Dialysis bag

Pichia pastoris culture containing recombinant xylanases (FGSG_03624, FGSG_10999, and FGSG_11487), recombinant XIs (TAXI–IA, TAXI–IAb, TAXI–IIA, TAXI–IIAb, TAXI–III, and TAXI–IV) and pPIC9K (CK) were centrifuged at 10,000×g for 10 min. The supernatant was filtered successively through the filter membrane (Rasco, China) with pore sizes of 0.8, 0.45, and 0.22 μm, then the protein was precipitated overnight with 75% ammonium sulfate at 4°C. After precipitation, the precipitate was recovered at 10,000×g for 30 min at 4°C. The precipitate was dissolved in deionized water, and the recovered supernatant was dialyzed and desalted with a dialysis bag (YuanYe, China) at 4°C for 12 h. Finally, the protein solution obtained was stored in the refrigerator at −80°C.
The extracted xylanase was incubated with xylanase inhibitor or P. pastoris secretion (CK) for 10 min at 30°C. Xylanase, xylanase inhibitor, P. pastoris secretion (CK), xylanase-inhibitor mixture, and xylanase-CK mixture were injected into leaves of Nicotiana benthamiana (6–8 weeks) with 1 ml syringe without a needle, respectively. The symptoms of leaves were observed and recorded for 3–5 days after infiltration. Each infiltration experiment was repeated three times.

The organism suspension above was centrifuged by refrigerated centrifuge at a speed of 8,000 × g for 45 min, the supernatant was stored at 4°C as crude enzyme.
The crude enzyme solution was precipitated by ammonium sulfate, then ammonium sulfate was slowly added to 60% saturation, stirring until completely dissolved and resting overnight. After the refrigerated centrifugation at a speed of 8000 × g for 45 min, the precipitate was discarded and the supernatant was then slowly added with ammonium sulfate to 90% saturation, stirred until dissolved and rested overnight. The above refrigerated centrifugation (8,000 × g, 45 min) was repeated, the supernatant discarded, and the precipitate added to 60 mL acetic acid buffer solution (0.02 M, pH=5) to make a suspension. Finally, the suspension was transferred to a dialysis bag (shanghai yuanye Bio-Technology Co., Ltd. interception molecular weight of 4,000) and dialyzed against 4°C distilled water overnight.