4 nitrophenyl α d galactopyranoside

Recombinant α-galactosidase and its C494N mutant form were obtained according to a previously described procedure [60 (link),62 (link)]. Enzymes and substrate 4-nitrophenyl-α-D-galactopyranoside (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 0.05 M sodium phosphate buffer, pH 7.0. To estimate the influence of targeting extracts on enzymatic activity, 0.05 mL of an enzyme solution (0.02 ± 0.001 units of α-galactosidase or 0.002 unit of C494N mutant) was mixed with 0.025 mL of the extract (5 mg/mL) and incubated for 30 min at room temperature. Then, 0.075 mL substrate (stock concentration of 1.65·mM) was added. The reaction mixture was incubated at 20 °C for 4 min for α-galactosidase and 10 min for C494N. The reaction was stopped by the addition of 0.15 mL of 1 M Na₂CO₃. Hydrolytic activity of galactosidases was spectrophotometrically measured (ε₄₀₀ = 18,300 M⁻¹cm⁻¹). One unit of activity (U) was determined as the amount of enzyme that released 1 µmol of 4-nitrophenol per 1 min at 20 °C in 0.05 M sodium phosphate buffer at pH 7.0. After extract addition, residual enzymatic activity was measured as U/U₀ and represented in percentage, where U₀ is the standard activity of α-galactosidases without extracts.

Besides barley β-glucan, other substrates including sodium carboxyl methyl cellulose (CMC-Na), 4-nitrophenyl α-d-galactopyranoside (pNPG), 4-nitrophenyl β-cellobioside (pNPC), birchwood xylan, Avicel, phosphoric acid swollen cellulose (PASC) and locust bean galactomannan were all purchased from Sigma-Aldrich. Lichenin was purchased from Megazyme (Wicklow, Ireland). Whatman Grade 1 filter paper was purchased from General Electric Company, United Kingdom. The enzyme activities of Cel7A towards various substrates (1.0%, w/v) were assessed by using the standard methods. Except for filter paper that required a longer incubation period (30 min), other substrates were incubated with Cel7A for 10 min. All enzyme activity measurements were carried out three times.
The kinetic parameters K_m and V_max were determined in 100 mM McIlvaine buffer (pH 5.0) containing 0.5−10.0 mg ml⁻¹ barley β-glucan at 60°C for 5 min. The K_m and V_max values were calculated according to the Lineweaver-Burk method.

MacConkey agar media (lactose-free) was purchased from Difco. Unlabeled melibiose and 4-nitrophenyl-α-D-galactopyranoside (α-NPG) were purchased from Sigma-Aldrich. Maltose was purchased from Acros Organics (Fisher Scientific). [1-³H]Melibiose (5.32 Ci/mmol) was custom synthesized by PerkinElmer. Detergents undecyl-β-D-maltopyranoside (UDM) and n-dodecyl-β-D-maltopyranoside (DDM) were purchased from Anatrace. E. coli lipids (Extract Polar, 100600) were purchased from Avanti Polar Lipids, Inc. Oligodeoxynucleotides were synthesized by Integrated DNA Technologies. LC-MS grade water, LC-MS 0.1% formic, LC-MS grade acetonitrile with 0.1% formic acid in water were purchased from Fisher Scientific (Hampton, NH). Guanidine hydrochloride, citric acid, and Zirconium (IV) oxide 5-μm powder were purchased from Sigma-Aldrich (St. Louis, MO). Deuterium oxide (99⁺% D) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA). All other materials were reagent grade and obtained from commercial sources.