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BACKGROUND: Fructo‐oligosaccharide (FOS) belongs to the group of short inulin‐type fructans and is one of the most important non‐digestible bifid‐oligosaccharides capable of biotransforming sucrose using fructosyltransferase (FTase). However, there are no immobilized FTase products that can be successfully used industrially. In this study, diatomite was subjected to extrusion, sintering and granulation to form diatomaceous earth particles that were further modified via chitosan aminomethylation for modification. FTase derived from Aspergillus oryzae was successfully immobilized on the modified support via covalent binding. RESULTS: The immobilized enzyme activity was 503 IU g−1 at an enzyme concentration of 0.6 mg mL−1, immobilization pH of 7.0 and contact time of 3 h. Additionally, the immobilization yield was 56.91%. Notably, the immobilized enzyme was more stable under acidic conditions. Moreover, the half‐life of the immobilized enzyme was 20.80 and 10.96 times as long as that of the free enzyme at 45 and 60 °C, respectively. The results show good reusability, as evidenced by the 84.77% retention of original enzyme activity after eight cycles. Additionally, the column transit time of the substrate was 35.56 min when the immobilized enzyme was applied in a packed‐bed reactor. Furthermore, a consistently high FOS production yield of 60.68% was achieved and maintained over the 15‐day monitoring period. CONCLUSIONS: Our results suggest that immobilized FTase is a viable candidate for continuous FOS production on an industrial scale. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The whole cells of Aspergillus niger with high yield of fructosyltransferase were immobilized by using the embedding method. Then, the sodium alginate-chitosan was used as the carrier, and the optimum immobilization conditions were obtained by using the single factor and orthogonal experiment optimization, respectively. The single-factor optimization test results showed that: the optimal concentration of sodium alginate was 2%, the optimal quantity of bacteria embedded was 150 g·L−1, and the optimal concentrations of chitosan and CaCl2 were both 4%. The results of orthogonal test showed that when the concentration of sodium alginate was 3%, the quantity of bacteria embedded was 200 g·L−1, the concentration of chitosan was 5%, and the concentration of CaCl2 was 3%, the recovery of enzyme activity could reach the optimal 74.7%. The validation test showed that the immobilized cells were catalyzed for the synthesis of fructooligosaccharides in seven consecutive batches, and still had 96.85% residual activity, which had good potential for application and provided a new technical route for the synthesis of fructooligosaccharides. [ABSTRACT FROM AUTHOR]

Fructooligosaccharides (FOS) are fructose oligomers beneficial to human health and nutrition for prebiotic sugars. Their production occurs by a transfructosylation reaction in sucrose molecules catalyzed by fructosyltransferase enzymes (FTase, E.C.2.4.1.9) adhered to microbial cells. The purpose of this work was to study the preparation, enzymatic activity, and stability of glutaraldehyde-crosslinked Aspergillus oryzae IPT- 301 cells used as a biocatalyst for the transfructosylation reaction of sucrose in a packed bed reactor (PBR), aiming at FOS production. The highest transfructosylation activity (AT) was presented by the biocatalyst prepared by cross-linking at 200 rpm and 45 min. The highest AT in the PBR was obtained at 50 °C, with flow rates from 3 mL min-1 to 5 mL min-1 and sucrose concentrations of 473 g L-1 and 500 g L-1. The enzymatic kinetics was described using the Michaelis-Menten model. Finally, the biocatalyst showed constant AT of approximately 75 U g-1 and 300 U g-1 for 12 h of reaction in the PBR operating in continuous and discontinuous flow, respectively. These results demonstrate a high potential of glutaraldehyde- crosslinked A. oryzae IPT-301 cells as heterogeneous biocatalysts for the continuous production of FOS in PBR reactors. [ABSTRACT FROM AUTHOR]

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The recombinant sucrose:sucrose 1-fructosyltransferase from Schedonorus arundinaceus (Sa1-SSTrec) produces short-chainfructooligosaccharides (scFOS). In this work, Sa1-SSTrec was covalently or hydrophobic immobilized on Sepabeads® and ReliZyme™ supports. The possible clusters and the parameters related to the immobilization process were predicted by rational design of immobilized derivatives (RDID). Immobilization was performed at pH 7.0 and the three immobilized derivatives with higher experimental enzymatic activity (one of each epoxy-activated, amino-activated and hydrophobic supports: Sepabeads® EC-HFA/S, ReliZyme™ EA403/M and Sepabeads® EC-BU/M, respectively) were selected. The mismatch between some predictions and experimental results were explained by probable limitations of the RDID strategy. The thermodynamic parameters determined at 40, 50 and 60ºC, under not reactive conditions (i.e. in absence of sucrose), showed high thermal stability of Sa1-SSTrec after immobilization on the Sepabeads®EC-HFA/S. Batch experiments with this support, at 45ºC reached similar total scFOS (1-kestotriose and 1,1-kestotetraose) than the soluble enzyme, but scFOS productivity was higher for the immobilized biocatalyst. Operational stability of the immobilized biocatalyst after 4 cycles dropped to about 60% and 25% of its initial value at 30 and 45ºC, respectively, probably by denaturation at these temperatures. The obtained results suggest that the immobilized enzyme could be effectively exploited for industrial production of 1-kestotriose. [Display omitted] • A fructosyltransferase was efficiently immobilized on Sepabeads® and ReliZyme™. • Immobilization was predicted by rational design and compared to practical results. • Immobilization improved thermostability and allowed reusability. • Immobilization opens new opportunities for fructooligosaccharides production. [ABSTRACT FROM AUTHOR]

The increasing demand for healthier food products has prompted the food industry to seek innovative production methods. Fructo-oligosaccharides (FOS), valued for their prebiotic properties, have been incorporated into various foods. This study explores a novel approach involving the conversion of sucrose from strawberry preparations into FOS in situ. Four commercial enzyme blends, Viscozyme® L, Pectinex® Ultra SP-L, Catazyme® 25 L, and Novozym® 960, were assessed. Novozym® 960 exhibited the highest transferase activity (12.506 ± 0.053 U·mL−1), and under optimal reaction conditions (47.4 °C, pH 5.4, enzyme-to-substrate ratio of 1:99 v/v) produced a total FOS yield of 0.646 ± 0.006 gFOS·ginital sucrose −1 after a 1.25 h reaction. The industrial potential of this in situ process was demonstrated in a 1.5 L bioreactor, yielding a strawberry preparation containing 56.3 % w/w prebiotic FOS, with an 85.4 % reduction in sucrose content and an 18.8 % decrease in caloric value. While the consistency of the preparations was slightly altered, the phenolic content and antioxidant capacity remained unaffected by the enzymatic treatment. These findings highlight the promising application of in situ enzymatic treatment in developing products with prebiotic properties and reduced sugar and caloric content.

