LEVANSUCRASES OF PSEUDOMONAS BACTERIA AS PRODUCERS OF LEVAN, FRUCTOOLIGOSACCHARIDES AND HETEROOLIGOFRUCTANS

Several soil- and plant-associated bacteria synthesize extracellular enzymes – levansucrases. These enzymes belonging to GH68 family according to CAZy database split sucrose and carry out transfructosylation reaction of another sucrose molecule producing polymeric levan and fructooligosaccharides (FOS) of varied degree of polymerization (DP). Regarding Pseudomonas bacteria, single levansucrase, LscA, is present in P. chlororaphis subsp. aurantiaca whereas Pseudomonas syringae pathovars have up to three levansucrase genes. The genome of P. syringae pv. tomato encodes three functional levansucrase proteins: Lsc1, Lsc2 and Lsc3 [1]. We will present data on polymerizing properties of Lsc2, Lsc3 and LscA proteins. Lsc2 and Lsc3 were purified by us from E. coli as a heterologous His-tagged or tagless proteins and LscA was purchased from Sigma-Aldrich. All three enzymes acted on sucrose, raffinose and sugar beet molasses, producing FOS and levan. Nano ESI MS analysis of reaction products of Lsc3 and LscA detected FOS with DP up to 6. Additionally, TLC analysis revealed FOS of higher DP, up to 10, for LscA. The LscA and Lsc3 were studied from the aspect of synthesis of heterooligofructans (HOF) as products of novel applications. D-xylose, D-fucose, L- and D-arabinose, D-ribose, D-sorbitol, xylitol, xylobiose, D-mannitol, D-galacturonic acid and methyl-α-D-glucopyranoside were used as nonconventional fructosyl acceptors by Lsc3. As a result, a mixture of conventional FOS and HOF was produced [2]. A high-throughput microplate-based assay was conducted to optimise reaction conditions for Lsc3 protein to produce a high amount of desired product, FOS or levan, or both.

[1] Visnapuu, T. et al, 2008, Process Biochem 43, 414-42.

[2] Visnapuu, T. et al, 2011, J Biotechnol 155, 338-49.