Synthesis of fructooligosaccharides and oligolevans by the combined use of levansucrase and endo-inulinase in one-step bi-enzymatic system

Highlights

• Levansucrase/endoinulinase bienzymatic system was developed to produce scFOSs and oligolevans.

• Bi-enzymatic process showed higher yield and productivity than the current available ones.

• Contribution of endo-inulinase to the efficiency of bi-enzymatic system was higher.

• Production of intermediate levans with appropriate MW by levansucrase was a prerequisite.

• Use of immobilized levansucrase resulted in higher yield of levan as compared to free one.

Abstract

Levansucrase from Bacillus amyloliquefaciens and endo-inulinase from Aspergillus niger were used in a one-step reaction to produce short chain fructooligosaccharides (scFOSs) and oligolevans from sucrose. Levansucrase catalyzed the synthesis of levan, while endo-inulinase regulated the product molecular size. The bi-enzymatic system showed higher yield and productivity (67% w/w; 96 g/L/h) than the levansucrase enzymatic system (3.0% w/w; 0.8 g/L/h) alone. The contribution of endo-inulinase to the formation of scFOSs and oligolevans through its hydrolytic activity was higher than that of levansucrase through its acceptor reaction; however, the production of intermediate levans with appropriate MW by levansucrase was prerequisite. The maximal concentration of scFOSs was higher as compared to that of oligolevans. Among scFOSs, 6-kestose was the most dominant product. The use of immobilized levansucrase resulted in a lower production of scFOSs and higher yield of levan. The current study is the first to highlight the potential of levansucrase/endo-inulinase bi-enzymatic system.

Industrial relevance

Functional food products are receiving a substantial amount of interest from consumers. Considering the size of this market, the food industry sector could benefit considerably from improvements in the functional ingredients used to support the development of these health promoting food products. In this context, fructooligosaccharides (FOSs), a class of ingredients whose potential health benefits in terms of supporting intestinal health and reducing the risk of cancers are increasingly being recognized. However, commercially available FOSs are exclusively β-(2→1)-inulin-type prebiotics with short chains, which are mainly absorbed in the small intestine. β-(2→6) and neolevan-type-FOSs have shown prebiotic activities that surpass the current β-(2→1)-FOSs generation.

The development of innovative bi-enzymatic process for the production of FOSs and β-(2→6)-oligolevans in the present study is of high interest. The developed bienzymatic system showed high yield and productivity for the production of FOSs and oligolevans from sucrose as abundant starting materials. These products being of high-degree of polymerization are expected to exhibit an increased colonic prebiotic persistence and reach the distal intestinal region where most of the chronic diseases are originated.

Introduction

Fructooligosaccharides (FOSs) have attracted greater attention due to the increased demand for functional ingredients. In addition to their low caloric value and non-cariogenic properties, FOSs promote the intestinal health by stimulating the growth/activity of beneficial lactic acid bacteria and enhance the immune response (Fanaro et al., 2005). Commercially available FOSs for human consumption are exclusively β-(2→1)-inulin-type prebiotics that are mainly produced from sucrose by the action of fructofuranosidases (Mussatto, Rodrigues, & Teixeira, 2009). The β-(2→6) and neolevan-type-FOSs have shown prebiotic activities that surpass the current FOS generation (Kilian, Kritzinger, Rycroft, Gibson, & du Preez, 2002). In addition, β-(2→6)-levan-type-hetero-FOSs may have new anti-adhesive activity against pathogens and immunomodulatory capacity (Shibata et al., 2009). Levansucrases have been investigated by our group and others as potential biocatalysts for the synthesis of β-(2→6)-levan-type-FOSs (van Hijum et al., 2004, Homann et al., 2007, Beine et al., 2008, Tian and Karboune, 2012).

Levansucrase (EC 2.4.1.10), which belongs to the glycoside hydrolase family 68 (GH68), catalyzes the synthesis of FOSs and high-molecular weight β-(2→6)-levan from sucrose (van Hijum et al., 2001, Meng and Fütterer, 2003). Levansucrases from Gram-negative bacteria Gluconacetobacter diazotrophicus (Hernandez et al., 1995) and Zymomonas mobilis (Crittenden & Doelle, 1993) synthesize mostly FOSs and low amounts of levan; while levansucrases from Gram-positive bacteria, Bacillus subtilis (Euzenat, Guibert, & Combes, 1997), Bacillus megaterium (Homann et al., 2007), Lactobacillus reuteri 121 (Ozimek, Kralj, Van der Maarel, & Dijkhuizen, 2006), Microbacterium laevaniformans (Park et al., 2003) and Bacillus amyloliquefaciens (Tian, Inthanavong, & Karboune, 2011), synthesize dominantly high-molecular weight levan with a molecular mass up to 10⁴ kDa. In recent years, some structural elements of levansucrases that govern their polymer versus oligomer synthesis have been identified (Meng and Fütterer, 2003, Ozimek et al., 2006, Homann et al., 2007). Indeed, residues Asn²⁴² and its corresponding Asn²⁵² at the + 2 sugar binding subsites of levansucrases from Gram-positive B. subtilis (Beine et al., 2008) and B. megaterium (Homann et al., 2007), respectively, were reported to be critical in the synthesis of levan. Mutations of His³²¹ and Thr³⁰² located at the + 1 sugar binding subsite of levansucrase from Pseudomonas syringae pv. changed the product pattern towards the production of short-chain FOSs (scFOSs) than levan and long-chain FOSs (Visnapuu et al., 2011). Recently, a surface platform, made of residues Asn²⁵², Lys³⁷³, and Tyr²⁴⁷, has been identified of great importance for the product spectrum of levansucrase from B. megaterium (Strube et al., 2011).

In spite of the progress that has been made, the yields of FOSs produced through levansucrase-catalyzed transfructosylation reactions are still low and their chain lengths are short (Crittenden and Doelle, 1993, Hernandez et al., 1995). The production of controlled molecular size FOSs and oligolevans (degree polymerization, DP ≥ GF₅ and levanohexaose F₆) would result in an increased colonic persistence of the prebiotic effect and thereby reduce the risk of chronic diseases in the distal intestinal region (Wichienchot et al., 2006, van de Wiele et al., 2007). Indeed, short chain scFOSs (GF₂–GF₃) are mainly absorbed in the small intestine. As part of ongoing research, the combined use of levansucrase and fructanase was investigated in the present study for the synthesis of FOSs (scFOSs and β-(2→6)-FOSs) and oligolevans (DP ≥ GF₅–F₆) using sucrose as an abundant substrate. Levansucrase from B. amyloliquefaciens catalyzes the synthesis of levan and FOSs from sucrose, while fructanase hydrolyses fructan polymer into scFOSs and oligolevans. These transfructosylation products may be further hydrolyzed by fructanase or serve as acceptors for levansucrase. The understandings of the synergistic mechanism by which levansucrase and fructanase function and the parameters that affect their thermodynamic relationships and the availability of their substrates and acceptors are important for the synthesis of FOSs and oligolevans of controlled sizes. In this context, the specific objectives of the present study were (1) the evaluation of selected fructanases (endo-inulinase and fructanase® from Aspergillus niger) for the hydrolysis of levan; (2) the investigation of the combination of levansucrase and endo-inulinase in a one-step enzymatic reaction; (3) the study of the effect of levansucrase to endo-inulinase ratio and sucrose concentration on the product spectrum and productivity; and (4) the determination of the effect of immobilization of levansucrase on the efficiency of the bi-enzymatic system.