In the quest for maximum conversion of sucrose into 1-kestose using fructosyltransferases in a continuous reactor, consideration of the reversible reactions at competing concentrations of sucrose and 1-kestose is crucial. In this study, a kinetic model based on a combined approach of rapid equilibrium and steady-state kinetics was developed to predict the profile of FOS production using recombinant sucrose: sucrose 1-fructosyltransferase from Festuca arundinacea produced in Pichia pastoris. The purified enzyme selectively converted sucrose into 1-kestose with a final yield of 0.66 g/g sucrose consumed. The reaction rate laws adequately described the kinetic profile of the substrate and the products and accurately predicted the composition of the reaction mixture. The model was fitted to experimental data obtained at three enzyme dosages and three initial sucrose concentrations. The model could predict the effect of substrate concentration on initial rate kinetics, half-saturation constant, and transferase to hydrolase ratio. For the known values of initial sucrose and enzyme concentration, the optimum reaction time and the required dilution rate were predicted for the development of a continuous process to produce 1-kestose-enriched short-chain FOS. A volumetric productivity of ∼110 g/L/h of 1-kestose with 40 % carbohydrate composition in the permeate flux was demonstrated.

Inulosucrase from Lactobacillus reuteri 121 (LrInu) exhibits promise in the synthesis of prebiotic inulin and fructooligosaccharides. However, for its use in industry, LrInu's thermostability is a crucial consideration. In this study, the computational program FireProt was used to predict the thermostable variants of LrInu. Using rational criteria, nine variants were selected for protein expression and characterization. The G237P variant was determined to be the greatest designed candidate due to its greatly enhanced stability and activity in comparison to the wild-type enzyme. The optimum temperature of G237P increased from 50 to 60°C, with an over 5-fold increase in the half-life. Spectroscopy studies revealed that the G237P mutation could prevent the structural change in LrInu caused by heat or urea treatment. Molecular dynamics (MD) simulations showed that the enhanced thermostability of the G237P variant resulted from an increase in structural rigidity and the number of native contacts within the protein molecule. In addition, G237P variant synthesizes inulin with greater efficiency than WT. Key points: • Thermostable inulosucrase variant(s) were designed by Fireprot server. • G237P variant showed significantly improved thermostability compared to the wild type. • Inulin is synthesized more efficiently by G237P variant. [ABSTRACT FROM AUTHOR]

Fructans are fructose-based polymers, defined as fructooligosaccharides (FOS), when they possess a short chain. These molecules are highly appreciated in the food and pharmaceutical international market and have an increasing demand worldwide, mainly for their prebiotic activity and, therefore, for all their health benefits to those who consume them constantly. Thus, new natural or alternative FOS production systems of industrial scale are needed. In this regard, microorganisms (prokaryotes and eukaryotes) have the potential to produce them through a wide and diverse number of enzymes with fructosyltransferase activity, which add a fructosyl group to sucrose or FOS molecules to elongate their chain. Microbial fructosyltransferases are preferred in the industry because of their high FOS production yields. Some of these enzymes include levansucrases, inulosucrases, and β-fructofuranosidases obtained and used through biotechnological tools to enhance their fructosyltransferase activity. In addition, characterizing new microorganisms with fructosyltransferase activity and modifying them could help to increase the production of FOS with a specific degree of polymerization and reduce the FOS production time, thus easing FOS obtention. Therefore, the aim of this review is to compile, discuss, and propose new perspectives about the microbial potential for FOS production through enzymes with fructosyltransferase activity and describe the modulation of FOS production yields by exogenous stimuli and endogenous modifications. [ABSTRACT FROM AUTHOR]

Fructosyltransferases (FTs) are an important group of enzymes involved in the biosynthesis of fructans in some plants and in a large number of microorganisms. Several studies have shown that FTs play a key role in regulating the levels of fructan molecules during abiotic stress tolerance of certain plant species. Because of their diverse range of functional characteristics, efforts have increased to improve fructans applications in food quality and in added health benefits of plants. For these reasons, interest on the search for more direct and cost-effective routes to producing fructans have also increased. In this paper, we review recent research on plant FTs and summarize their role in the regulation of fructan biosynthesis and their use in producing fructans at an industrial scale. We also present the challenges and opportunities that FTs will present in the future.

Cooking with fire produces foods containing carbohydrates that are not naturally occurring, such as α-Dfructofuranoside found in caramel. Each of the hundreds of compounds produced by caramelization reactions is considered to possess its own characteristics. Various studies from the viewpoints of biology and biochemistry have been conducted to elucidate some of the scientific characteristics. Here, we review the composition of caramelized sugars and then describe the enzymatic studies that have been conducted and the physiological functions of the caramelized sugar components that have been elucidated. In particular, we recently identified a glycoside hydrolase (GH), GH172 difructose dianhydride I synthase/hydrolase (αFFase1), from oral and intestinal bacteria, which is implicated in the degradation of oligosaccharides in caramel. The structural basis of αFFase1 and its ligands provided many insights. This discovery opened the door to several research fields, including the structural and phylogenetic relationship between the GH172 family enzymes and viral capsid proteins and the degradation of cell membrane glycans of acid-fast bacteria by some αFFase1 homologs. This review article is an extended version of the Japanese article, Identification and Structural Basis of an Enzyme Degrading Oligosaccharides in Caramel, published in SEIBUTSU BUTSURI Vol. 62, p. 184-186 (2022). [ABSTRACT FROM AUTHOR]

