Synthesis of fructooligosaccharides and oligolevans by the combined use of levansucrase and endo-inulinase in one-step bi-enzymatic system
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.
Section snippets
Materials
d-(−)-Fructose, d-(+)-glucose, d-(+)-raffinose, sucrose, endoinulinase from A. niger and dextran standards (50 to 270 kDa) were all purchased from Sigma Chemical Co. (St-Louis, MO). Fructanase® was purchased from Megazyme (Bray, Co. Wicklow, Ireland). Carbohydrate standards, 1-kestose, nystose and 1F-fructofuranosyl-nystose, were obtained from Wako Pure Chemical (Japan) while levanbiose was provided by Jinan Haohua Industry (China). Chemical reagents including acetic acid, 3,5-dinitrosalicylic
Hydrolytic catalytic efficiency of endo-inulinase and fructanase® on levan
To evaluate the hydrolytic catalytic efficiency of endo-inulinase and fructanase® from A. niger, the time courses for the hydrolysis of levan into scFOSs (DP ≤ GF4, levanopentaose) and oligolevans (DP ≥ GF5, levanohexaose F6) were investigated using low (5.5 ± 0.5 kDa) and high (3000 ± 10 kDa) MW-levans. These levans were synthesized through B. amyloliquefaciens levansucrase-catalyzed transfructosylation reaction of sucrose and their linkages were characterized of being mainly β-(2→6)-linked type (Tian
Conclusions
A bi-enzymatic system using levansucrase and endo-inulinase in one-step reaction was developed for the synthesis of scFOSs and oligolevans. The availability of levans, synthesized by levansucrase, at the appropriate amount and MW was prerequisite. The contribution of endo-inulinase to the production of the transfructosylation products through its hydrolytic activity was significant. The use of immobilized levansucrase resulted in different product profiles and highly increased the levan
Acknowledgments
This research is supported by funds from the Natural Sciences and Engineering Research Council (NSERC) of Canada. Financial infrastructure support from Canada Foundation for Innovation (CFI) is gratefully acknowledged.
References (37)
- et al.
Synthesis of novel fructooligosaccharides by substrate and enzyme engineering
Journal of Biotechnology
(2008) - et al.
Immobilization of levansucrase on calcium-phosphate gel strongly increases its polymerase activity
Carbohydrate Research
(1993) - et al.
Production of fructo-oligosaccharides by levansucrase from Bacillus subtilis C4
Process Biochemistry
(1997) - et al.
Synthesis of isomaltooligosaccharides and oligodextrans by the combined use of dextransucrase and dextranase
Enzyme and Microbial Technology
(2004) - et al.
Levan production by use of the recombinant levansucrase immobilized on titanium-activated magnetite
Process Biochemistry
(2001) - et al.
Increase in the fructooligosaccharides yield and productivity by solid state fermentation with Aspergillus japonicus using agro-industrial residues as support and nutrient source
Biochemical Engineering Journal
(2010) - et al.
Occurrence of 2 forms of extracellular endoinulinase from Aspergillus niger mutant-817
Journal of Fermentation and Bioengineering
(1994) - et al.
Enzymatic synthesis of fructosyl oligosaccharides by levansucrase from Microbacterium laevaniformans ATCC 15953
Enzyme and Microbial Technology
(2003) - et al.
Cloning and characterization of a levanbiohydrolase from Microbacterium laevaniformans ATCC 15953
Gene
(2002) - et al.
Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs
Journal of Biological Chemistry
(2011)
Enzymatic synthesis of fructooligosaccharides by levansucrase from Bacillus amyloliquefaciens: Specificity, kinetics, and product characterization
Journal of Molecular Catalysis. B, Enzymatic
Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced
FEMS Microbiology Letters
Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: Substrate specificity, polymerizing properties and usage of different acceptors for fructosylation
Journal of Biotechnology
Structural identification of oligosaccharides produced by Zymomonas mobilis levansucrase
Biotechnology Letters
Enzymic synthesis of levan and fructo-oligosaccharides by Bacillus circulans and improvement of levansucrase stability by carbohydrate coupling
World Journal of Microbiology and Biotechnology
Immobilisation of Bacillus subtilis NRC33a levansucrase and some studies on its properties
Brazilian Journal of Chemical Engineering
Galacto-oligosaccharides and long-chain fructo-oligosaccharides as prebiotics in infant formulas: A review
Acta Paediatrica
Production of fructooligosaccharides by beta-fructofuranosidase from Aspergillus sp 27H
Journal of Chemical Technology and Biotechnology
Cited by (30)
Cross-linked enzyme aggregates (combi-CLEAs) derived from levansucrase and variant inulosucrase are highly efficient catalysts for the synthesis of levan-type fructooligosaccharides
2023, Molecular CatalysisCitation Excerpt :An alternative method to produce the LFOS is to use the hydrolysis activity of endolevanase or endoinulinase to control the size of LFOS. These enzymes can be used to partially hydrolyse the synthetic levan (two-step reaction) or directly added into the levansucrase reaction mixture [19,20]. Nevertheless, the hydrolysis activity of endolevanase is difficult to control and to obtain the desired size of LFOS, the duration of bi-enzymatic reaction has to be strictly regulated [19].
Comprehensive utilization of sucrose resources via chemical and biotechnological processes: A review
2022, Biotechnology AdvancesValorization of renewable resources to functional oligosaccharides: Recent trends and future prospective
2022, Bioresource TechnologyEffect of metal ions on levan synthesis efficiency and its parameters by levansucrase from Bacillus subtilis
2019, International Journal of Biological Macromolecules