL enzyme using a boiled enzyme control (95 , 15 min) added to substrate for background subtraction. The pH optimum of every single enzyme was measured making use of 1 mg/mL cGM (LsGH5_7A), wAX (LsGH10A), or bMLG (LsGH5_5A, TlGH12A) inside a collection of buffers (citrate,Supplementary InformationThe online version includes supplementary material accessible at doi. org/10.1186/s1306802202107z. Additional file 1. Proteomic hit information and facts for cellulase DYRK4 Synonyms pulldown from A. biennis secretomes. Additional file 2. Proteomic hit information for cellulase pulldown from F. fomentarius secretomes. Additional file three. Proteomic hit facts for cellulase pulldown from H. MAP3K5/ASK1 site nitida secretomes. Extra file 4. Proteomic hit facts for cellulase pulldown from L. sp. 1048 secretomes.McGregor et al. Biotechnology for Biofuels and Bioproducts(2022) 15:Page 12 ofAdditional file five. Proteomic hit info for cellulase pulldown from T. menziesii secretomes. Added file 6. Proteomic hit info for cellulase pulldown from P. brumalis secretomes. Additional file 7. Proteomic hit information and facts for cellulase pulldown from P. sanguineus secretomes. More file 8. Proteomic hit info for cellulase pulldown from T. gibbosa secretomes. Added file 9. Proteomic hit info for cellulase pulldown from T. ljubarskyi secretomes. Further file ten. Proteomic hit data for cellulase pulldown from T. meyenii secretomes. Added file 11. Supplementary synthetic methods, figures, and tables. Acknowledgements The authors thank Dan Cullen (Forest Product Laboratory, USDA, Madison, WI, USA) to get a sample of Wileymilled aspen (Populus grandidentata). Authors’ co