Abstract Due to the health-promoting effects and functional properties of inulin-type fructooligosaccharides (I-FOS), the global market for I-FOS is constantly growing. Hence, there is a continuing demand for new, efficient biotechnological approaches for I-FOS production. In this work, crude inulosucrase InuGB-V3 from Lactobacillus gasseri DSM 20604 was used to synthesize I-FOS from sucrose. Supplementation with 1 mM CaCl2, a pH of 3.5–5.5, and an incubation temperature of 40 °C were found to be optimal production parameters at which crude inulosucrase showed high conversion rates, low sucrose hydrolysis, and excellent stability over 4 days. The optimal process conditions were employed in cell-free bioconversion reactions. By elevating the substrate concentration from 570 to 800 g L−1, the I-FOS concentration and the synthesis of products with a low degree of polymerization (DP) could be increased, while sucrose hydrolysis was decreased. Bioconversion of 800 g L−1 sucrose for 20 h resulted in an I-FOS-rich syrup with an I-FOS concentration of 401 ± 7 g L−1 and an I-FOS purity of 53 ± 1% [w/w]. I-FOS with a DP of 3–11 were synthesized, with 1,1-kestotetraose (DP4) being the predominant transfructosylation product. The high-calorie sugars glucose, sucrose, and fructose were removed from the generated I-FOS-rich syrup using activated charcoal. Thus, 81 ± 5% of the initially applied I-FOS were recovered with a purity of 89 ± 1%.

Fructosyltransferase was a key catalyst for the enzymatic production of fructooligosaccharides. The immobilized enzyme had the advantages of stable catalytic properties > repeated use and saving the enzyme cos匚 The study on the catalytic properties of immobilized fructosyltransferase could provide data basis for its industrial application. The fructosyltransferase was immobilized by using the embedding method with sodium alginate as the carrier. Then, the catalytic characteristics such as the optimum catalytic temperature, thermal stability, optimum reaction pH, pH stability and enzymatic kinetics were investigated. The application of immobilized fructosyltransferase in the catalytic synthesis of fructooligosaccharides was also preliminarily studied. The results showed that the optimum catalytic temperature of fructosyltransferase immobilized by the sodium alginate embedding method was 50°C, the pH value was 5. 0, and the michaelis-menten constant (K,n) of enzymatic kinetics was 0. 03 mg • mL_1. 48. 03% purity of fructooligosaccharides could be prepared by using the immobilized enzyme and sucrose as the substrate. Compared with the soluble enzyme, the sodium alginate immobilized fructosyltransferase had better compatibility with sucrose substrate, better temperature and pH stability, and had better potential for the industrial application. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fructosyltransferase (FTase) enzyme is an important biocatalyst for fructooligossaccaride (FOS) production, but it has low stability in soluble form. The aim of this work was to analyze the stability and kinetics of an fructosyltransferase (FTase E.C.2.4.1.9) from Aspergillus oryzae IPT‐301 immobilized on polyhydroxybutyrate (PHB) and glutaraldehyde‐activated polyhydroxybutyrate (GLU‐PHB). RESULTS: The immobilization yields of the FTase on PHB and GLU‐PHB were about 41% and 55%, respectively. The recovery activities of the FTase immobilized on PHB and GLU‐PHB were about 17% and 11%, respectively. At 30 °C, the FTase immobilized on GLU‐PHB showed a half‐life 2.6 and 1.4 times higher than the soluble FTase and the FTase immobilized on PHB, respectively. The FTase immobilized on PHB and GLU‐PHB retained more than 40% and 55% of their initial activity after six sequential reaction batches, respectively. Both immobilized enzymes showed kinetic behaviors that were fitted by the Hill model, with transfructosylation activity maximum at concentrations between 400 g substrate L−1 and 500 g substrate L−1. CONCLUSION: These results demonstrate that the FTase immobilized on PHB and, especially, on GLU‐PHB, are promising biocatalysts for FOS production in heterogeneous reaction systems. © 2022 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Fructans are fructose-based polymers, defined as fructooligosaccharides (FOS), when they possess a short chain. These molecules are highly appreciated in the food and pharmaceutical international market and have an increasing demand worldwide, mainly for their prebiotic activity and, therefore, for all their health benefits to those who consume them constantly. Thus, new natural or alternative FOS production systems of industrial scale are needed. In this regard, microorganisms (prokaryotes and eukaryotes) have the potential to produce them through a wide and diverse number of enzymes with fructosyltransferase activity, which add a fructosyl group to sucrose or FOS molecules to elongate their chain. Microbial fructosyltransferases are preferred in the industry because of their high FOS production yields. Some of these enzymes include levansucrases, inulosucrases, and β-fructofuranosidases obtained and used through biotechnological tools to enhance their fructosyltransferase activity. In addition, characterizing new microorganisms with fructosyltransferase activity and modifying them could help to increase the production of FOS with a specific degree of polymerization and reduce the FOS production time, thus easing FOS obtention. Therefore, the aim of this review is to compile, discuss, and propose new perspectives about the microbial potential for FOS production through enzymes with fructosyltransferase activity and describe the modulation of FOS production yields by exogenous stimuli and endogenous modifications.

