V 500 spectrophotometer

Chemical composition of raw material, unbleached and bleached pulps was determined according to NREL/TP-510-42618: Laboratory Analytical Procedures for determination of structural carbohydrates and lignin in biomass [15 ]. In brief, extractives were estimated as the soluble fraction after extensive Soxhlet extraction with ethanol. Then, carbohydrate composition was determined by a quantitative acid hydrolysis in two steps performed over the free-extractive samples. An Agilent Technologies 1260 HPLC with refractive index detector (Agilent, Waldbronn, Germany) was employed to quantify carbohydrate concentration in the hydrolyzed liquids, using an Agilent Hi-PlexH column at 65 °C, with 5-mM sulfuric acid as mobile phase (0.6 mL min⁻¹). Finally, Klason lignin was calculated as the solid residue left after the acid hydrolysis and acid soluble lignin was determined by UV–Visible spectrometry at 205 nm using a Jasco V-500 spectrophotometer (Jasco, Japan).

Both β-M-Ce6 and β-G-Ce6 were dissolved individually in DMSO to prepare stock solutions (5 × 10⁻³ M). The stock solution was diluted with a 1:1 1-octanol/acetone mixed solution, which was characterized by UV-vis spectroscopy (V-500 spectrophotometer, JASCO, Tokyo, Japan) to determine the molar absorbance coefficient in the 1:1 1-octanol/acetone-mixed solution. Similarly, the molar absorbance coefficient in a 1:1 PBS/acetone-mixed solution was determined. A 1:1 1-octanol/PBS-mixed solution was prepared. From the solution, the 1-octanol phase (600 μL) and PBS phase (600 μL) were collected and combined. A stock solution (3 μL) was added, mixed using a vortex mixer, and then centrifuged to obtain the partition solution. The 1 − octanol phase (500 μL) was collected from the partition solution and diluted with 1-octanol (500 μL) and acetone (1.00 mL), which was characterized by UV-vis spectroscopy to determine the concentration in the 1:1 1-octanol/acetone-mixed solution ([C_1−octanol]). Similarly, the PBS phase (500 μL) was collected from the partition solution and diluted with PBS (500 μL) and acetone (1.00 mL), which provided the concentration in the 1:1 1-PBS/acetone-mixed solution ([C_PBS]). The log([C_1−octanol]/[C_PBS]) values were calculated to provide the log P-values.

The LOX-1 activity for solutions was determined by a modified method of Axelrod et al. [18 ]. The activity of LOX-1 was determined via the increase in absorbance at 234 nm using a JASCO V-500 spectrophotometer at 25°C as described previously [19 ] after addition of linoleic acid in borate buffer containing the enzyme. Shortly, in the cuvette containing 0.84 mL 0.2 M borate buffer (pH = 9) and 0.16 mL standard enzyme (1 : 10 containing 46.000 units/mg solid and 63.500 units/mg protein), 0.0084 mL of substrate solution (sodium linoleate 10 mM) were rapidly added and mixed, and the increase in absorbance (A) versus the blank was recorded. The blank contained 0.84 mL 0.2 M borate buffer (pH = 9) and 0.16 mL standard enzyme (1 : 10).
The time course of the reaction was registered in each case and the enzymatic specific activity ESA—the variation of the product formation (absorption increase at 234 nm) per time unit and mg enzyme—was determined (AU/sec/mg protein). Each measurement was done in triplicate. For each experimental variant the amount of pure protein taken into reaction was 11.6 × 10⁻³mg.