*3.2. Separation and Purification of SPA*

Using the methods described by Liang et al. [21] with minor modification. SPA was separated and purified with the following steps. Fresh sweet potatoes were washed and sliced into chips. 100 g sweet potatoes were weighed out, and 200 mL distilled water was added for pulverization in a pulverizer (SQ2002, Shanghai Shuaijia Electronic Technology Company, China) for 1 min. The sample was filtered through 40 mesh sieve, and the resulting filtrate was placed in a refrigerated centrifuge at 4 ◦C for centrifugation at 4000 rpm for 15 min. The supernatant was obtained, and ammonium sulfate was added to the supernatant to 70% saturation. The resulting solution was stored in a refrigerator at 4 ◦C for 4 h. The centrifuge parameters were set at 4 ◦C and 8000 rpm for refrigerated centrifugation for 15 min. The resulting precipitate was collected, and dissolved in a bufferA. Ammonium sulfate was desalinated by using 1 kDa ultrafiltration membrane for 4 h. Protein purifier (AKTA purifierTM10, General Electric Company, Boston, Massachusetts, MA, USA) was used to purify the enzyme, and Mono Q anion exchange chromatographic column and SuperdexTM75 gel column were adopted for separation and purification with detection wavelength set at 280 nm. The buffers for purification were as follows. Buffer A: 20 mM pH 5.8 disodium-hydrogen phosphate-citric acid, Buffer B: 1 mol/L NaCl. The buffers were filtered through a 0.22 μm membrane, ultrasound treated for 20 min, and stored in a refrigerator at 4 ◦C. Buffer A was used to equilibrate the chromatographic column. The sample was injected after equilibration of buffer to the baseline. The column was washed with 5 column volumes of buffer A, and then eluted with a gradient from 0–100% Buffer B at the flow rate of 1.0 mL/min, and maximum back-pressure of 4 MPa for 30 min. A collector was used to obtain 1 mL fractions. After the elution was complete, the enzymes in the collection tube corresponding to the peak position were collected, frozen and dried in a vacuum freeze dryer (Thermo Savan, Thermo Electron Co., Waltham, MA, USA) and stored at 4 ◦C for later usage. The separation and purification of mPEG5000-β-SPA: 70% ammonium sulfate was added into the reaction liquid to precipitate. The rest steps were the same as those of the β-amylase separation and purification.

The molecular mass and the purity of the enzyme was conducted by SDS-PAGE according to the Laemmli [22] method, and 15% (*w*/*v*) polyacrylamide gel was adopted.