Due to the health-promoting effects and functional properties of inulin-type fructooligosaccharides (I-FOS), the global market for I-FOS is constantly growing. Hence, there is a continuing demand for new, efficient biotechnological approaches for I-FOS production. In this work, crude inulosucrase InuGB-V3 from Lactobacillus gasseri DSM 20604 was used to synthesize I-FOS from sucrose. Supplementation with 1 mM CaCl2, a pH of 3.5–5.5, and an incubation temperature of 40 °C were found to be optimal production parameters at which crude inulosucrase showed high conversion rates, low sucrose hydrolysis, and excellent stability over 4 days. The optimal process conditions were employed in cell-free bioconversion reactions. By elevating the substrate concentration from 570 to 800 g L−1, the I-FOS concentration and the synthesis of products with a low degree of polymerization (DP) could be increased, while sucrose hydrolysis was decreased. Bioconversion of 800 g L−1 sucrose for 20 h resulted in an I-FOS-rich syrup with an I-FOS concentration of 401 ± 7 g L−1 and an I-FOS purity of 53 ± 1% [w/w]. I-FOS with a DP of 3–11 were synthesized, with 1,1-kestotetraose (DP4) being the predominant transfructosylation product. The high-calorie sugars glucose, sucrose, and fructose were removed from the generated I-FOS-rich syrup using activated charcoal. Thus, 81 ± 5% of the initially applied I-FOS were recovered with a purity of 89 ± 1%. [ABSTRACT FROM AUTHOR]

[Display omitted] • The erythritol titer was increased by 1.5 times by fermentation process optimization. • Co-expression of HGT1 and APC11 genes shortened the fermentation time by 16.7%. • The optimal conversion condition (60℃, pH 6) of FOS was achieved. • The highest FOS yield of 65% was obtained by surface display in Y.lipolytica. Currently, fructooligosaccharides (FOS) are converted from sucrose by purified enzymes or fungal cells, but these methods are costly and time-consuming. Here, the optimal fermentation conditions for strain E326 were determined through fermentation optimization: initial glucose 200 g/L, NaCl 25 g/L, inoculum volume 20 %, dissolved oxygen 20–30 %, pH 3, and glucose feeding concentration 100 g/L, which increased erythritol titer by 1.5 times. The co-expression of HGT1 and APC11 genes alleviated the erythritol synthesis stagnation, shorten the fermentation time by 16.7 %, and increased the erythritol productivity by 17.2 %. The episomal plasmids based on yeast mitochondrial replication origins (mtORIs) were constructed to surface display fructosyltransferase, effectively utilizing waste yeast cells generated during erythritol fermentation. Under the conditions of 60℃ and pH 6, the FOS yield reached 65 %, which to our best of knowledge is so-far the highest yield of FOS obtained. These findings will contribute to the industrial production of erythritol and FOS. [ABSTRACT FROM AUTHOR]

Maple syrup, a popular natural sweetener has a high content of sucrose, whose consumption is linked to different health issues such as obesity and diabetes. Hence, within this paper, the conversion of sucrose to prebiotics (fructo-oligosaccharides, FOS) was proposed as a promising approach to obtaining a healthier, value-added product. Enzymatic conversion was optimized with respect to key experimental factors, and thereafter derived immobilized preparation of fructosyltransferase (FTase) from Pectinex® Ultra SP-L (FTase-epoxy Purolite, 255 IU/g support) was successfully utilized to produce novel functional product in ten consecutive reaction cycles. The product, obtained under optimal conditions (60 °C, 7.65 IU/mL, 12 h), resulted in 56.0% FOS, 16.7% sucrose, and 27.3% monosaccharides of total carbohydrates, leading to a 1.6-fold reduction in caloric content. The obtained products' prebiotic potential toward the probiotic strain Lactobacillus plantarum 299v was demonstrated. The changes in physico-chemical and sensorial characteristics were esteemed as negligible. [Display omitted] • Maple syrup modification to value-added product with prebiotic potential. • In situ enzymatic transformation of sucrose to fructo-oligosaccharides (FOS). • Decrease in sucrose concentration (82%) led to a 1.6-fold reduction in caloric value. • Product retained original physico-chemical and sensorial properties. • Product remains stable for at least one year without FOS degradation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fructooligosaccharides (FOS) are prebiotic sugars used in the production of functional foods. These sugars can be produced by the transfructosylation reaction of sucrose catalyzed by fructosyltransferase enzymes (FTase, E.C.2.4.1.9). The aim of this work was to evaluate the use of cells of Aspergillus oryzae IPT‐301 as biocatalysts for the sucrose transfructosylation reaction in a packed bed reactor (PBR). RESULTS: The morphology of the cells was observed by scanning electron microscopy, and BET specific surface areas were examined by nitrogen physisorption The highest transfructosylation activity (660 U g−1) of the cells was obtained at 25 min of reaction by adopting a reaction temperature of 50 °C, sucrose concentration of 470 g L−1, and volumetric flow of 15 mL min−1. The rise in volumetric flow provided an increase in the transfructosylation activity, but also a faster deactivation of the biocatalyst. Enzyme kinetics was described using the Michaelis–Menten model, with a Vmax of 632.8 U g−1 and a km of 160.7 g L−1. The cells showed constant enzymatic activity for 12 h of reaction in the PBR operated with discontinuous flow at flow rates of 5 mL min−1 and 11.5 mL min−1, and in the PBR operated with continuous flow at a flow rate of 5 mL min−1. The transition from diffusional to kinetic regimes was observed starting from 11.5 mL min−1. CONCLUSION: The results obtained suggest a high potential of application of the whole A. oryzae IPT‐301 cells for continuous FOS production in PBR. © 2022 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

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 (GF 2), 1,1-kestotetraose (GF 3), and 1,1,1-kestopentaose (GF 4), produced by fructosyltransferases (FTases) or β-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 GF 2 without affecting that of GF 3. 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. • Fructosyltransferase (Ftase) from A. oryzae N74 heterologously expressed in K. phaffii GS115. • Three independent single-point mutations introduced into A. oryzae N74 FTase. • Mutation R163A virtually abolished transfructosylating activity. • FTase activity improved by introduction of mutations V242E or F254H. • FTase-V242E led to the highest production of 1-kestotriose. [ABSTRACT FROM AUTHOR]

