Sephadex g 200

The cells were harvested using centrifugation at 12,000 x g for 20 min at 4°C. The pellets were stored at -80°C before purification. Cell pellets from 1L LB culture were thawed and resuspended in 10 mL extraction buffer (50 mM Tris-HCl, 200 mM NaCl, and 2 mM β-mercaptoethanol, at pH 8.0) and DNase I (50 units). The cell pellet was disrupted with a cell disrupter (Constant System Ltd, USA). Two cycles of pressure (30 GPa) were applied to destroy the cells. Following removal of the insoluble portion of the crude cell lysate with an ultra-centrifuge (20 min, 75,000 x g, 4°C), the supernatant was passed through a membrane (0.45 μm) and loaded onto an anion- exchange column (Q-column) (GE Healthcare Life Science, USA). The column was washed with 100 mL buffer (25 mM Tris-HCl, at pH 7.0), and eluted with the elution buffer (25 mM Tris-HCl, 500 mM NaCl, at pH 7.0). The active fractions were collected and concentrated with an ultracentrifuge, and further purified with a size-exclusion column (Sephadex G-200, GE Healthcare Life Science, USA). The protein concentration was determined with the Bradford method (Bio-Rad Laboratories, Inc., USA). The aliquot protein was frozen in liquid nitrogen and stored at -80°C.

The CAT–VEGF conjugate was separated using Sephadex G-200 (GE Healthcare, Uppsala) gel filtration column equilibrated with 100 mM PB (pH 7.0) at a flow rate of 0.6 mL min⁻¹ under the monitoring of A₂₈₀ via an ultraviolet spectrometer. Aliquots of 300 µL of each fraction were collected, and the CAT activity was examined via CL-based CAT assay using H₂O₂-sensitive TGA-CdTe quantum dots assay [56 ]. The protein components of effective fractions were analyzed by 8% native-PAGE and stained by Coomassie Brilliant Blue.

The LSEP was extracted using the method mentioned in a previous study [34 (link)]. After fermentation for 144 h, the culture broth was centrifuged at 12,000× g for 10 min to obtain a cell-free supernatant. Subsequently, trichloroacetic acid (4%, w/v) was added to the supernatant and incubated at 4 °C for 6 h to remove the protein. To precipitate the LSEP, pre-cooled 98% ethanol (1:3 volumetric ratio) was mixed and kept at 4 °C for 12–14 h. Furthermore, the LSEP obtained by alcohol precipitation was dialyzed in deionized water using a dialysis bag (Mw cutoff 20,000 Da). After dialysis, the retentate was freeze-dried to obtain LSEP.
The LSEP solution (100 mg/mL) was subjected to a DEAE-Sepharose Fast Flow column (2.6 × 20 cm, GE Healthcare, Chicago, IL, USA) and eluted with step gradient of sodium chloride (0–0.5 M) at the flow rate of 1 mL/min. The total sugar contents of the collected fractions were measured by the phenol-sulfuric acid method [64 (link)]. The selected fractions were pooled, desalted, and further purified using gel-filtration chromatography with a Sephadex-G 200 (1.6 × 50 cm, GE Healthcare, Chicago, IL, USA). The collection of fractions was carried out at a flow rate of 0.5 mL/min using deionized water and the total sugar content in the eluate fractions was tested using the phenol-sulfuric acid method. The purified LSEP was dried and stored for further use.

Oat bran was purchased from Tong Yuan Gong He Co., Ltd. (Yantai, China). TermamylSC and amyloglucosidase were obtained from Novozymes Co., Ltd. (Bagsvaerd, Denmark). Pepsin and trypsin were obtained from Amresco Co., Ltd. (Solon, OH, USA). Lichenase was purchased from Megazyme (Wicklow, Ireland). Mannose, glucose, galactose, xylose, arabinose, and fucose standard were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). DEAE52 and SephadexG200 were purchased from GE Healthcare. All other chemicals used were of analytical grade.