title: Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemical-reducing-end assays.
abstract: In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates.
authors.affiliation: Department of Science, Systems, and Models (NSM), Biomaterials, Roskilde University, DK-4000 Roskilde, Denmark.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Baumann
authors.forename: Leigh
authors.initials: L
authors.lastname: Murphy
authors.forename: Nina
authors.initials: N
authors.lastname: Lei
authors.forename: Kristian B R M
authors.initials: KB
authors.lastname: Krogh
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D001230:Aspergillus / Q000201:enzymology; D052160:Benzothiazoles / Q000737:chemistry / Q000378:metabolism; D002151:Calorimetry / Q000379:methods*; D003373:Coumaric Acids / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D006868:Hydrolysis; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011804:Quinolines / Q000378:metabolism; D020156:Salicylic Acid / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D014990:Xylans / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D003373:Coumaric Acids; D062385:Hydroxybenzoates; D011804:Quinolines; D014990:Xylans; C024795:bicinchoninate reagent; C058836:benzothiazoline; C002429:4-hydroxybenzoic acid hydrazide; C004999:ferulic acid; D043364:Endo-1,4-beta Xylanases; D020156:Salicylic Acid