In the present study, generation of prebiotic fructooligosaccharides (FOS) using Aspergillus tamarii FTase was optimized by applying response surface methodology. Optimal FOS (251 g L−1) was generated at 28.4°C, pH 7.0 and 50% (w/v) sucrose leading to 1.97‐fold yield enhancement. The m‐FTase was purified using ultrafiltration followed by HiTrap Q HP anion exchange chromatography resulting in 2.15‐fold purified FTase with 12.76 U mg–1 specific activity. Purified FTase (75 kDa) had Km and Vmax values of 1049.717 mM and 2.094 µmol min–1 mg–1, respectively. FOS incorporation led to upregulation of caspase 3, caspase 9, and Bax genes suggesting mitochondrial apoptosis activation in cancer cells. The study describes characteristics of purified FTase from A. tamarii, production optimization of FOS and unravels the role of FOS in anticancer activity against HT‐29 cells. Practical Application: This study provides detailed insights of kinetic and thermodynamic characteristics of purified FTase, a prebiotic FOS‐generating enzyme. Moreover, the role of the apoptotic genes involved in anticancer activity, and the prebiotic potential of FOS is also investigated. These findings are important in the context of FOS applications, and the optimized production strategies make it useful for industrial application. [ABSTRACT FROM AUTHOR]

The obvious benefits of employing prebiotics as functional components in many foods and feed products have resulted in higher demand for their industrial production, necessitating the development of more efficient and cost-effective manufacturing procedures. As a result, the goal of this study was to synthesize confirmed prebiotics, namely fructo-oligosaccharides (FOS), using sucrose as a substrate, since it allows the synthesis of oligosaccharides with lower polymerization degree, and consequently, a more pronounced prebiotic effect. Due to its availability, low market price, and high stability under industrial conditions, a commercial enzymatic mixture, Pectinex® Ultra SP-L, is used as a source of enzyme - fructosyltransferase (FTase). By varying key experimental conditions such as pH, temperature, enzyme and substrate concentrations, as well as the duration of the process, the composition of the FOS mixture can be adjusted to fit the potential applications. It was found that by performing the reaction in an aqueous medium (pH 7), at a temperature of 50 °C using an enzyme concentration of 1% (v/v) and any sucrose concentration in the range of 200-700 g/L, it was possible to achieve maximum FOS yield of 60% of total carbohydrates within a 24 h. The produced syrup with a high content of FOS can be further used as an adequate food additive, or else, developed processes should be used for the transformation of various food products (such as juices, jams, fillings, candies, cakes, etc.) in which sucrose dominates, creating products with lower caloric and higher functional value.

Functional foods are essential food products that possess health-promoting properties for the treatment of infectious diseases. In addition, they provide energy and nutrients, which are required for growth and survival. They occur as prebiotics or dietary supplements, including oligosaccharides, processed foods, and herbal products. However, oligosaccharides are more efficiently recognized and utilized, as they play a fundamental role as functional ingredients with great potential to improve health in comparison to other dietary supplements. They are low molecular weight carbohydrates with a low degree of polymerization. They occur as fructooligosaccharide (FOS), inulooligosaccharadie (IOS), and xylooligosaccahride (XOS), depending on their monosaccharide units. Oligosaccharides are produced by acid or chemical hydrolysis. However, this technique is liable to several drawbacks, including inulin precipitation, high processing temperature, low yields, and high production costs. As a consequence, the application of microbial enzymes for oligosaccharide production is recognized as a promising strategy. Microbial enzymatic production of FOS and IOS occurs by submerged or solid-state fermentation in the presence of suitable substrates (sucrose, inulin) and catalyzed by fructosyltransferases and inulinases. Incorporation of FOS and IOS enriches the rheological and physiological characteristics of foods. They are used as low cariogenic sugar substitutes, suitable for diabetics, and as prebiotics, probiotics and nutraceutical compounds. In addition, these oligosaccharides are employed as anticancer, antioxidant agents and aid in mineral absorption, lipid metabolism, immune regulation etc. This review, therefore, focuses on the occurrence, physico-chemical characteristics, and microbial enzymatic synthesis of FOS and IOS from coprophilous fungi. In addition, the potential health benefits of these oligosaccharides were discussed in detail.

Fructosyltransferases (FTases) are drawing increasing attention due to their application in prebiotic fructooligosaccharide (FOS) generation. FTases have been reported to occur in a variety of microorganisms but are predominantly found in filamentous fungi. These are employed at the industrial scale for generating FOS which make the key ingredient in functional food supplements and nutraceuticals due to their bifidogenic and various other health-promoting properties. This review is aimed to discuss recent developments made in the area of FTase production, characterization, and application in order to present a comprehensive account of their present status to the reader. Structural features, catalytic mechanisms, and FTase improvement strategies have also been discussed in order to provide insight into these aspects. Although FTases occur in several plants and microorganisms, fungal FTases are being exploited commercially for industrial-scale FOS generation. Several fungal FTases have been characterized and heterologously expressed. However, considerable scope exists for improved production and application of FTases for cost-effective production of prebiotic FOS. Fructosyltrasferase (FTase) is a key enzyme in fructo-oligosaccharide (FOS) generation Developments in the production, properties, and functional aspects of FTases Molecular modification and immobilization strategies for improved FOS generation Fructosyltransferases are innovation hotspots in the food and nutraceutical industries [ABSTRACT FROM AUTHOR]

Several archaea harbor genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure–function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition, and pharmaceutical applications. In this study, 3D structure of an inulin‐type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form and with bound substrate (sucrose) molecule and first transglycosylation product (1‐kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five‐bladed β‐propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram‐negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram‐positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram‐negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1‐kestose indicates that the residues D287 in the 4B‐4C loop, Y330 in 4D‐5A, and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure–function studies of FTF enzymes, particularly those from archaea. [ABSTRACT FROM AUTHOR]

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: β-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 β-galactosidases and fructosyltransferases, but there is still great place for innovative developments. [ABSTRACT FROM AUTHOR]

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 sugarcanemolasses. 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. [ABSTRACT FROM AUTHOR]

