Fructooligosaccharides (FOS) are fructose oligomers linked to a sucrose skeleton by different β(2→1) or β(2→6) glycosidic bonds [1
]. In addition to their prebiotic properties—which promote the development of bifidobacteria and lactobacillus in the gastrointestinal tract [2
]—and their low glycemic index, FOS may exert other benefits in human health, including a better gut absorption of Ca2+
, a reduction of blood lipid levels and a reduced risk of suffering colon cancer [3
]. FOS can be synthesized from sucrose by a transfructosylation reaction [5
]. Commercial FOS possess an inulin-type structure containing β(2→1) linked fructose units [6
]. However, it has been reported that neo-FOS, in which one fructosyl moiety is β(2→6) linked to the glucose unit of sucrose, could display improved prebiotic and physicochemical properties with regard to inulin-type FOS [7
The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous
(formerly Phaffia rhodozyma
) is a dimeric glycoprotein with a molecular mass of 320–380 kDa, which belongs to the glycoside hydrolase (GH) family 32 [9
]. Like other β-fructofuranosidases, Xd-INV catalyzes both the hydrolysis of sucrose and the synthesis of FOS [11
]. However, Xd-INV is unique in its ability to catalyze the transfer a fructosyl moiety to the 6-OH hydroxyl of glucose unit in sucrose. In fact, this is the most efficient enzyme reported for the production of neo-FOS (basically neokestose and neonystose) [11
]. Xd-INV is an attractive enzyme not only for the production of neo-FOS, but also for the preparation of novel fructosylated derivatives [13
]. Recently we successfully expressed this enzyme in Pichia pastoris
(pXd-INV) yielding a significant volumetric activity [12
Despite the enormous potential of biocatalytic processes [14
], the industrial application of enzymes is often hampered by a lack of long-term operational stability and the difficulties to recover and reuse the biocatalysts [15
]. Enzyme immobilization can help to overcome these drawbacks, since it allows the easy separation of the biocatalyst facilitating product recovery, which is commonly accompanied by the stabilization effect towards denaturation by high temperatures, extreme pHs or organic cosolvents [16
Immobilization methodologies for industrial biotransformations should be relatively simple, inexpensive and provide active biocatalysts with substantial stability [15
]. The strategies for enzyme immobilization are commonly classified into three groups [18
]: support binding (by adsorption or covalent linkages), entrapment and cross-linking. For reactions involving the transformation of carbohydrates, covalent binding is preferred over adsorption to avoid enzyme leakage [19
], but most of the commercial activated carriers are expensive [20
]. Cross-linking gives rise to biocatalysts with highly concentrated enzyme activity, significant stability and low production costs due to the absence of carrier, although the recovery of activity is commonly low [14
]. Entrapment is an efficient and inexpensive technique, which is very useful when substrates and products have low molecular sizes and high diffusion rates, as occurs with simple sugars [14
]. The entrapment in hydrogels can be combined with cross-linking in order to provide more resistant biocatalysts [25
In this work, we have investigated the immobilization of recombinant pXd-INV to facilitate its industrial application in the production of neo-FOS and other fructosylated derivatives. Considering that the size of Xd-INV is significantly large (it is a dimeric enzyme with an average molecular mass of 360 kDa and dimensions 135 × 75 × 45 Å [10
]), we believed that entrapment methodologies could be appropriate for this enzyme as the leakage through pores should be restricted Our focus was to evaluate polyvinyl alcohol (PVA) entrapment as an immobilization strategy. PVA is cheap, mechanically robust and nontoxic to organisms [27
]. The efficiency of this methodology was assessed in terms of the recovered activity and operational stability. The resulting biocatalysts were applied to the production of neo-FOS.
3. Materials and Methods
Sucrose was from Scharlau. Polyvinyl alcohol (PVA) (99% hydrolyzed, average MW 130,000) was purchased from Sigma Aldrich (Madrid, Spain). Fructose was from Merck and 1-kestose was from TCI. Neokestose, 6-kestose, neonystose and blastose were synthesized according to previous works [9
]. All other reagents and solvents were of the highest purity available.
3.2. β-Fructofuranosidase Activity Source
The β-fructofuranosidase from Xanthophyllomyces dendrorhous
ATCC MYA-131 (Xd-INV) was expressed in Pichia pastoris
as previously reported [12
]. Basically, the gene Xd-INV
(GenBank accession no. FJ539193.2) fused to the Saccharomyces cerevisiae
MFα secretion signal sequence was cloned in plasmid pIB4 (construction QDNS-pIB4) and included in P. pastoris
. Transformants were grown in 50 mL of Buffered Minimal Glycerol (BMG), yeast nitrogen base w/o amino acids 1.34%, biotin 4 × 10−5
%, glycerol 1%, 50 mM potassium phosphate buffer, pH 6.0) during 24 h and then in 400 mL of Buffered Minimal Methanol (BMM) the same as BMG but containing 0.5% methanol instead of glycerol) for 35 h, giving approximately 21 U/mL of β-fructofuranosidase activity per mL of culture. The extracellular β-fructofuranosidase activity (pXd-INV) was purified by tangential concentration followed by DEAE-Sephacel chromatography. Active fractions were concentrated using Microcon YM-10 (Amicon) filters (0.7 mL; 4220 U/mL; 5.8 mg/mL) and stored at −70 °C.
