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Fructo-oligosaccharides (FOS) are one of the most well-studied and commercialized prebiotics. FOS can be obtained either by controlled hydrolysis of inulin or by sucrose transfructosylation. FOS produced from sucrose are typically classified as short-chain FOS (scFOS), of which the best known are 1-kestotriose (GF2), 1,1-kestotetraose (GF3), and 1,1,1-kestopentaose (GF4), produced by fructosyltransferases (FTases) or ß-fructofuranosidases. In previous work, FOS production was studied using the Aspergillus oryzae N74 strain, its ftase gene was heterologously expressed in Komagataella phaffii (Pichia pastoris), and the enzyme's tertiary structure modeled. More recently, residues that may be involved in protein-substrate interactions were predicted. In this study, the aim was to experimentally validate previous in silico results by independently producing recombinant wild-type A. oryzae N74 FTase and three single-point mutations in Komagataella phaffii (Pichia pastoris). The R163A mutation virtually abolished the transfructosylating activity, indicating a requirement for the positively charged arginine residue in the catalytic domain D. In contrast, transfructosylating activity was improved by introducing the mutations V242E or F254H, with V242E resulting in higher production of GF2 without affecting that of GF3. Interestingly, initial sucrose concentration, reaction temperature and the presence of metal cofactors did not affect the enhanced activity of mutant V242E. Overall, these results shed light on the mechanism of transfructosylation of the FTase from A. oryzae and expand considerations regarding the design of biotechnological processes for specific FOS production.

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Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

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OBJECTIVE: Extracellular fructosyltransferase (FTase, E.C.2.4.1.9) from Aspergillus oryzae IPT-301 was immobilized on silica gel by adsorption and biochemically characterized aiming at its application in the transfructosylation reaction of sucrose for the production of fructooligossaccarides (FOS). RESULTS: The transfructosylation activity (AT) was maximized by the experimental design in function of the reaction pHs and temperatures. The AT of the immobilized enzyme showed the kinetics behavior described by the Hill model. The immobilized FTase showed reuse capacity for six consecutive reaction cycles and higher pH and thermal stability than the soluble enzyme. CONCLUSION: These results suggest a high potential of application of silica gel as support for FTase immobilization aiming at FOS production.

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The highly demanding conditions of industrial processes may lower the stability and affect the activity of enzymes used as biocatalysts. Enzyme immobilization emerged as an approach to promote stabilization and easy removal of enzymes for their reusability. The aim of this review is to go through the principal immobilization strategies addressed to achieve optimal industrial processes with special care on those reported for two types of enzymes: ß-galactosidases and fructosyltransferases. The main methods used to immobilize these two enzymes are adsorption, entrapment, covalent coupling and cross-linking or aggregation (no support is used), all of them having pros and cons. Regarding the support, it should be cost-effective, assure the reusability and an easy recovery of the enzyme, increasing its stability and durability. The discussion provided showed that the type of enzyme, its origin, its purity, together with the type of immobilization method and the support will affect the performance during the enzymatic synthesis. Enzymes' immobilization involves interdisciplinary knowledge including enzymology, nanotechnology, molecular dynamics, cellular physiology and process design. The increasing availability of facilities has opened a variety of possibilities to define strategies to optimize the activity and re-usability of ß-galactosidases and fructosyltransferases, but there is still great place for innovative developments.

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OBJECTIVE: Fructooligosaccharides (FOS) are prebiotic substances that have been extensively incorporated in different products of food industry mostly for their bifidogenic properties and economic value. The main commercial FOS production comes from the biotransformation of sucrose and intracellular and extracellular microbial enzymes-fructosyltransferases (FTase). Aspergillus oryzae IPT-301 produces FTase. In order to increase its production, this study focuses on evaluating the effects of different agitation speed and aeration rates which affect yields in a stirred tank bioreactor. RESULTS: Agitation had more influence on cell growth than aeration. The maximum intracellular FTase activity and the volumetric productivity of total intracellular FTase were obtained at 800 rpm and 0.75 vvm, and reached values of 2100 U g-1 and 667 U dm-3 h-1, respectively. The agitation speed had a strong influence on the activity of extracellular FTase produced which reached the maximum amount of 53 U cm-3. The higher value of total activity obtained was 22,831 U dm-3 at 0.75 vvm and 800 rpm. CONCLUSION: Aeration rates and agitation speed showed strong influence upon the growth and production of fructosyltransferase from Aspergillus oryzae IPT-301 in media containing sucrose as carbon source. The control of aeration rate and agitation speed can be a valuable fermentation strategy to improve enzyme production.

