Fructooligosaccharides (FOS) are leading prebiotics that help keep the gut healthy and aid wellness by stimulating the growth and activity of beneficial intestinal bacteria. The best-studied FOS are inulin-type FOS, mainly oligosaccharides with β-Fruf-(2→1)-Fruf linkages, including 1-kestose [β-Fruf-(2→1)-β-Fruf-(2↔1)-α-Glcp] and nystose [β-Fruf-(2→1)-β-Fruf-(2→1)-β-Fruf-(2↔1)-α-Glcp]. However, the properties of other types of FOS-levan-type FOS with β-Fruf-(2→6)-Fruf linkages and neo-type FOS with β-Fruf-(2→6)-Glcp linkages-remain ambiguous because efficient methods have not been established for their synthesis. Here, using site-saturation mutation of residue His79 of β-fructofuranosidase from Zymomonas mobilis NBRC13756, we successfully obtained a mutant β-fructofuranosidase that specifically produces neo-type FOS. The H79G enzyme variant loses the native β-Fruf-(2→1)-Fru-transfer ability (which produces 1-kestose), and instead has β-Fruf-(2→6)-Glc-transfer ability and produces neokestose. Its hydrolytic activity specific to the β-Fruf-(2↔1)-α-Glcp bond of neokestose then yields blastose [β-Fruf-(2→6)-Glcp]. The enzyme produces 0.4 M blastose from 1.0 M sucrose (80 % of the theoretical yield). The production system for blastose established here will contribute to the elucidation of the physiological functions of this disaccharide.

The present study focused on evaluating the sequential fermentation (SF) method for FFase production from Aspergillus tamarii URM4634 using soybean bran as substrate. The SF was performed using soybean bran as substrate at 72 h and 30 °C and the maximum hydrolytic activity (44.00 U mL-1), corresponding to an increase of 2.98-fold to about SmF using sucrose as substrate. Already the maximum transfructosylating activity was 26.10 U mL-1. The FFase presents maximum hydrolytic activity at pH 5.0-6.0 and transfructosylating at pH 6.0 and 60 °C for both enzyme activities. The enzyme showed a typical hydrolytic kinetic profile evidenced by more affinity by sucrose hydrolysis reaction than the fructosyl transfer one. From kinetic and thermodynamic data of thermal denaturation, it was observed that the enzyme presents suitable at 55 °C, evidenced by the large half-life (990.21 min) and D values (3289.41 min). The maximum release of reducing sugars (8.45 g L-1) was obtained in hydrolysis of 20% sucrose during 180 min. The results obtained for FFase production by SF proved that this method can be used satisfactorily for sucrose-degrading enzymes and can contribute to the development of the SF technique to produce different industrial-interest enzymes.

BACKGROUND: Aspergillus niger ATCC 20611 is an industrially important fructooligosaccharides (FOS) producer since it produces the β-fructofuranosidase with superior transglycosylation activity, which is responsible for the conversion of sucrose to FOS accompanied by the by-product (glucose) generation. This study aims to consume glucose to enhance the content of FOS by heterologously expressing glucose oxidase and peroxidase in engineered A. niger.
RESULTS: Glucose oxidase was successfully expressed and co-localized with β-fructofuranosidase in mycelia. These mycelia were applied to synthesis of FOS, which possessed an increased purity of 60.63% from 52.07%. Furthermore, peroxidase was expressed in A. niger and reached 7.70 U/g, which could remove the potential inhibitor of glucose oxidase to facilitate the FOS synthesis. Finally, the glucose oxidase-expressing strain and the peroxidase-expressing strain were jointly used to synthesize FOS, which content achieved 71.00%.
CONCLUSIONS: This strategy allows for obtaining high-content FOS by the multiple enzymes expressed in the industrial fungus, avoiding additional purification processes used in the production of oligosaccharides. This study not only facilitated the high-purity FOS synthesis, but also demonstrated the potential of A. niger ATCC 20611 as an enzyme-producing cell factory.

Inulin is widely used as a prebiotic and emerging as a priming compound to counteract plant diseases. We isolated inulin-degrading strains from the lettuce phyllosphere, identified as Bacillus subtilis and Priestia megaterium, species hosting well-known biocontrol organisms. To better understand their varying inulin degradation strategies, three intracellular β-fructofuranosidases from P. megaterium NBRC15308 were characterized after expression in Escherichia coli: a predicted sucrose-6-phosphate (Suc6P) hydrolase (SacAP1, supported by molecular docking), an exofructanase (SacAP2), and an invertase (SacAP3). Based on protein multiple sequence and structure alignments of bacterial glycoside hydrolase family 32 enzymes, we identified conserved residues predicted to be involved in binding phosphorylated (Suc6P hydrolases) or nonphosphorylated substrates (invertases and fructanases). Suc6P hydrolases feature positively charged residues near the structural catalytic pocket (histidine, arginine, or lysine), whereas other β-fructofuranosidases contain tryptophans. This correlates with our phylogenetic tree, grouping all predicted Suc6P hydrolases in a clan associated with genomic regions coding for transporters involved in substrate phosphorylation. These results will help to discriminate between Suc6P hydrolases and other β-fructofuranosidases in future studies and to better understand the interaction of B. subtilis and P. megaterium endophytes with sucrose and/or fructans, sugars naturally present in plants or exogenously applied in the context of defense priming.

