Additional file 1 of A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase-based HRP colorimetric method for assaying lytic polysaccharide monooxygenase activity
Creators: name: Wu, Shuaishuai
name: Tian, Juan
name: Xie, Ning
name: Adnan, Muhammad
name: Wang, Juan
name: Liu, Gang
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Cancer
subject: Plant Biology

description: Additional file 1: Figure S1. Effects of chromogenic substrate concentrations on A515 values in HRP assay. A: Effect of 4-AAP concentrations on A515 OD values, in which the DCHBS concentration was set at 10.0 mM; B: Effect of DCHBS concentrations on A515 OD values, in which the 4-AAP concentration was set at 0.1 mM. In both of the experiments, 0.1 mM cellobiose was used as substrate, and 100 nM recombinant SsGOOX and 50 U/ml HRP were used as catalysts. Error bars show standard deviation (n = 3; independent experiments). Table S1. The correlated parameters of the modified Hill’s model. Figure S2. Effect of pH and temperature on recombinant SsGOOX activity. A: Enzyme activity at different temperatures. The activity was assayed at different temperatures in Britton-Robison buffer solution at pH 7.0. The enzyme activity at 50oC was defined as 100%. B: Enzyme activity at different pH values. The activity was assayed in Britton-Robison buffer solutions at different pH values (from pH 2.0 to pH 12.0), at 50. The enzyme activity at pH 9.0 was defined as 100%. Error bars show standard deviation (n = 3; independent experiments). Figure S3. Effect of temperature on SsGOOX stability. A: The residual activity of SsGOOX after incubation at different temperatures for 1 h. B: The residual activity of SsGOOX after incubation at 60. for different time intervals. Error bars show standard deviation (n = 3;independent experiments). Table S2. The DNA sequence of SsGOOX after codon optimization. Table S3. Links of TtAA9 genes in NCBI database. Figure S4. HPAEC-PAD analysis of the cello-oligosaccharide mixture standard before and after oxidation by SsGOOX. Lower line: analysis of the cello-oligosaccharide mixture (G1 to G5); upperline: analysis of the GOOX oxidized cello-oligosaccharide mixture (G1 to G5). In the SsGOOX oxidation reaction, the concentration of each cello-oligasaccharides was 1mg/ml (the molar concentrations are: G1 5.55 mM, G2 2.92 mM, G3 1.98 mM, G4 1.50 mM, G5 1.20 mM), the reaction time is 25 min. Upon action of SsGOOX, cello-oligosaccharides have been completely converted within 25 min. Figure S5. HRP colorimetric analysis of the amount of H2O2 generated by SsGOOX oxidation towards LPMO lytic products and futile formation of H2O2 by LPMO reaction. Futile formation of H2O2 was detected using SsGOOX based HRP colorimetric assay of which SsGOOX solution was replaced by heat deactivated SsGOOX solution. A: TtAA9F, B: TtAA9G. ■: the amount of H2O2 generated by SsGOOX oxidation indicated by absorbance at 515 nm, □: futile formation of H2O2 by LPMO reaction indicated by absorbance at 515 nm. The futile formation of H2O2 is magnified and shown in the inset. Error bars show standard deviation (n=3; independent experiments).