**2. Materials and Methods**

#### *2.1. Chemicals, Reagents, and Strains*

*Escherichia coli* DH5α and BL21 (DE3) cells, Isopropyl β-D-1-thiogalactopyranoside (IPTG), ampicillin sodium, Luria–Bertani (LB) medium, and other chemicals of analytical grade were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Standards, including sucrose, glucose, and fructose, were purchased from Sigma (St. Louis, MO, USA) for high-performance liquid chromatography (HPLC) analysis.

#### *2.2. Expression and Purification of Psor-LS*

The genomic DNA of *P*. *orientalis* is available on the NCBI database with the GenBank accession number ASM385204v1, which revealed a putative gene encoding levansucrase. The gene was fused with the pET-22(+) vector using two restriction sites *Nde* I and *Xho* I, at the 5 - and 3 - terminus, which were commercially synthesized by Generay Biotech Co., Ltd. (Shanghai, China). A 6 × histidine-tag was designed at the 3 -terminus for purification via Ni2+ affinity chromatography.

For levansucrase expression, the recombinant plasmid was transformed into competent *E. coli* BL21 (DE3) cells, which were then inoculated into 200 mL of LB broth containing ampicillin (100 μg/mL broth) for the selection of transformants. The cells were cultivated at 37 ◦C with shaking at 200 rpm until the optical density at 600 nm (OD600) reached 0.6–0.8.

Then, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into the broth with the final concentration of 1 mM, and the target enzyme expression was performed at 28 ◦C with shaking at 200 rpm for 6~7 h.

The bacteria were collected by centrifugation at 6000× *g* for 5 min, then disrupted in 15 mL lysis buffer (50 mM sodium phosphate buffer with 100 mM NaCl, pH 7). After being disrupted by ultrasonication, the supernatant was centrifuged at 8000× *g* and 4 ◦C for 10 min and filtered through a 0.45 μm Millipore filter. The enzyme was purified via Ni2+ affinity chromatography using Ni-IDA-Sefinose resin (Sangon Biotech Co. Ltd., Shanghai, China). A binding buffer (50 mM sodium phosphate buffer with 100 mM NaCl, pH 7.0), washing buffer (50 mM sodium phosphate buffer with 100 mM NaCl and 50 mM imidazole, pH 7.0), and elution buffer (50 mM sodium phosphate buffer with 100 mM NaCl and 500 mM imidazole, pH 7.0) were used to equilibrate the chromatography column, wash out non-specific proteins and elute target proteins, respectively. Then, the collected enzyme solution was dialyzed against 50 mM sodium phosphate buffer (pH 7.0) for more than 18 h to remove imidazole. SDS-PAGE examined the subunit molecular mass of Psor-LS with 12% (*w*/*v*) separation gel and 5% stacking gel, and the protein bands were stained with Coomassie Brilliant Blue R250. The Bradford assay [18] estimated the protein concentration using bovine serum albumin as the standard.