Carbohydrate-active enzymes are accountable for the synthesis and degradation of glycosidic bonds among diverse carbohydrates. Fructosyl-transferases represent a subclass of these enzymes, employing sucrose as a substrate to generate fructooligosaccharides (FOS) and fructan polymers. This category primarily includes levansucrase (LS, EC 2.4.1.10), inulosucrase (IS, EC 2.4.1.9), and β-fructofuranosidase (Ffase, EC 3.2.1.26). These three enzymes possess a similar five-bladed β-propeller fold and employ an anomer-retaining reaction mechanism mediated by nucleophiles, transition state stabilizers, and general acids/bases. However, they exhibit distinct product profiles, characterized by variations in linkage specificity and molecular mass distribution. Consequently, this article comprehensively explores recent advancements in the catalytic characteristics, structural features, reaction mechanisms, and product specificity of levansucrase, inulosucrase, and β-fructofuranosidase (abbreviated as LS, IS, and Ffase, respectively). Furthermore, it discusses the potential for modifying catalytic properties and product specificity through structure-based design, which enables the rational production of custom fructan and FOS.

A great volume of carrots is discarded daily worldwide because they do not meet the required shape and size standards. However, they have the same nutritional characteristics as those commercialized, and can be used in different food products. Carrot juice is an excellent matrix for the development of functional foods with prebiotic compounds, such as fructooligosaccharides (FOS). In this work, the production of FOS in situ in carrot juice was evaluated using a fructosyltransferase from Aspergillus niger, produced by solid-state fermentation on carrot bagasse. The enzyme was partially purified 12.5-fold with a total yield of 93 %, and specific activity of 59 U/mg of protein by Sephadex G-105 molecular exclusion chromatography. It was identified by nano LC-MS/MS as a β-fructofuranosidase with a 63.6 kDa MW and it allowed obtaining a FOS yield of 31.6 % in carrot juice. The result was a prebiotic juice with a final concentration of 32.4 mg/mL of FOS. Using the commercial enzyme Viscozyme L a higher yield of FOS (39.8 %) was obtained in carrot juice, corresponding to a total amount of FOS of 54.6 mg/mL. This circular economy scheme allowed the obtention of a functional juice, that may contribute to improve health of consumers.

β-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and can be used in the production of invert sugar and fructo-oligosaccharides (FOS). This last is an important prebiotic extensively used in the food industry. In the present study, the FFase production by Aspergillus tamarii Kita UCP 1279 was assessed by solid-state fermentation using a mixture of wheat and soy brans as substrate. The FFase presents optimum pH and temperature at 5.0-7.0 and 60 °C, respectively. According to the kinetic/thermodynamic study, the FFase was relatively stable at 50 °C, a temperature frequently used in industrial FOS synthesis, using sucrose as substrate, evidenced by the parameters half-life (115.52 min) and D-value (383.76 min) and confirmed by thermodynamic parameters evaluated. The influence of static magnetic field with a 1450 G magnetic flux density presented a positive impact on FFase kinetic parameters evidenced by an increase of affinity of enzyme by substrate after exposition, observed by a decrease of 149.70 to 81.73 mM on Km. The results obtained indicate that FFases present suitable characteristics for further use in food industry applications. Moreover, the positive influence of a magnetic field is an indicator for further developments of bioprocesses with the presence of a magnetic field.

BACKGROUND: Filamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications.
RESULTS: A novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%.
CONCLUSIONS: This PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications.

Lactosucrose (LS) is a prebiotic trisaccharide enzymatically synthesized by transglycosylation from lactose and sucrose with beneficial health effect. The β-fructofuranosidase used for synthesis of LS was produced from Bacillus methanolicus LB-1, which was isolated from traditional rice wine. A maximal yield of 8.63 U/mL of the enzyme was obtained by fermentation with B. methanolicus LB-1 under the optimized conditions: 10 g/L of glucose, 5 g/L of yeast extract, initial medium pH at 7.0, 37 °C, 24 h. The enzyme was purified and identified by ammonium sulfate fractional precipitation, Sephadex G-75 gel filtration chromatography and LC-MS, and SDS-PAGE of the purified enzyme showed a major protein band at 45 kDa. Biosynthesis of LS was performed using the purified β-fructofuranosidase, and production of LS reached 110 g/L under the optimized reaction conditions: pH at 7.0, 37 °C, 6.0 U/g sucrose of enzyme, 15% of sucrose, 15% of lactose, 28 h. HPLC analysis of the reaction products showed a distinct peak for LS at about 30 min of elution, confirming that B. methanolicus LB-1 β-fructofuranosidase had effective transfructosylation activity. Therefore, this new microbial source of β-fructofuranosidase may be a candidate with potential application prospect in biosynthesis of prebiotic LS.

Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.