Regenerated amorphous Cellulose

Regenerated amorphous cellulose (RAC) preparation

Microcrystalline cellulose Avicel PH105 (20 um) – FMC (Philadelphia,
PA, USA).

Approximately 0.2 g of microcrystalline cellulose and 0.6 mL distilled water were added into a 50-mL centrifuge tube to form a cellulose-suspension slurry. Ten milliliters of ice-cold 86% H3 PO4 (i.e., commercial 85% grade) was slowly added into the slurry with vigorous stirring so that the final phospho-ric acid concentration was ca. 83.2%. The cellulose mixture turned transparent within several minutes. It stood for ca. an hour in an ice bath with occasional stirring. Approximately 40 mL of ice-cold water was added at a rate of approximately 10 mL per addition with vigorous stirring between additions, resulting in a white cloudy precipitate. The precipitated cellulose was centrifuged at ∼10,000 × g and 4 ◦ C for 20 min. The pellet was suspended in ice-cold water, followed by centrifugation to remove the phosphoric acid from the supernatant, four times. Approximately 0.5 mL of 2 M Na2CO3 and 40 mL of ice- cold distilled water were used to neutralize and suspend the cellulose pellet. After centrifugation, the pellet was washed by distilled waters twice or until pH 5–7. The carbohydrate concentration of RAC was calibrated by the phenol-H2SO4 method [28]. Other cellulose powders such as SigmaCell can be used to replace FMC PH105. The RAC slurry can be stored as a ∼10 g RAC L −1 suspension solution at 4 ◦ C in the presence of 0.2% (w/v) sodium azide for at least 1 year.

Protein Purification

was mixed with a cellulosic adsorbent—RAC slurry. The RAC amount used was estimated at a ratio of 200 mg of target protein to 1 g of RAC (i.e., RAC was in excess to adsorb
>90% of the target protein), where the amount of target protein was roughly estimated by the enzyme activity or SDS-PAGE. After adsorption at room temperature for ∼10–15 min followed by centrifugation, the RAC pellet was suspended in 20 mL of 50 mM Tris–HCl buffer (pH 8.0) to remove impure proteins in the RAC matrix. After centrifugation, the RAC pellet containing the adsorbed CBM-target protein was suspended in 4 RAC pellet volumes of 100% ethyl glycol (EG), i.e., the final EG concentration was ∼80% (v/v). After centrifugation, the purified Cellulose-binding module (CBM)-target protein was obtained in the supernatant. The purified protein can be stored in EG solution at −20 ◦ C. In order to obtain high concentration protein, a low volume of EG can be used. In order to obtain high protein yields, the pellet can be washed several times by a small volume of EG. Ethylene glycol can be removed by dialysis, and then the dilute protein can be re-concentrated by Pall ultra-filtration centrifugal tubes. Similarly, glycerol can be used to replace EG for elution if EG inhibits enzyme activity.