Synthesis of Fructooligosaccharides by Isla4, a Truncated Inulosucrase

Peña-Cardeña et al. BMC Biotechnology (2015) 15:2 DOI 10.1186/s12896-015-0116-1 RESEARCH ARTICLE Open Access Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum Arlen Peña-Cardeña, María Elena Rodríguez-Alegría, Clarita Olvera and Agustín López Munguía* Abstract Background: IslA4 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. Results: Screening 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. Conclusions: Domain 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. Keywords: Fructosyltransferase, Fructooligosaccharides, Inulin, Inulosucrase, Leuconostoc citreum Background [5-7]. FTFs (EC 2.4.1.-) are enzymes that catalyze the Fructooligosaccharides (β2-1, FOS) with a degree of transfer of the fructosyl moiety of sucrose to different polymerization (DP) in the range of 2–10, are one of the acceptors, resulting in the synthesis of fructans with dif- most important sources of carbon and energy for the ferent molecular weights depending on the specificity of beneficial microbiota (probiotics) that exist in the intes- the enzyme. The fructosyl unit can also be transferred tinal lumen of humans [1]. FOS are generally consumed to water, resulting in the hydrolysis of sucrose. Accord- directly as part of a healthy diet, including fruit and veg- ing to the classification system for carbohydrate-active etables. FOS can also be consumed indirectly through enzymes, bacterial FTFs belong to family 68 of the functional foods, which can be enriched with FOS ob- glycoside hydrolases (GH68). Most FTFs are 45 to tained by the hydrolysis of chicory inulin or produced 64 kDa in length and consist of a single catalytic domain industrially through enzymatic processes with commer- with a five-bladed β-propeller fold that encloses a cial fructosyltransferases (FTFs) from fungal species like funnel-like central cavity where the conserved catalytic Aspergillus [2]. In the latter case, fungal enzymes are the residues are located. FTFs can be classified as inulosu- preferred choice of enzyme because of their high specifi- crases (EC 2.4.1.9), which synthesize β2-1 linked fruc- city towards low molecular weight inulins, low hydro- tans (inulin), or levansucrases (EC 2.4.1.10), which lytic activity and high stability [3,4]. In contrast, bacterial produce fructans with β2-6 linkages (levan) [8]. inulosucrases can be used to synthesize high molecular We previously reported the isolation of inulosucrase weight inulin with low specificity towards FOS production from Leuconostoc citreum CW28. This particular enzyme was designated IslA and was found to synthesize high- * Correspondence: [email protected] molecular-weight inulin. IslA is a multidomain enzyme Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos C.P. 62210, México © 2015 Peña-Cardeña et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Peña-Cardeña et al. BMC Biotechnology (2015) 15:2 Page 2 of 9 containing additional regions at both the amino- and Results and discussion carboxyl-terminals of its catalytic domain, which are Influence of substrate and enzyme concentration on IslA4 similar to those found in glucosyltransferases [9]. In this reaction specificity context, IslA4 is a truncated form of IslA that retains A common characteristic of fructosyltransferases is their only the five-bladed β-propeller catalytic domain. IslA4 ability to transfer the fructosyl moieties of a substrate to is therefore similar to several other fructosyltransferases an acceptor molecule (the fructan growing chain) or previously reported in the literature, including the levan- water, leading to the hydrolysis of the substrate. The sucrases from Bacillus subtilis (SacB) and Gluconaceto- effects of the IslaA4 and sucrose concentrations on the bacter diazotrophicus, and the InuJ inulosucrase from transfer and hydrolysis reactions of the fructosyl moiety Lactobacillus johnsonii NCC 533. We previously studied are shown in Figure 1. As in many other FTF cases, the effects of the additional domains of IslA on its over- transferase activity is favored by high substrate concen- all properties and found that IslA4 was similar to IslA, trations, because of the higher amount of sucrose mole- in the sense that it produced mainly high molecular cules available with respect to water for the initial weight inulin. However, IslA4 exhibits a much higher transfer of the fructosyl residue [17]. Similar behavior hydrolytic activity than IslA under the same reaction has also been reported for levansucrase from B. subtilis, conditions [10]. Interestingly, IslA developed a high where almost 80% of the sucrose was used for transfer- level of hydrolytic activity following the elimination of ase reactions at sucrose concentrations greater than some of its additional domains, and achieved similar 1,750 mM [18]. In contrast, SacB becomes predomin- activity to a single domain fructosyltransferase, such as antly hydrolytic in terms of its activity at low sucrose levansucrase SacB, which can hydrolyze as much as 80% concentrations (12 mM) [19]. Furthermore, levansucrase of the sucrose substrate [11]. It has been demonstrated from G. diazotrophicus displayed only hydrolytic activity that reaction specificity (i.e., hydrolysis or transferase) at sucrose concentrations lower than 60 mM [20]. An as well as product specificity (i.e., type and size of fructan inverse reaction specificity effect was observed for the or FOS) in fructosyltransferases is strongly dependent on enzyme concentration of IslA4, where an increase in the − the reaction conditions, including the substrate concentra- enzyme concentration from 1 to 10 U mL 1 led to an in- tion and temperature [12,13], the form in which the crease in the level of hydrolytic activity, regardless of enzyme is applied, such as free or immobilized [14], the the sucrose concentration. Reactions involving a low presence of organic solvents or co-solvents [15], and the enzyme concentration and high substrate concentration − sourceoftheenzyme[16]. (e.g., 1 U mL 1 and 2,046 M, respectively) were there- In this study, we have evaluated the effect of the reac- fore determined to be suitable reaction conditions for tion conditions on the specificity of IslA4 and the trun- high transferase efficiencies, despite the lengthy reac- cated form of inulosucrase IslA, in an attempt to tion times required. In contrast, a high IslA4 concentration − identify efficient enzymes for the synthesis of inulin- (10 U mL 1) coupled with a low sucrose concentration type FOS. (292 mM) led to 90% of the sucrose being hydrolyzed. Figure 1 Transferase efficiency of IslA4 as a function of enzyme and substrate concentration. All reactions are carried out at 30°C in 50 mM phosphate (pH 6.0) containing 1 mM CaCl2 and allowed to proceed until they reached a sucrose conversion of approximately 90%. Peña-Cardeña et al. BMC Biotechnology (2015) 15:2 Page 3 of 9 Similar behavior has also been reported for several the synthesis of high molecular weight inulin, the com- other enzymes, such as SacB from B. subtilis,which bination of a truncated domain with the appropriate transferred 78% of 1,750 mM sucrose at low enzyme reaction conditions can results in the synthesis of a wide concentrations [18]. variety of FOS as the major transferase products. This With regard to other inulosucrases, it has been re- effect can be readily visualized by TLC analysis, as ported that the transfructosylation activity of L. reuteri shown in Figure 2. In this particular case, the FOS and 121 increased when the substrate and enzyme concen- inulin could both be clearly seen amongst the final reac- tration were increased from 200 to 1800 mM and 40 to tion products when the products of reactions containing − − 130 U mg 1, respectively, with FOS being identified as 1UmL 1 of enzyme activity are analyzed at three differ- the main transferase product together with a relatively ent initial sucrose concentrations.

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