Production and anti-inflammatory evaluation of β-(2 → 6)-linked levan synthesized by a recombinant levansucrase from Streptococcus salivarius SY511
摘要
Levansucrase catalyzes the synthesis of levan, a β-(2 → 6)-linked fructan that has antioxidant, prebiotic, and film-forming properties and is a promising biopolymer for applications in the food, cosmetic, and biomedical industries. We isolated a fructosyltransferase (FTF) gene from Streptococcus salivarius SY511 (a strain generally regarded as safe), induced heterologous expression in Escherichia coli, and purified the recombinant enzyme using nickel affinity chromatography. The molecular weight of the enzyme was approximately 150 kDa, optimal transfructosylation activity was at pH 6.0 and 37 °C, activity was greater with the addition of Ca2+, and activity was less with the addition of Cu2+ or Fe2+. The enzyme retained over 60% of its activity when incubated at 30 °C for 4 h, but higher incubation temperatures greatly decreased activity. Maximum polysaccharide production was achieved by incubation of 18 U levansucrase/g sucrose with 300 mg/L sucrose for 10 h. Structural analysis using 13C NMR revealed chemical shift differences (3.45 ppm for C-1 vs. C-6 and 1.1 ppm for C-3 vs. C-4) that were consistent with the β-(2 → 6) bonds of levan. The levan synthesized by the recombinant protein had excellent biocompatibility based on measurements of the anti-inflammatory and cytotoxic effects in RAW 264.7 macrophages. These findings conclusively define the recombinant FTF as a levansucrase and support its potential for scalable production of functional levan.