3.3. Entrapment of β-Fructofuranosidase in PVA Lenses
The PVA solution (10% w
) was prepared in 100 mM sodium acetate buffer (pH 5.0) at 90 °C under magnetic stirring for 45 min [53
]. Enzyme entrapment was carried out at room temperature by directly adding the enzyme to the PVA solution under magnetic stirring. Two different enzyme loadings were assayed (7.1 and 16.9 enzyme units per mL of PVA solution). The amount of enzyme was adjusted by diluting the enzyme stock solution (in 20 mM Tris-HCl pH 7.0) with 100 mM sodium acetate buffer (pH 5.0) and measuring the activity by the 3,5-dinitrosalicylic acid (DNS) assay. Lenses were produced by pumping the mixture through a syringe pump volume dispenser (NewERA model NE-300) into a 96-well microplate. Lenses were made by dripping 4 drops of the PVA solution in each well and then dried overnight at 50 °C. After that, lenses were hydrated in 100 mM sodium acetate buffer (pH 5.0) until constant weight. The average volume of each lens was calculated according to the total number of drops dispensed, the drops used for the production of each lens and the total volume of solution dispensed. Lens volume was determined using the following equation:
Lens volume (µL) = (volume dispensed/number of drops dispensed) × number of drops in each lens
3.4. Enzyme Activity Assay
β-Fructofuranosidase activity was determined by detection of reducing sugars with a modified 3,5-dinitrosalicylic acid (DNS) method adapted to a 96-well microplate scale [37
]. The reaction mixture contained 45 µL of a 100 mg/mL sucrose solution in 100 mM sodium acetate buffer (pH 5.0) and 5 µL of a conveniently diluted enzyme solution. The reaction was incubated at 50 °C for 20 min, and then stopped by adding 50 µL of 3,5-dinitrosalicylic acid (DNS). The quantification of reducing sugars was carried out with a calibration curve of d
-glucose, and one unit of activity (U) corresponded to the release of one µmol of reducing sugars per minute. The apparent activity of the immobilized biocatalysts was determined using a methodology developed in our group [36
]. Basically, the lens-shaped PVA particles was incubated with 500 µL of 100 g/L sucrose solution in a micro-centrifuge filter tube (Spin-X®
, 0.45 µm, Costar, Corning Inc., Corning, NY, USA) at the desired temperature for 60 min under vigorous agitation (900 rpm). The reaction mixture was separated from the biocatalyst by centrifugation at 2000× g
. Inactivation of the possible lixiviated enzyme was carried out by adding 500 µL of 0.4 M sodium carbonate. Finally, reducing sugars were measured by the DNS assay as described above.
3.5. Microscale Assay for the Operational Stability of Immobilized pXd-INV
The operational stability of the immobilized biocatalysts was assayed following a previously described microscale assay [36
]. One PVA lens was placed in a filtered micro-centrifuge tube (Spin-X®
, 0.45 µm, Costar, Corning Inc., Corning, NY, USA) with 500 µL of a 100 mg/mL sucrose solution. Reactions were carried out at 50 °C and pH 5.0 for 20 min. Centrifugation was carried out at 2000× g
to separate the lens from the reaction medium. The amount of reducing sugars was measured by the DNS method, as described before. The biocatalysts were washed three times with 100 mM sodium acetate buffer (pH 5.0) between cycles. Experiments were performed in triplicate to calculate the standard deviations.
3.6. Thermostability of the Immobilized PVA Particles
The thermostability of the lens-shaped PVA particles was analyzed by incubating the immobilized biocatalyst at different temperatures (4–60 °C) for 24 h in 100 mM sodium acetate buffer (pH 5.0). Residual activity was measured using the DNS assay under standard conditions (50 °C, pH 5.0). Experiments were performed in triplicate to calculate the standard deviations.
3.7. Analysis of Fructooligosacharides by HPAEC-PAD
The identification and quantification of FOS was carried out by High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD, Dionex ICS3000 system, Sunnyvale, CA, USA) and a CarboPack PA-1 column (4 × 250 mm) connected to a PA-1 guard column. The method was adapted from Campbell et al. [54
]. Initial mobile phase was 100 mM NaOH at 1 mL/min and it was maintained for 8 min. Then, a gradient from 100 to 88% 100 mM NaOH and from 0 to 12% 100 mM NaOH/600 mM sodium acetate was performed in 22 min. These conditions were kept for 6 min and then the eluents concentration was changed to 50% 100 mM NaOH and 50% 100 mM NaOH/600 mM sodium acetate. Eluents were degassed by flushing with helium and peaks were analyzed using Chromeleon software.
3.8. Production of Fructooligosacharides by Immobilized pXd-INV
Lenses of immobilized pXd-INV were added to a 600 mg/mL sucrose solution in 100 mM sodium acetate buffer, pH 5.0, until reaching a final activity of 1 U/mL. The reaction mixture was incubated at 30 °C in an orbital stirrer, and aliquots (100 µL) were taken out at different times and inactivated with 0.4 M Na2CO3. The formation of the different FOS was analyzed by HPAEC-PAD.
3.9. Operational Stability of Immobilized pXd-INV for Neo-FOS Production
The operational stability of the immobilized biocatalysts was assayed following neo-FOS production. One lens-shaped PVA particle (0.3 U) was placed in a filtered micro-centrifuge tube (Spin-X®, 0.45 µm, Costar, Corning Inc, Corning, NY, USA) with 340 µL of a 600 mg/mL sucrose solution in 100 mM sodium acetate buffer (pH 5.0). The mixture was incubated at 30 °C for 26 h in a Thermoshaker (model TS-100, bioSan, Nebikon, Switzerland) at 900 rpm. The tube was then centrifuged at 2000× g for 2 min to separate the supernatant. To inactivate any possible lixiviated enzyme, the supernatant was diluted with 340 µL of 0.4 M sodium carbonate solution. Samples were diluted 1:500 before analyzing the FOS composition by HPAEC-PAD as described before. Experiments were performed in triplicate to calculate the standard deviations. Between cycles, the lens-shaped biocatalysts were washed three times with 100 mM sodium acetate buffer (pH 5.0) followed by centrifugation at 2000× g for 2 min.