Fungal beta glucanase units

DSF (53.4% of crude protein, 36.3% of carbohydrate, and 7.5% of moisture) ground from defatted soy meal was obtained from Shandong Wonderful Industrial Group Co., Ltd. (Kenli, China). 98% of the DSF was passed through a 200-mesh screen. Hydrochloric acid, ferric chloride, and sodium hydroxide and other chemical reagents used in this study were analytical grade and purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China). Viscozyme^® L (from Aspergillus aculeatus) with 100 fungal beta-glucanase units (FBG) per gram (FBG/g) of enzyme activity was obtained from Novozymes (Bagsværd, Denmark). Commercial diglycidyl ether of bisphenol-A (DGEBA) epoxy resin (E-51, with epoxide number 0.50–0.54) was purchased from Sanmu Group (Wuxi, China). Pinus massoniana veneers (30 cm × 30 cm in size, 1.2 to 1.3 mm in thickness, and 10% to 12% of moisture contents) were supplied by Jianyang Luban Wood Industry Co., Ltd. (Jianyang, China).

[¹⁴C]-labeled products, formed in in vitro enzyme assays, were dried completely under a nitrogen gas flow and resuspended in 30 μl of 50 mM citrate buffer (pH 4.7). Following addition of 1 μl of Viscozyme L (0.121 Fungal Beta-Glucanase units; Novozymes), samples were incubated (3 h, 55 °C) and reactions terminated by adding 90 μl of methanol. The Viscozyme-treated samples were separated by TLC using the solvent system dichloromethane:methanol:formic acid (7:2:2, by volume). [¹⁴C]-labeled products were visualized by phosphorimaging. [¹⁴C]Linamarin was produced enzymatically by using a recombinant S-tagged cassava UGT, UGT85K4 (accession no. AEO45781) synthesized in Escherichia coli. Crude E. coli lysate containing 0.5 μg of S-tagged UGT85K4 was incubated in an assay mixture of 20 μl composed of 100 mM Tris-HCl (pH 7.5), 3.3 μM [¹⁴C]UDP-glucose (specific activity: 302 Ci mmol⁻¹), and 5 mM acetone cyanohydrin. The reaction was incubated (0.5 h, 30 °C) and terminated by adding 2 μl 10% (v v⁻¹) acetic acid^{41 (link)}. The produced [¹⁴C]Linamarin was dried completely under a nitrogen gas flow prior to Viscozyme treatment.