Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum

Abstract

BackgroundIslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme.ResultsScreening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS.ConclusionsDomain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.

The fructan syndrome: Evolutionary aspects and common themes among plants and microbes

“…Microbial levans are produced by a wide range of different gram‐negative and gram‐positive bacteria (see Table below). On the other hand, microbial inulins (a backbone of β‐2,1 linkages between fructofuranosyl units with β‐2,6 branches) are only produced by a limited number of gram‐positive bacteria belonging to genera of Lactobacillus , Leuconostoc , Streptococcus , and Weissella (Anwar, Kralj, van der Maarel, & Dijkhuizen, ; Malang, Maina, Schwab, Tenkanen, & Lacroix, ; Peña‐Cardeña, Rodríguez‐Alegría, Olvera, & Munguía, ; Rosell & Birkhed, ), and the literature is very scarce as compared to levan. In fact, levan production by different microbes has been studied well for over a few decades, and it was revealed that levan shows significantly different physicochemical properties such as branching degree, molecular weight, intrinsic viscosity, and functional properties such as adhesive strength and immunogenic activity, depending on the microbial strains used and various fermentation conditions.…”

Understanding the transfer reaction network behind the non-processive synthesis of low molecular weight levan catalyzed by Bacillus subtilis levansucrase

“…The presence of three products (6-kestose, 1-kestose and neo-kestose) reflects the low regioselectivity of levansucrase in the first fructosyl transfer to a sucrose molecule. The synthesis of these three kestoses has been reported in reactions catalyzed by both levansucrases and inulosucrases 24 , 27 , 31 , suggesting that this lack of regioselectivity when sucrose is the acceptor molecule may be a common characteristic among fructansucrases. The relaxed regiospecificity is consistent with the result of structure-function analysis that locate bond type specificity of fructansucrases in residues further away from the +2 substrate subsite 32 .…”

Extracellular polysaccharides produced by bacteria of the Leuconostoc genus

“…Compared to levan, both the whole culture method and the enzymatic synthesis so far are even less frequently used for the production of microbial inulin, as currently only a few sequences of bacterial inulosucrases have been fully described (Anwar et al 2008(Anwar et al , 2010Ortiz-Soto et al 2004;van Hijum et al 2002) and none of them have reached an application status neither for inulin synthesis nor for production of related oligosaccharides (FOS) (Vallejo-García et al 2019). However, inulin and FOS synthesized by microbial processes remain still considered (Vallejo-García et al 2019) to be a viable alternative to the fructans of vegetable origin that have been dominant so far (Olivares-Illana et al 2002;Mensink et al 2015;Vallejo-García et al 2019) and some promising results have also been achieved (Ozimek et al 2006;Peña-Cardeña et al 2015;Wada et al 2005). Only one strain L. citreum CW28 of Leuconostoc spp.…”

“…Only one strain L. citreum CW28 of Leuconostoc spp. has been studied very comprehensively and currently reported as capable of producing inulosucrase with the required characteristics for the enzymatic synthesis of inulin and/or FOS (Olivares-Illana et al 2002Oritz-Soto et al 2004;Peña-Cardeña et al 2015;Vallejo-García et al 2019). A bacterial strain from Leuconostoc spp.…”

“…According to the whole culture method in a 30 L pilot plant with 250 g/L sucrose and using the cell-associated inulosucrase 76% transformation of substrate and concentration of inulin 95 g/L (2.375 g/L/h) were achieved (Ortiz-Soto et al 2004), indicating a relatively efficient and productive process of enzymatic synthesis. In recent years research has focused more on the production of FOS than on inulin itself using different but equally efficient approaches (Peña-Cardeña et al 2015;Vallejo-García et al 2019). Thus, cloning and expression in E. coli of the truncated form, retaining only the catalytic domain islA4 of the L. citreum CW28 inulosucrase gene islA, has been shown to inhibit inulin formation, increase the hydrolytic activity of the enzyme and shift the specifity of the reaction towards FOS synthesis if high sucrose concentrations (400 ÷ 700 g/L) and an appropriate enzyme activity (1÷5 U/mL) from the E. coli cell-free extract are used (Peña-Cardeña et al 2015).…”