Enzymatic Process Yielding a Diversity of Inulin-Type Microbial Fructooligosaccharides

Abstract

The specificity of fructooligosaccharides as prebiotics depends on their size and structure, which in turn depend on their origin or the synthesis procedure. In this work we describe the application of an inulosucrase (IslA) from Leuconostoc citreum CW28 to produce high molecular weight inulin from sucrose alongside a commercial endoinulinase (Novozym 960) produced by Aspergillus niger for a simultaneous or sequential reaction to synthesize fructooligosaccharides (FOS). The simultaneous reaction resulted in a higher substrate conversion and a wide diversity of FOS when compared to the sequential reaction. A shotgun MS analysis of the commercial endoinulinase preparation surprisingly revealed an additional enzymatic activity: a fructosyltransferase, responsible for the synthesis of FOS from sucrose. Consequentially, the range of FOS obtained in reactions combining inulosucrase from Ln. citreum with the fructosyltransferase and endoinulinase from A. niger with sucrose as substrate may be extended and regulated.

Extracellular polysaccharides produced by bacteria of the Leuconostoc genus

“…Another fructose polymer inulin (Fig. 1) is formed in a similar way, by the related enzyme inulosucrase 4 (EC 2.4.1.9) although with a different structure and linkage type, containing β-(2,1) linked fructose molecules in the main chain and β-(2,6) ramifications (Vallejo-García et al 2019;Mensink et al2015). In addition, the presence of inulosucrase has been reported only in some genera of gram-positive bacteria, including Leuconostoc spp., which may additionally contain levansucrases (Table 1).…”

“…Although recombinant levansucrases are primarily used to produce levan and related fructooligosaccharides (FOS), other applications are possible, for example the production of melibiose from raffinose, by the enzyme expressed in E. coli according the levansucrase gene (Gen-Bank accession No AY665464) of L. mesenteroides (Xu et al 2017). 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. 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).…”

“…In addition, the presence of inulosucrase has been reported only in some genera of gram-positive bacteria, including Leuconostoc spp., which may additionally contain levansucrases (Table 1). while the occurrence of levansucrases is widespread in gram-positive and gram-negative bacteria (Anwar et al 2010;Olvera et al 2007;Öner et al 2016;Vallejo-García et al 2019).…”