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Pectinex Ultra SP-L, a commercial enzyme preparation with fructosyltransferase activity, was successfully immobilized by covalent binding to Fe3O4-chitosan- magnetic nanoparticles. Immobilization carried out according to a 23-full factorial design where glutaraldehyde concentration, activation time and time of contact between enzyme and support were selected as the independent variables and immobilization yield as the response. The highest immobilization yield (94.84%) was obtained using 3.0% (v/v) glutaraldehyde and activation and contact times of 180 and 30 min, respectively. The immobilized biocatalyst, which showed for both hydrolytic and transfructosylating activities optimum pH and temperature of 7.0 and 60 °C, respectively, retained 70 and 86% of them after 6 cycles of reuse. A kinetic/thermodynamic study focused on thermal inactivation of the immobilized construct indicated high thermostability at temperatures commonly used for fructo-oligosaccharides (FOS) production. Maximum FOS concentration obtained in lab-scale experiments was 101.56 g L-1, with predominant presence of 1-kestose in the reaction mixture. The results obtained in this study suggest that the immobilized-enzyme preparation may be effectively exploited for FOS production and easily recovered from the reaction mixture by action of a magnetic field.

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Aguamiel is a natural sap produced by some species of agave plants, such as Agave salmiana, A. atrovirens, or A. angustifolia. It is a product with a high concentration of fructose, glucose or sucrose, although its composition may vary depending on the season in which it is produced, and may also contain agave fructans (or agavins) or fructooligosaccharides (FOS). It has been reported that FOS can be produced by enzymes that act on sucrose or inulin, transfructosylating or hydrolyzing these materials, respectively. Due to the sugar content in aguamiel, the application of an enzymatic complex produced by Aspergillus oryzae DIA MF was carried out. This complex was characterized by 1-D electrophoresis SDS-PAGE, and its transfructosylation and hydrolysis activities were determined by HPLC. In order to determine the conditions at which the concentration of FOS in this beverage increased, kinetics were carried out at different temperatures (30, 50, and 70°C) and times (0, 1, 2, 3, 4, 5, 10, and 15 h). Finally, the antioxidant and prebiotic activities were evaluated. FOS concentration in aguamiel was increased from 1.61 ± 0.08 to 31.01 ± 3.42 g/ L after 10 h reaction at 30°C applying 10% enzymatic fraction-substrate (v/v). Antioxidant activity was highly increased (34.81-116.46 mg/eq Trolox in DPPH assay and 42.65 to 298.86 mg/eq Trolox in FRAP assay) and growth of probiotic bacteria was higher in aguamiel after the enzymatic treatment. In conclusion, after the application of the enzymatic treatment, aguamiel was enriched with FOS which improved antioxidant and prebiotic properties, so it can be used as a functional food.

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Enzymatic fructosylation of organic acceptors other than sugar opens access to the production of new molecules that do not exist in nature. These new glycoconjugates may have improved physical-chemical and bioactive properties like solubility, stability, bioavailability, and bioactivity. This review focuses on different classes of acceptors including alkyl alcohols, aromatic alcohols, alkaloids, flavonoids, and xanthonoids, which were tested for the production of fructoderivatives using enzymes from the glycoside hydrolase (GH) families 32 and 68 that use sucrose as donor substrate. The enzymatic strategies and the reaction conditions required for the achievement of these complex reactions are discussed, in particular with regard to the type of acceptors. The solubility and pharmacokinetic and antioxidant activity of some of these new ß-D-fructofuranosides in comparison is reviewed and compared with their glucoside analogs to highlight the differences between these molecules for technological applications.

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The aim of this work was to improve the production of fructosyltransferase (FTase) by Solid-State Fermentation (SSF) using aguamiel (agave sap) as culture medium and Aspergillus oryzae DIA-MF as producer strain. SSF was carried out evaluating the following parameters: inoculum rate, incubation temperature, initial pH and packing density to determine the most significant factors through Box-Hunter and Hunter design. The significant factors were then further optimized using a Box-Behnken design and response surface methodology. The maximum FTase activity (1347U/L) was obtained at 32°C, using packing density of 0.7g/cm(3). Inoculum rate and initial pH had no significant influence on the response. FOS synthesis applying the enzyme produced by A. oryzae DIA-MF was also studied using aguamiel as substrate.

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Fructooligosaccharides (FOS) production was carried out in a batch basket reactor with immobilized fructosyltransferase from Rhodotorula sp. from 500×10(3) g m(-3) of sucrose in 50 mM sodium acetate buffer at pH 6.0, 48 °C at 85 rpm and with an activity of 22.44×10(3) U m(-3). The experimental data were well adjusted to the mathematical model for FOS production using SIMULINK(®) (MATLAB(®)). The highest regression coefficient (R(2)>90%) and the lowest percentual residual standard deviation (%RSD<4.0) and chi-square (χ(2) <1.0) were obtained for sucrose (GF), kestose (GF2) and total FOS. The mass transfer coefficient (kL) was determined as 5.6×10(-5) m h(-1) and the diffusivity (DS) was 2.11×10(-11) m(2) s(-1). The best predicted FOS yield (after 96 h) was 60.62%, with an equivalent productivity of 3.16×10(3) g m(-3) h(-1). These results reaffirm the good potential of this enzyme for industrial application and, in addition, are in conformation to other studies conducted with the same enzyme from the same and different microbial sources.

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