β galactosidase from aspergillus oryzae

Galacto-oligosaccharides derived from lactulose (Lu) were synthesized using a commercial lactulose preparation (670 g of lactulose per liter; Duphalac, Abbott Biologicals BV, Olst, Netherlands) and β-galactosidase from Aspergillus oryzae (16 U/mL; Sigma, St. Louis, MO, United States) (López-Sanz et al., 2015 (link)). The enzymatic reaction took place at pH 5.4, achieved after the addition of 3 mL of KOH 2M at 800 mL of Duphalac, and 50°C in an orbital shaker at 300 rpm for 24 h. Afterwards, the enzymatic reaction was stopped by heating at 110°C for 10 min. The resulting mixture contained 66% (w:w) of total carbohydrates.
The carbohydrate fraction was qualitatively and quantitatively determined by gas chromatography-flame ionization detector (GC-FID) as trimethyl silylated oxime (TMSO) derivatives following previous approaches (Cardelle-Cobas et al., 2009 (link); Hernández-Hernández et al., 2012 (link)). The carbohydrate composition of GOS-Lu, whose main involved glycosidic linkage was β(1→6), was as follows: fructose (19.5%), galactose (12.4%), glucose (1.2%), lactulose (24.7%), GOS-Lu disaccharides (13.6%), GOS-Lu trisaccharides (22.6%), GOS-Lu tetrasaccharides (5.1%) and GOS-Lu pentasaccharides (1.0%).

Poly(methacrylic acid-co-ethyl acrylate) in 1:1 ratio, commercially known as Eudragit® L100-55 (hereafter referred to as L100), was obtained from Evonik Canada Inc. (Burlington, Ontario, Canada). 2-Nitrophenyl β-D-galactopyranoside, β-galactosidase from Aspergillus Oryzae, galactose and lactose assay kit, sodium dodecyl sulfate, acetonitrile, and disodium hydrogen phosphate were acquired from Sigma-Aldrich (St Louis, Missouri, USA). Yellow-green fluorescent beads with different sizes (100 nm, 1 µm, and 4 µm) were purchased from Life Technologies (Carlsbad, CA, USA).

The enzymatic modification of XG was achieved by following the protocol of Brun-Graeppi et al. using β-Galactosidase from Aspergillus oryzae which was purchased from Sigma-Aldrich (Saint Louis, MI, USA) [30 (link)]. Enzymatic modifications were achieved on XG/CNC complexes at 50 °C with an enzyme/substrate ratio of 0.37 U/mg XG. The mixtures were stirred and left to react for 22 h. The mixtures were heated at 90 °C for 5 min to deactivate the enzyme. The mixtures were then cooled and kept at 4 °C.

For cargo release studies, 2 mg of GosNP(rho), GosNP(icg), GalNP(rho), GalNP(dox), or GalNP(nav) nanoparticles were suspended in 5 ml of water at pH 4.5, and β‐galactosidase from Aspergillus oryzae (Sigma, #G5160) was added at 1,000 ppm (5 mg). Suspensions were stirred, and different aliquots were taken and centrifuged at different time points. The amount of cargo released was determined in a JASCO spectrofluorometer FP‐8300 by monitoring the emission (for rho, icg, dox) or the absorption (for nav) of the dye or drug in the aqueous solution, as a function of time (rho λ_ex = 550 nm, rho λ_em = 580 nm; icg λ_ex = 775 nm, icg λ_em = 799 nm; dox λ_ex = 495 nm, dox λ_em = 556 nm; nav λ_em = 356 nm). The same procedure was performed without adding the enzyme to the nanoparticles suspension, as a “blank” control.

GALNS enzyme activity was determined in plasma and tissues [2 (link)]. Frozen tissues were homogenized by Bead Mill Homogenizer (OMNI International, Kennesaw, GA, USA) in 25 mmol/L Tris-HCl (pH 7.2) and 1 mmol/L phenylmethylsulphonyl fluoride. Then, homogenates were centrifuged for 30 min at 4 °C, and the supernatant was collected to a new tube and assayed for enzyme activity. Either tissue lysate or plasma (2 μL) was used for the enzymatic reaction with 22 mM 4-methylumbelliferyl-β-galactopyranoside-6-sulfate (Research Products International, Mount Prospect, IL, USA) in 0.1 M NaCl/0.1 M sodium acetate (pH 4.3) and incubated at 37 °C for 16 h. After incubation, 10 mg/mL β-galactosidase from Aspergillus oryzae (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M NaCl/0.1 M sodium acetate (pH 4.3) was added and incubated for additional 2 h at 37 °C. The reaction was stopped with 1 M glycine NaOH (pH 10.5) solution and read at excitation 366 nm and emission 450 nm by FLUOstar Omega plate reader (BMG LABTECH Inc., Cary, NC, USA). Activity is shown as nanomoles of 4-methylumbelliferone released per hour per microliter of plasma or milligram of protein. Protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

The previously described protocols [51 (link)] determined activity levels of GALNS. Frozen tissues were homogenized with 25 mmol/L Tris-HCl (pH 7.2) and 1 mmol/L phenylmethylsulfonyl fluoride homogenization buffer. The tissue lysate or previously collected plasma was combined with 22 mM 4-methylumbelliferyl-β-galactopyranoside-6-sulfate (Research Products International, Mount Prospect, IL, USA) in 0.1 M NaCl/0.1 M sodium acetate (pH 4.3) in a 96-well plate and incubated at 37 °C for 16 h. 10 mg/mL β-galactosidase from Aspergillus oryzae (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M NaCl/0.1 M sodium acetate (pH 4.3) was added to the reaction mixture and incubated at 37 °C for an additional 1 h. 1 M glycine, NaOH (pH 10.5) was then added to arrest the reaction. Plates were transferred to a PerkinElmer Victor X4 plate reader (PerkinElmer, Waltham, MA, USA) and excited at 366 nm with an emission read at 450 nm. Results were recorded in nanomoles of 4-methylumbelliferone released per hour per microliter of plasma or milligram of protein. Conversion of plasma to protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).