# Enzymatic Degradation of _Gracilariopsis lemaneiformis_
## 2 Results and Discussion
### Single-Factor Experiment  
The optimal hydrolysis conditions of \(\beta\)-dextranase were screened through single-factor and orthogonal optimization experiments. Enzyme activity is affected by hydrolysis time, temperature, and pH [11]. In the present study, the hydrolysis conditions of GLP, including time, temperature, pH, and enzyme dosage, were optimized, and the hydroxyl radical scavenging, DPPH radical scavenging, reducing, and total antioxidant abilities were measured for the assessment of the antioxidant activity of GDP obtained at different hydrolysis conditions. The antioxidant activities of GDP improved at its concentration increased from 0.2 to 1.0 mg/mL in the present study (Figures S2-S5).  
The effects of enzymolysis time (60, 90, 120, and 150 min) on GLP degradation were studied at a hydrolysis temperature of 50 \({}^{\circ}\)C, pH of 5.0, and enzyme dosage of 300,000 U/g (Figure S2). At 0.2-1.0 mg/mL, the DPPH radical scavenging ability, reducing ability, and total antioxidant ability of the degradation products increased significantly at an enzymatic hydrolysis time of 90 min, whereas decreased from 90 to 150 min (Figure S2). These results may be explained by the complete degradation of GLP with the extension of time at the initial stage of degradation, and the excessive degradation of polysaccharides may have occurred after 90 min of degradation, leading to structural changes and decreased activity of the polysaccharides [32; 33]. Therefore, the optimal hydrolysis time for the production of antioxidant polysaccharides from _G. lemaneiformis_ was 90 min.  
Temperature is an important factor affecting enzyme activity [11; 34]. The effects of hydrolysis temperature on the antioxidant activity of GDP in vitro are displayed in Figure S3. Scavenging abilities against hydroxyl and DPPH radicals, reducing capacity, and total antioxidant capacity of GDP increased from 30 to 40 \({}^{\circ}\)C (Figure S3). A high temperature can accelerate the molecular movement and ensure full contact between a substrate and enzyme [35]. When the temperature exceeded 40 \({}^{\circ}\)C, the antioxidant activity of GDP decreased, possibly because of the degradation and inactivation of the enzyme at a high temperature [36]. Therefore, 40 \({}^{\circ}\)C was regarded as the best temperature for GLP degradation.  
pH has a great influence on enzyme activity. Enzymes have catalytic activities only within a certain pH range [37]. As shown in Figure S4, the antioxidant activity of GDP improved with increasing pH, and the strongest antioxidant activity of GDP was observed at pH 5.0. It was inferred that pH exceeding 5.0 was beyond the suitable pH range of \(\beta\)-dextranase and led to decreases in enzyme activity and poor degradation effect. Thus, pH 5.0 was considered optimal in the present study.  
Figure 1: Antioxidant activities of GLP and different enzymatic hydrolysis products at a concentration of 1.0 mg/mL; scavenging ability of hydroxyl radical (**A**), scavenging ability of DPPH radical (**B**), reducing capacity (**C**), and total antioxidant capacity (**D**). Vertical bars represent means \(\pm\) SD (n = 3), and different letters indicate significant difference (\(p<0.05\)).  
The effects of enzyme dosages of 200,000-350,000 U/g on the antioxidant activity of GDP were studied. GLP was hydrolyzed by \(\beta\)-dextranase effectively, and enzyme dosage is vital for the antioxidant activity of polysaccharide hydrolyses (Figure S5). Antioxidant activity increased at enzyme dosages of 200,000-300,000 U/g (Figure S5) but decreased rapidly when the enzyme dosage exceeded 300,000 U/g, possibly because of the limited substrate concentration, which limited the interaction between substrates and enzymes or because the polysaccharide was wrapped in the enzyme [38].