Novel Cold-Active Levansucrase (SacBPk) from Priestia koreensis HL12 for Short-Chain Fructooligosaccharides and Levan Synthesis

Abstract

Levansucrases are key enzymes responsible for the synthesis of β-2,6-linked fructans, found in plants and microbes, especially in bacteria. Levansucrases have been applied in the production of levan biopolymer and fructooligosaccharides (FOSs) using sucrose as a substrate as well as in reducing sugar levels in fruit juice. As a result, levansucrases that are active at low temperatures are required for industrial applications to maintain product stability. Therefore, this work firstly reports the novel cold-active levansucrase (SacBPk) isolated from a sucrolytic bacterial strain, P. koreensis HL12. The SacBPk was classified into glycoside hydrolase family 68 subfamily 1 (GH68_1) and comprised a single catalytic domain with the Asp104/Asp267/Glu362 catalytic triad. Interestingly, the recombinant SacBPk demonstrated cold-active levansucrase activity at low temperatures (on ice and 4–40 °C) with the highest specific activity (167.46 U/mg protein) observed at 35 and 40 °C in 50 mM sodium phosphate buffer pH 6.0. SacBPk mainly synthesized levan polymer as the major product (129 g/L, corresponding to 25.8% of total sugar) with a low number of short-chain FOSs (GF2–4) (12.8 g/L, equivalent to 2.5% of total sugar) from 500 g/L sucrose after incubating at 35 °C for 48 h. These results demonstrate the industrial application potential of SacBPk levansucrase for levan and FOSs production.