Dataset Information

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae ?-Galactosidase.

ABSTRACT: ?-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP)???3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae ?-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP???3 production. Assays with ?-galactosidase immobilized onto a glutaraldehyde-agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP???3 were 50?°C, pH 6.5, 450?g/L of lactulose, and 8?U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6'-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60?°C, pH 4.5, 450?g/L of lactulose and 8?U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae ?-galactosidase immobilized on a support of glutaraldehyde-agarose constitutes an efficient and cost-effective alternative to the use of soluble ?-galactosidases for the synthesis of prebiotic OsLu mixtures.

SUBMITTER: Cardelle-Cobas A

PROVIDER: S-EPMC4780266 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase.

Cardelle-Cobas Alejandra A Olano Agustin A Irazoqui Gabriela G Giacomini Cecilia C Batista-Viera Francisco F Corzo Nieves N Corzo-Martínez Marta M

Frontiers in bioengineering and biotechnology 20160307

β-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 productio ...[more]

PMID: 27014684

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Project description:BackgroundContinuous processing with enzyme reuse is a well-known engineering strategy to enhance the efficiency of biocatalytic transformations for chemical synthesis. In one-pot multistep reactions, continuous processing offers the additional benefit of ensuring constant product quality via control of the product composition. Bottom-up production of cello-oligosaccharides (COS) involves multistep iterative β-1,4-glycosylation of glucose from sucrose catalyzed by sucrose phosphorylase from Bifidobacterium adeloscentis (BaScP), cellobiose phosphorylase from Cellulomonas uda (CuCbP) and cellodextrin phosphorylase from Clostridium cellulosi (CcCdP). Degree of polymerization (DP) control in the COS product is essential for soluble production and is implemented through balance of the oligosaccharide priming and elongation rates. A whole-cell E. coli catalyst co-expressing the phosphorylases in high yield and in the desired activity ratio, with CdP as the rate-limiting enzyme, was reported previously.ResultsFreeze-thaw permeabilized E. coli cells were immobilized in polyacrylamide (PAM) at 37-111 mg dry cells/g material. PAM particles (0.25-2.00 mm size) were characterized for COS production (~ 70 g/L) in mixed vessel with catalyst recycle and packed-bed reactor set-ups. The catalyst exhibited a dry mass-based overall activity (270 U/g; 37 mg cells/g material) lowered by ~ 40% compared to the corresponding free cells due to individual enzyme activity loss, CbP in particular, caused by the immobilization. Temperature studies revealed an operational optimum at 30 °C for stable continuous reaction (~ 1 month) in the packed bed (volume: 40 mL; height: 7.5 cm). The optimum reflects the limits of PAM catalyst structural and biological stability in combination with the requirement to control COS product solubility in order to prevent clogging of the packed bed. Using an axial flow rate of 0.75 cm- 1, the COS were produced at ~ 5.7 g/day and ≥ 95% substrate conversion (sucrose 300 mM). The product stream showed a stable composition of individual oligosaccharides up to cellohexaose, with cellobiose (48 mol%) and cellotriose (31 mol%) as the major components.ConclusionsContinuous process technology for bottom-up biocatalytic production of soluble COS is demonstrated based on PAM immobilized E. coli cells that co-express BaScP, CuCbP and CcCdP in suitable absolute and relative activities.

Project description:BackgroundCellodextrin phosphorylase (CdP; EC 2.4.1.49) catalyzes the iterative β-1,4-glycosylation of cellobiose using α-D-glucose 1-phosphate as the donor substrate. Cello-oligosaccharides (COS) with a degree of polymerization (DP) of up to 6 are soluble while those of larger DP self-assemble into solid cellulose material. The soluble COS have attracted considerable attention for their use as dietary fibers that offer a selective prebiotic function. An efficient synthesis of soluble COS requires good control over the DP of the products formed. A mathematical model of the iterative enzymatic glycosylation would be important to facilitate target-oriented process development.ResultsA detailed time-course analysis of the formation of COS products from cellobiose (25 mM, 50 mM) and α-D-glucose 1-phosphate (10-100 mM) was performed using the CdP from Clostridium cellulosi. A mechanism-based, Michaelis-Menten type mathematical model was developed to describe the kinetics of the iterative enzymatic glycosylation of cellobiose. The mechanistic model was combined with an empirical description of the DP-dependent self-assembly of the COS into insoluble cellulose. The hybrid model thus obtained was used for kinetic parameter determination from time-course fits performed with constraints derived from initial rate data. The fitted hybrid model provided excellent description of the experimental dynamics of the COS in the DP range 3-6 and also accounted for the insoluble product formation. The hybrid model was suitable to disentangle the complex relationship between the process conditions used (i.e., substrate concentration, donor/acceptor ratio, reaction time) and the reaction output obtained (i.e., yield and composition of soluble COS). Model application to a window-of-operation analysis for the synthesis of soluble COS was demonstrated on the example of a COS mixture enriched in DP 4.ConclusionsThe hybrid model of CdP-catalyzed iterative glycosylation is an important engineering tool to study and optimize the biocatalytic synthesis of soluble COS. The kinetic modeling approach used here can be of a general interest to be applied to other iteratively catalyzed enzymatic reactions of synthetic importance.

Dataset Information

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae ?-Galactosidase.

Publications

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase.

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