The global demand for therapeutic prebiotics persuades the quest for novel exopolysaccharides that can retard the growth of pathobionts and healthcare-associated pathogens. In this regard, an exopolysaccharide (3.69 mg/mL) producing strain showing prebiotic and antibiofilm activity was isolated from indigenous pineapple pomace of Tripura and identified as Bacillus subtilis PR-C18. Zymogram analysis revealed EPS PR-C18 was synthesized by levansucrase (∼57 kDa) with a maximal activity of 4.62 U/mg. Chromatography techniques, FTIR, and NMR spectral data revealed the homopolymeric nature of purified EPS with a molecular weight of 3.40 × 104 Da. SEM and rheological study unveiled its microporous structure and shear-thinning effect. Furthermore, EPS PR-C18 showed remarkable emulsification, flocculation, water retention, water solubilization, and antioxidant activity. DSC-TGA data demonstrated its high thermostability and cytotoxicity analysis verified its nontoxic biocompatible nature. In addition, the antibiofilm activity of EPS PR-C18 was validated using molecular docking, molecular simulation, MM-GBSA and PCA studies, which exhibited its strong binding affinity (−20.79 kcal/moL) with PelD, a virulence factor from Pseudomonas aeruginosa. Together, these findings support the future exploitation of EPS PR-C18 as an additive or adjuvant in food and pharmaceutical sectors. [Display omitted] [ABSTRACT FROM AUTHOR]

In this study, the preparation, characterization, and evaluation of the cross-linked enzyme aggregates (CLEAs) of fructosyltransferase isolated from the mutant-type Aureobasidium pullulans NAC8 for the production of fructooligosaccharides (FOS) with prebiotic properties were investigated. The reaction conditions were optimized statistically by using response surface methodology. CLEAs were characterized by using both photo-microscopy and scanning electron microscopy energy-dispersive X-ray spectroscopy (SEM-EDX). The secondary structure of the protein was predicted by using Fourier transform infrared spectroscopy (FTIR). FOSs produced as a result of the biotransformation of sucrose was quantified by using a chemometric analysis of FTIR spectral data. The prepared FOSs were investigated for the prebiotic effects under anaerobic conditions using a standard strain of Lactobacillus sp. Following were the statistically determined, optimum conditions for the production of FOSs: 5% (v/v) glutaraldehyde, pH 5.5, and a temperature of 32°C. The values of R 2 , F value, adjusted precision, and coefficient of variation were found to be 0.94, 11.50, 9.34, and 35.34%, respectively. Validation of the model resulted in the reusability of the CLEAs in six reaction cycles as it retained 70% of the residual activity. The SEM-EDX and photomicrographic analysis showed that the sizes of CLEAs varied, which ranged between 10 and 40 μm. Aggregation leads to the formation of a higher number of beta sheets in the CLEAs than that in the free enzyme. The total FOSs produced by the CLEAs was around 57% (w/v). A. pullulans FOS supplemented to the medium had a stimulatory effect on the growth of bi dobacteria compared with the control medium. The growth curves of Lactobacillus sp. reached the plateau phase afteR 2 4 h of cultivation with FOS. The CLEA of fructosyltransferase catalyzed the biocatalysis of sucrose to FOSs, a process with a biotechnological importance as prebiotics.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Asparagus (Asparagus officinalis L.) accumulates inulin and inulin neoseries-type fructans in root, which are synthesized by three fructosyltransferases—sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan:fructan 6G-fructosyltransferase (6G-FFT, EC 2.4.1.243). Fructans in roots are considered as energy sources for emerging of spears, and it has been demonstrated that a gradual decrease in root fructan content occurs during the spear harvesting season (budding and shooting up period). However, the roles of certain three fructosyltransferases during the harvest season have not yet been elucidated. Here, we investigated the variation in enzymatic activities and gene expression levels of three fructosyltransferases and examined sugar contents in roots before and during the spear harvest period. Two cDNAs, aoft2 and aoft3, were isolated from the cDNA library of roots. The respective recombinant proteins (rAoFT2 and rAoFT3), produced by Pichia pastoris , were characterized: rAoFT2 showed 1-FFT activity (producing nystose from 1-kestose), whereas rAoFT3 showed 1-SST activity (producing 1-kestose from sucrose). These reaction profiles of recombinant proteins were similar to those of native enzymes purified previously. These results indicate that aoft2 and aoft3 encoding 1-FFT and 1-SST are involved in fructan synthesis in roots. A gradual downregulation of fructosyltransferase genes and activity of respective enzymes was observed in roots during the harvest period, which also coincided with the decrease in fructooligosaccharides and increase in fructose due to fructan exohydrolase activity. These findings suggest that downregulation of fructosyltransferases genes during harvest time may contribute to efficient degradation of fructan required for the emergence of spears. • Two cDNAs involved in fructan biosynthesis were isolated from asparagus cDNA library. • Properties of recombinant proteins were similar to native asparagus fructosyltransferases. • Expression of these genes was reduced in roots during asparagus spear harvest. • Fructosyltransferase activities were also reduced in roots during asparagus spear harvest. • Reduction of fructosyltransferases may indirectly help in fructan degradation. [ABSTRACT FROM AUTHOR]

Mutation S164A largely affects the transfructosylation properties of Bacillus subtilis levansucrase (SacB). The variant uses acceptors such as glucose and short levans with an average molecular weight of 7.6 kDa more efficiently than SacB, leading to the enhanced synthesis of medium and high molecular weight polymer and a blasto-oligosaccharide series with a polymerization degree of 2–10. A 3-fold increase in blasto-oligosaccharides yield is provoked by the modified interplay between the variant and glucose. Despite its modified product specificity, protein-carbohydrate and protein-protein interactions are still a major factor affecting size and distribution of levan molecular weight. This study highlights the importance of critical factors such as protein concentration in the analysis of wild-type and mutagenized levansucrases. Docking experiments with the crystal structures of SacB and variant S164A – the latter obtained at a 2.6 Å resolution – identified unreported potential binding subsites for fructosyl moieties on the surface of both enzymes. • SacB variant S164A favors the synthesis of blasto-oligosaccharides. • The presence of short levan improves SacB S164A mutant for transfructosylation. • Protein-protein and levan-protein interactions may be critical for levan elongation. • Mutation S164A specifically affects polymerization rate. • SacB Wt and variant S164A share polymer elongation mechanism. [ABSTRACT FROM AUTHOR]

