*2.3. Enzyme Activity and the Ratio of Transfructosylation Activity to Hydrolytic Activity (T/H) Assay*

The reaction systems of Psor-LS were set as 1 mL containing 30% (*w*/*v*) sucrose and 10 μg enzyme. The activity of Psor-LS was determined at 65 ◦C for 15 min. The reaction was stopped in boiling water for 10 min. One unit of total and hydrolytic activity was designated as the amount of enzyme catalyzing the release of 1 μmol glucose and fructose per minute, respectively. The transfructosylation activity was the subtraction between total and hydrolysis activity.

The ratio of transfructosylation activity to hydrolytic activity (T/H) of Psor-LS was determined at different concentrations of substrate (from 10% to 60%), different pH (4.0–9.0), and different temperatures (30–80 ◦C). The glucose and fructose contents of the reaction mixture were analyzed by high-performance liquid chromatography (HPLC). The Waters e2695 system (Waters Corporation, MA, USA) has a Waters 2414 RI detector and a Sugar-Pak I column (6.5 mm × 300 mm, Waters, USA). The column temperature and the mobile phase velocity were set as 85 ◦C and 0.4 mL/min.

## *2.4. Biochemical Characterization*

Three types of buffers with different pH ranges were used to determine the influence of pH on the activity of Psor-LS: acetate buffer (pH 4.0–6.0, 50 mM), sodium phosphate buffer (pH 6.0–7.5, 50 mM), and Tris-HCl (pH 7.5–9.0, 50 mM). The interval of each pH was set as 0.5. The reaction conditions were the same as those for the enzymatic activity assay.

The reactions were performed at various temperatures within 35–80 ◦C at intervals of 5 ◦C to measure the optimal temperature of Psor-LS. The other reaction conditions were the same as the enzymatic activity determination except for the temperature. The enzyme was incubated at 45, 55, and 65 ◦C for different time intervals to determine the thermostability of Psor-LS. The initial activity without pre-incubation treatment was taken as 100%. The formula calculated the half-life (*t*1/2) value, t1/2 = ln2/*k*d, and the inactivation rate constant (*k*d) was determined by the linear regression with the equation: ln (At/A0) = *k*<sup>d</sup> × t (At: residual activity; A0: initial activity; t: incubation time).

Nano DSC III (TA Instrument, New Castle, DE, USA) was introduced to detect the denaturation temperature of this recombinant Psor-LS. The Nano-DSC was equipped with flow-through capillary cells loaded with a pipette mounted on both the inlet and outlet. The sodium phosphate (pH 7.0) buffer was used as the dialysis fluid overnight for the sample, and it was injected into the reference cell for buffer baseline determination. All samples were placed horizontally in a rack on an orbital shaker in a temperature-controlled

incubator with gentle agitation. All samples must be degassed under a vacuum and loaded into the DSC. Scans were conducted by elevating temperatures from 20 ◦C to 100 ◦C, with a scan rate of 1 ◦C min<sup>−</sup>1. To measure molar heat capacities (Cp), sample scans were obtained from the subtraction of the buffer scans to measure molar heat capacities (Cp), and the experimental thermograms with protein concentration and the volume of the calorimeter cell were also normalized. The apparent denaturation temperature Tm values of Psor-LS were determined, and experiment data from Nano DSC III were analyzed using the Nano Analyze software package.

The kinetic parameters for Psor-LS against sucrose were determined at 65 ◦C. The reactions were prepared in potassium phosphate buffer (pH 6.5) containing sucrose 10–900 mM. The apparent Michaelis−Menten constant (Km) and the turnover number (kcat) were obtained through the nonlinear least square regression method (nonlinear regression Michaelis and Menten (http://biomodel.uah.es/en/metab/enzimas/MM-regresion.htm).

#### *2.5. Optimization of Levan Production*

A reaction containing 10–60% sucrose (*w*/*v*) was carried out to determine the optimal sucrose concentration. Under optimized conditions, different enzyme dosages, ranging from 5 to 100 μg/mL, were added to the reaction mixture to investigate the optimal enzyme dosages. The highest enzyme activity determined the optimal sucrose concentration. Differently, the optimal enzyme dosage was decided upon the benefit ratio, which is the ratio of the product formed in 15 min (including FOS and levan) to enzyme dosage. The biological production of Psor-LS was studied by measuring different saccharides at specific time intervals (0.5, 1, 2, 3, and 6 h).

#### *2.6. Purification of Polysaccharide and FOSs*

#### 2.6.1. Purification of Polysaccharides

Sevag reagent (N-butanol: chloroform = 1:4, *v*/*v*) was used to remove protein from the reaction mixture to purify fructans. The polysaccharides in the system were separated by ethanol (final concentration 60%, *v*/*v*) precipitation. Subsequent alcohol precipitation was used to increase the purity of polysaccharides (until HPLC could detect no monosaccharide and sucrose). The samples obtained were then dried via lyophilization using a LABCONCO FreeZone (LABCONCO Co., Kansas City, MO, USA).
