Discovery of new levansucrase enzymes with interesting properties and improved catalytic activity to produce levan and fructooligosaccharides

Abstract

Levansucrases (EC 2.4.1.10, LS) are of high interest for the synthesis of novel prebiotic fructooligosaccharides. Hindered by the limited availability of LS enzymes and a propensity for performing the hydrolysis of sucrose rather than the transfructosylation reaction, a desire to find new LSs was fueled. Genome mining was employed to explore their biodiversity using 26 characterized LSs as a reference set for a sequence driven-approach leading to a collection of 32 enzymes representative of the biodiversity for which the gene was cloned and over-expressed in E. coli. These enzymes underwent an initial screening process based upon total activity, transfructosylation activity and levan forming ability which narrowed the candidates to 10 potential enzymes. These LS enzymes were found to have high levan production (33 ± 1 g levan per mg protein) and able to produce very large polymers (6986 kDa). The LS from Paraburkholderia graminis had a very high natural thermal stability with a half-life of 291 minutes at 50 °C. The full kinetic parameters of the top candidates were characterized for the enzymes with the highest potential. Enzymes with higher catalytic efficiency and activity for transfructosylation over hydrolysis were identified. The acceptor specificity of these new levansucrase enzymes was briefly explored. It showed wide specificity for each of the selected enzymes.