A low molecular weight intracellular and extracellular fructosyltransferase was purified from the wild (Wt) and improved mutant-type (Mt) Aureobasidium pullulans and its characteristics and thermodynamic properties determined. The Wt had been previously genetically modified by chemical mutagenesis. The purified fructosyltransferases from the Wt and Mt had subunit molecular weights between 13.4 and 17.0 kDa, respectively. The pH of the purified fructosyltransferase from the Wt and Mt were within 4.0–5.5. From the KM values, the fructosyltransferase from Mt showed higher affinity for sucrose than Wt fructosyltransferase. The optimum temperature obtained for the Mt intracellular and extracellular fructosyltransferase were 80 and 70 °C, respectively, while the Wt extracellular and intracellular fructosyltransferase was 60 °C. Most metals enhanced fructosyltransferase activity in a concentration-dependent manner except for mercury. Triton X-100 and Tween-80 enhanced the fructosyltransferase activity. Organic solvents such as acetone, ethanol, methanol, toluene and dichloromethane enhanced the enzyme activity while dimethylformamide inhibited the enzyme. From the thermodynamic and kinetic parameters ( t 1 / 2 , Δ H * , Δ S * , Δ G *) Mt fructosyltransferase was more stable to thermal inactivation than the Wt fructosyltransferase. Hence, it can be concluded that, after strain improvement of the Wt, the purified Mt fructosyltransferase was more stable to organic solvents, surfactants, and thermal denaturation, etc. compared to the Wt. This makes Mt fructosyltransferase more useful mostly in the food industry than the Wt. [ABSTRACT FROM AUTHOR]

Pectinex Ultra SP-L, a food-grade enzymatic preparation with high transfructosylating activity, was covalently immobilized on chitosan beads. The highest immobilization yield (95.9%) was obtained using 4.0% glutaraldehyde for 60 min and 80rpm at 2 5 ± 1 CC. The immobilized biocatalyst showed good operational stability, being able to retain, after the third cycle of reuse, no less than 100.0 and 73.9% of starting hydrolytic and transfructosylating activities, respectively. Results of residual activity experiments allowed estimating, for irreversible them al inactivation of both free and immobilized enzyme, the activation energy (E*d = 234.3 and 242.2kj-mol-1), enthalpy (234.4 < ΔH*d < 234.2 kj-mol-1 and 239.4 <ΔH*d < 239.3 kj-mol-1), entropy (381.2 < ΔS*d < 379.8J-mol-1. K-1 and 390.4 < ΔS*d < 389.7 J-mol-1) and Gibbs free energy (109.3 < ΔG*d < 103.9 kj-mol-1 and 111.3 < ΔG*d < 103.6 kJ-mol). The use of the immobilized enzyme in a packed bed reactor allowed obtaining, after 100 min, a fructooligosaccharide mixture containing more than 25 gL-1 1-kestose. [ABSTRACT FROM AUTHOR]

Pectinex Ultra SP-L, a commercial enzyme preparation with fructosyltransferase activity, was successfully immobilized by covalent binding to Fe 3 O 4 –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. • Pectinex was effectively immobilized on Fe 3 O 4 –chitosan-magnetic nanoparticles. • Immobilized biocatalyst easily separated of reaction medium by magnetic field. • Immobilized preparation presents good retention of activity after 6 reuses. • Immobilized enzyme preparation showed good thermostability. • FOS mixture mainly composed by 1-kestose was produced by immobilized preparation. [ABSTRACT FROM AUTHOR]

Purpose: A mutant BA-SacB-Del encoding BA-SacB minus K296-D320 region was constructed to analyze its effect on catalytic characteristics of the enzyme as well as help deepen understanding of the catalytic mechanism of BA-SacB and even proteins of GH68 family. Methods: Based on the comparison of levansucrases from Bacillus amyloliquefaciens (BA-SacB) and Sphingopyxis macrogoltabida (SM-Lev), a mutant BA-SacB-Del encoding BA-SacB minus K296-D320 region was constructed and its effect on catalytic characteristics of the enzyme was analyzed. Results: Deletion of this region would undoubtedly affect the conserved structure (i.e., central negatively charged cavity surrounded by five antiparallel β-strands) shared by the GH68 family. Therefore, Escherichia coli-expressing mutant BA-SacB-Del could more efficiently catalyze the production of levan in media containing high concentration of sucrose, which is unrealizable for BA-SacB. Conclusions: This result should be valuable for understanding this conditional lethal mechanism. Therefore, this study should be very valuable for understanding the catalytic mechanism of BA-SacB and even proteins of the GH68 family. More importantly, levan can be conveniently produced by direct fermentation of sucrose-containing media with E. coli-expressing BA-SacB-Del which is not sensitive to sucrose. [ABSTRACT FROM AUTHOR]

Levan is an enzymatically synthesized fructose polymer with widely reported structural heterogeneity depending on the producing levansucrase, the reaction conditions employed for its synthesis and the characterization techniques. We studied here the specific properties of levan produced by recombinant levansucrase from B. subtilis 168 (Bs -SacB), often characterized as a bimodal distribution, that is, a mixture of low and high molecular weight levan. We found significant differences between both levans in terms of the already reported molecular weight, size and morphology using different analytical methods. The low molecular weight levan consists of a non-uniform polymer ranging from 50 to 230 kDa, synthesized through a non-processive mechanism that can spontaneously form spherical nanoparticles in the reaction medium. In contrast, high molecular weight levan is a uniform polymer, most probably synthesized through a processive mechanism, with an average molecular weight of 30,750 kDa and a poorly defined nano-structure. This is the first report exploring differences in morphology between low and high molecular weight levans. Our findings demonstrate that only the low molecular weight levan forms spherical nanoparticles in the reaction medium and that high molecular weight levan is mainly composed of a 33,000 kDa fraction with a microgel behavior. [Display omitted] [ABSTRACT FROM AUTHOR]

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.

