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Figure 3
TtGH8 activity on polysaccharides was determined using the 3,5-dinitrosalicyclic acid (DNSA) reducing-sugar assay. (a) Photograph of the plate containing aliquots representing different reaction time points (0–25 min) and substrate concentrations [mixed-linkage xylan (MLX) and wheat arabinoxylan (WAX) at 0.5–2.0 mg ml−1]. Reaction of the DNSA agent with reducing sugars causes a visible colour change from light yellow to deep brown, with the amount of colour change measured by the absorbance at 510 nm being equivalent to the amount of reducing sugar (chain ends) produced during the enzyme reaction. Standard curves were calculated using the free xylose controls in the top and bottom rows of the plate. These were MLX (1 mg ml−1) + xylose (0–500 µg ml−1) and WAX (1 mg ml−1) + xylose (0–500 µg ml−1). TtGH8 is more active on MLX than on WAX as there is a greater colour change. Absorption measurements taken for each reaction aliquot can be converted into the amount of reducing sugar present using the standard curve [shown in (a)] and plotted against time to give a rate (µM min−1, not shown). The rates for each reaction condition are plotted against the substrate concentration, where the gradient divided by the enzyme concentration is equivalent to kcat/Km. (b) Activity of TtGH8 and TtGH8 catalytic mutant on MLX, (c) activity of TtGH8 on WAX and (d) activity of TtGH8 on BX.

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ISSN: 2059-7983
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