β glucosidase from almond

The plant extract was prepared following the protocol of [12 (link)] with slight modifications. Briefly, 50 mg of lyophilized powdered callus was mixed with 500µL of MeOH, followed sonication for 20 min at 45 °C and an ultrasonic frequency of 45 kHz in an ultrasonic bath (Prolabo). The sample was vortexed for 5 min and the overall procedure was repeated twice for efficient extraction of phytochemicals. Then, the sample was centrifuged at 10,000 rpm for 15 min and the supernatant was evaporated to dryness in a SpeedVac concentrator (Thermo Fisher) at 40 °C. The resulting pellet was resuspended in 500 µL of 0.1 M citrate-phosphate pH 4.8 buffer containing 5 unit/mL β-glucosidase from almonds (Sigma) for lignan and neolignan aglycone release for 4 h at 37 °C. The sample was finally centrifuged at 10,000 rpm for 15 min and the supernatant filtered (0.45 µm) prior to further analyses (phytochemical analysis and biological assays). Note that an aliquot of each extract was also analyzed by HPLC, without enzymatic treatment, to appreciate the relative proportion of aglycones vs. glycosides. The extracts were at −80 °C.

Poplar (Populus trichocarpa) used in this study was harvested at Oak Ridge National Laboratory, TN [19 (link)]. The biomass size was reduced in a Wiley mill to pass a 1 mm screen and then sieved to collect the fractions between 0.18 and 0.85 mm. The p-dioxane used in this study was distilled over sodium borohydride prior to use. Peracetic acid solution (32 wt% in dilute acetic acid), phenyl isocyanate (assay grade), and dichloromethane (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO). Anhydrous pyridine (EMD, Millipore) was purchased from VWR. Cellulase C1794 from Trichoderma sp. (3–10 units/mg) and β-glucosidase from almonds (10–30 units/mg) were purchased from Sigma-Aldrich (St. Louis, MO). All the reagents and chemicals, unless otherwise noted, were used as received.

Using previously described methods, the aforementioned Cln2^R207X/R207X and Cln2^+/+ total protein samples were evaluated for PPT1 enzyme activity [25 (link), 26 (link)]. The assays were conducted in a black, flat-bottom 96-well plate. 10 μg of total protein were loaded into each well. Each sample was done in quadruplicates. 20 μl of PPT1 substrate [0.64 mM 4-methylumbelliferyl-6-thiopalmitoyl-β-glucoside (Santa Cruz); 15 mM dithiothreitol with 2.3 mg/ml bovine serum albumin and 0.09% sodium azide; 0.2 M disodium phosphate with 0.1 M citric acid buffer pH 4.0; 0.945 mg/mL β-Glucosidase from almonds (Sigma Aldrich)] was added to each well. The plate was incubated in the dark at 37°C for 1h. 200 μl of stop buffer [0.5 M sodium bicarbonate with 0.5 M sodium carbonate pH 10.7; 0.025% Triton X-100] was added to each well. The plate was gently shaken and fluorescence was read using a BioTek Cytation 3 (Excite 355 nm/Emit 460 nm). Background fluorescence was accounted and removed from each sample. Relative fluorescence was calculated as a percentage of wild type fluorescence.

Tetraethylorthosilicate
(TEOS), urea, cetyltrimethylammonium bromide (CTAB), cyclohexane,
pentanol, 2-propanol, ethanol, hydrochloric acid solution (37.0% wt
in water), carboxymethylcellulose sodium salt (CMC), acetic acid (99.0%
wt), sodium acetate trihydrate, sodium hydroxide and glucose oxidase-peroxidase
(GOD-POD) assay kit, citric acid, trisodium citrate dihydrate, and
sulfuric acid (95.0–98.0% wt) were purchased from Sigma-Aldrich
(Milan, Italy). β-Glucosidase from almonds (molecular weight
of 135 kDa for the dimer, product number 49290, specific activity
of ≥4 U/mg, measured as micromole of glucose liberated per
minute at pH 5 and 37 °C with salicin as substrate) and cellulase
from T. reesei (product number C0615,
specific activity of ≥5 U/mg solid measured as micromole of
glucose liberated from cellulose per hour at 37 °C and pH 5)
were also acquired from Sigma-Aldrich. Sodium hypochlorite solution
(5.0% wt) was bought from a local supermarket. E. japonica (loquat) leaves were collected from a private garden in Caserta,
Italy.

All the chemicals used were analytical reagent grade. Acetic acid (C₂H₄O₂), dichloromethane (CH₂Cl₂), methanol (CH₄O), D(+)-glucose (C₆H₁₂O₆), stannous chloride (SnCl₂), o-xylene 98% (C₈H₁₀), calcium carbonate (CaCO₃), S. cerevisiae YSC2 (YSC₂), enzymatic digest of casein, meat extract, sodium acetate (C₂H₃NaO₂), diammonium citrate (C₆H₁₄N₂O₇), dipotassium phosphate (K₂HPO₄), magnesium sulphate (MgSO₄), iron (III) chloride hexahydrate (FeCl₃·6H₂O), 1-butanol (C₄H₁₀O), ammonium sulphate ((NH₄)₂SO₄), molecular sieve 3 Å (K_nNa_12-n[(AlO₂)₁₂(SiO₂)₁₂]·xH₂O), 3.5-dinitrisalycilic acid (DNS), and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany). Sodium chlorite (80%) (NaClO₂) was purchased from Alfa Aesar GmbH & Co. (Karlsruhe, Germany). Enzymes cellulase from Trichoderma reesei ATCC 26921 and β-glucosidase from almonds were purchased from Sigma-Aldrich (St. Louis, MO, USA). L. rhamnosus ATCC 7469 was purchased from Microbiologics (Cooper Avenue North, St. Cloud, MN, USA). All solutions were prepared by using ultrapure water (18.2 MΩcm⁻¹ at 20 °C) obtained from a Direct-Q3 UV Water Purification System (Millipore, Molsheim, France).

The root tissue was homogenized using steel beads and sonication. The coumarins were extracted at 4°C with 80% methanol. After 24 h two sets of methanol extracts were centrifuged for 20 min at 13000 rpm, one set was additionally subjected to enzymatic hydrolysis using β-glucosidase from almonds (Sigma-Aldrich) dissolved in acetate buffer according to modified protocol of [56 (link)].

All used chemicals were of analytical reagent grade. Acetic acid, dichloromethane, sulfuric acid (98%), sodium hydroxide, methanol, hydrochloric acid, suprapure nitric acid 65%, ethanol, toluene, acetone, hydrogen peroxide 30%, glucose, mannose, galactose, arabinose, pyridine, hydroxylamine hydrochloride (NH₂OH·HCl) and salicin were purchased from Merck (Darmstadt, Germany). Sodium chlorite (80%) was purchased from Alfa Aesar GmbH & Co (Karlsruhe, Germany). Enzymes cellulase from Trichoderma reesei ATCC 26921, β-glucosidase from almonds, S. cerevisiae YSC2, peptone, BSTFA, 5-hydroxymethylfurfural and furfural were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trichoderma reesei ATCC 26921 is a lyophilized powder with concentration ≥1.0 unit per mg of solid. All solutions were prepared by using ultrapure water (18.2 MΩcm⁻¹ at 20 °C) obtained from a Direct-Q3 UV Water Purification System (Millipore, Molsheim, France).