In this study, various levels of ultra-high pressure (UHP) were combined with the enzymatic synthesis of the fructooligosaccharide (FOS) using Pectinex Ultra SP-L and inulinase. The combination enhanced the FOS yields up to 2.5- and 1.5-fold, respectively, compared to atmospheric condition (0.1 MPa). However, the enzymatic reaction was dependent on the levels of pressure, the reaction times, and the initial sucrose concentrations. The combined UHP and inulinase showed that the maximum FOS yield (71.81%) was obtained under UHP at 200 MPa for 20 min with 300 g/L of initial sucrose as a substrate, while the FOS yield (57.13%) using Pectinex Ultra SP-L was obtained under UHP at 300 MPa for 15 min with 600 g/L of initial sucrose as a substrate. The FOS composition produced by Pectinex Ultra SP-L under the UHP was 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4), whereas the FOS produced by inulinase composed of only GF2 and GF3. The combined UHP is a useful tool in the industrial application for FOS production. UHP activated the activity of Pectinex Ultra SP-L yet inactivated inulinase Pressure level, time, and sucrose concentration significantly affect FOS yields under UHP UHP enhanced FOS production with time-saving benefits within 15–20 min [ABSTRACT FROM AUTHOR]

Drawbacks of industrially-used fructosyltransferases (FTs) such as low optimum temperature and low fructooligosaccharides (FOS) yield necessitates the search for engineered FTs that are highly thermostable and active. With the availability of the first plant FT crystal structure from Pachysandra terminalis (PDB ID: 3UGH), computer-aided protein engineering of plant FT is now feasible. To obtain insights on the effect of specific mutations i.e. disulfide bridge introduction, wild-type and mutant FTs were subjected to a 15 μs Martini Coarse-grained Molecular Dynamics (CGMD) simulations at 303 K and 334 K. We report here the five mutants, M31C-Q49C, L144C-S193C, P34C-W300C, S219C-L226C and V470C-S498C with enhanced thermostabilities and/or activities relative to the wild type. Interestingly, M31C-Q49C, which is located within the catalytic-carrying blade of the catalytic domain, has an activity enhancement at both temperatures. At 334 K, three mutations, L144C-S193C, P34C-W300C and V470C-S498C, achieved thermostability relative to the wild type. Intriguingly, both activity and stability enhancement exhibited only at 334 K can be achieved provided that the mutation is located either on the catalytic-carrying residue blade of the catalytic domain or on the non-catalytic domain. Our results suggest that V470C-S498C and L144C-S193C are promising mutants and that domain-specific approach may be exploited to customize enzyme properties. Image 1 • Coarse grained MD simulations were implemented on five interesting mutants to determine the effect of the introduction of disulfide bridge to the enzyme's thermostability and activity properties. • Two promising mutants having both improved thermostability and activity located on the non-catalytic and catalytic domain, respectively. • A domain-specific approach should be exploited to come up with a customized enzyme properties. [ABSTRACT FROM AUTHOR]

Abstract Erwinia tasmaniensis is an epiphytic bacterium related to the plant pathogen Erwinia amylovora , the etiological agent of fire blight. In this study the levansucrase from E. tasmaniensis (EtLsc) has been compared with the homologous enzyme from E. amylovora (EaLsc). We characterized the enzymatic activity and compared the products profile of both enzymes by High Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detector (HPAEC-PAD). Moreover we determined the crystal structure of EtLsc to understand the structural peculiarity causing the different product profiles of the two homologues. EtLsc exhibits increased efficiency in the production of FOS, resulting in a better catalyst for biotechnological synthesis than EaLsc. Based on our results, we propose that the role of this enzyme in the life cycle of the two bacteria is most likely related to survival, rather than linked to pathogenicity in E. amylovora. [ABSTRACT FROM AUTHOR]

In this study, the preparation, characterization, and evaluation of the cross-linked enzyme aggregates (CLEAs) of fructosyltransferase isolated from the mutant-type Aureobasidium pullulans NAC8 for the production of fructooligosaccharides (FOS) with prebiotic properties were investigated. The reaction conditions were optimized statistically by using response surface methodology. CLEAs were characterized by using both photo-microscopy and scanning electron microscopy energy-dispersive X-ray spectroscopy (SEM-EDX). The secondary structure of the protein was predicted by using Fourier transform infrared spectroscopy (FTIR). FOSs produced as a result of the biotransformation of sucrose was quantified by using a chemometric analysis of FTIR spectral data. The prepared FOSs were investigated for the prebiotic effects under anaerobic conditions using a standard strain of Lactobacillus sp. Following were the statistically determined, optimum conditions for the production of FOSs: 5% (v/v) glutaraldehyde, pH 5.5, and a temperature of 32°C. The values of R², F value, adjusted precision, and coefficient of variation were found to be 0.94, 11.50, 9.34, and 35.34%, respectively. Validation of the model resulted in the reusability of the CLEAs in six reaction cycles as it retained 70% of the residual activity. The SEM-EDX and photomicrographic analysis showed that the sizes of CLEAs varied, which ranged between 10 and 40 µm. Aggregation leads to the formation of a higher number of beta sheets in the CLEAs than that in the free enzyme. The total FOSs produced by the CLEAs was around 57% (w/v). A. pullulans FOS supplemented to the medium had a stimulatory effect on the growth of bi dobacteria compared with the control medium. The growth curves of Lactobacillus sp. reached the plateau phase after 24 h of cultivation with FOS. The CLEA of fructosyltransferase catalyzed the biocatalysis of sucrose to FOSs, a process with a biotechnological importance as prebiotics. [ABSTRACT FROM AUTHOR]

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies. [ABSTRACT FROM AUTHOR]