Rapid evaluation of 1-kestose producing β-fructofuranosidases from Aspergillus species and enhancement of 1-kestose production using a PgsA surface-display system

1-Kestose is a key prebiotic fructooligosaccharide (FOS) sugar. Some β-fructofuranosidases (FFases) have high transfructosylation activity, which is useful for manufacturing FOS. Therefore, obtaining FFases that produce 1-kestose efficiently is important. Here, we established a rapid FFase evaluation method using Escherichia coli that display different FFases fused to a PgsA anchor protein from Bacillus subtilis. E. coli cell suspensions expressing the PgsA-FFase fusion efficiently produce FOS from sucrose. Using this screening technique, we found that the E. coli transformant expressing Aspergillus kawachii FFase (AkFFase) produced a larger amount of 1-kestose than those expressing FFases from A. oryzae and A. terreus. Saturation mutagenesis of AkFFase was performed, and the mutant G85W was obtained. The E. coli transformant expressing AkFFase G85W markedly increased production of 1-kestose. Our results indicate that the surface display technique using PgsA is useful for screening of FFases, and AkFFase G85W is likely to be suitable for 1-kestose production.

Abbreviations: AkFFase: Aspergillus kawachii FFase; AoFFase: Aspergillus oryzae FFase; AtFFase: Aspergillus terreus FFase; FFase: β-fructofuranosidase; FOS: fructooligosaccharide; fructosylnystose: 1^F-β-fructofuranosylnystose     

Effective Isolation and Identification of Toluene-tolerant Pseudomonas Strains

A total of 1752 strains of osmophilic yeasts were isolated from honey and pollens. Forty-three strains of osmophilic yeasts produced polyols, among which 6 strains produced erythritol in good yields. On the other hand, 52 osmophilic yeasts converted sucrose to fructooligosaccharides, among which 8 strains produced both extra and intracellular β-fructofuranosidase, which converted sucrose to fructooligosaccharides. This investigation concluded that osmophilic yeasts converted sucrose not only to polyols, but also to fructooligosaccharides in good yields.     

Comparison of Fructooligosaccharide Utilization by Lactobacillus and Bacteroides Species

The utilization of 1-kestose (GF₂) and nystose (GF₃), the main components of fructooligosaccharides (FOS), by Lactobacillus and Bacteroides species was examined. Of seven Lactobacillus and five Bacteroides strains that utilized FOS, L. salivarius, L. rhamnosus, L. casei, and L. gasseri utilized only GF₂, whereas L. acidophilus and all the Bacteroides strains utilized both GF₂ and GF₃. Only the strains able to utilize both GF₂ and GF₃ had β-fructosidase activity in the culture supernatants. The culture supernatants of the Lactobacillus strains had higher β-fructosidase activity for GF₂ than for GF₃, whereas those of the Bacteroides strains had higher activity for GF₃ than for GF₂. Furthermore, β-fructosidase activity of the culture supernatants of the Lactobacillus cells grown in the GF₃ medium was much higher than that of the cells grown in the GF₂ medium, whereas the activity of the culture supernatants of the Bacteroides cells grown in the GF₃ medium was almost the same as that of the cells grown in the GF₂ medium. These results indicate that Lactobacillus species metabolize FOS in a different way from that of Bacteroides species.     

Structural analysis and optimised production of fructo- oligosaccharides by levansucrase from Acetobacter diazotrophicus SRT4

Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C-NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40°C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.     

Crystal structure of a β-fructofuranosidase with high transfructosylation activity from Aspergillus kawachii

β-Fructofuranosidases belonging to glycoside hydrolase family (GH) 32 are enzymes that hydrolyze sucrose. Some GH32 enzymes also catalyze transfructosylation to produce fructooligosaccharides. We found that Aspergillus kawachii IFO 4308 β-fructofuranosidase (AkFFase) produces fructooligosaccharides, mainly 1-kestose, from sucrose. We determined the crystal structure of AkFFase. AkFFase is composed of an N-terminal small component, a β-propeller catalytic domain, an α-helical linker, and a C-terminal β-sandwich, similar to other GH32 enzymes. AkFFase forms a dimer, and the dimerization pattern is different from those of other oligomeric GH32 enzymes. The complex structure of AkFFase with fructose unexpectedly showed that fructose binds both subsites ?1 and +1, despite the fact that the catalytic residues were not mutated. Fructose at subsite +1 interacts with Ile146 and Glu296 of AkFFase via direct hydrogen bonds.     

1-Kestose consumption during pregnancy and lactation increases the levels of IgA in the milk of lactating mice

To examine the effect of dietary supplementation with 1-kestose on the IgA levels in milk, BALB/c mice were fed diets with or without 5% 1-kestose during pregnancy and lactation. The total and specific IgA levels in the milk were measured at 7 and 14?days after delivery. A two-way ANOVA with repeated measures resulted in a significant effect of 1-kestose-supplementation on total IgA concentrations (p?<?0.05) and the level of anti-Bacteroides IgA (p?<?0.05). A significant positive correlation was found between the mean count of Bacteroides spp. in maternal feces and the total IgA concentration in maternal milk (r?=?0.55, p?<?0.05), suggesting a potential link between the gut and mammary gland immune system. In conclusion, this study demonstrated the effects of dietary prebiotics on milk IgA production.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor

* Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose. * Here we report on complexes between chicory (Cichorium intybus) 1-FEH IIa with the substrate 1-kestose and the inhibitors sucrose, fructose and 2,5 dideoxy-2,5-imino-D-mannitol. Comparisons with other family GH32 and 68 enzyme-substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1-FEH IIa). * Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H-linkage with the acid-base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1-kestose forms an intramolecular H-linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor. * The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose-inhibited FEHs show a K(i) (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Colonization of Aspergillus japonicus on synthetic materials and application to the production of fructooligosaccharides

The ability of Aspergillus japonicus ATCC 20236 to colonize different synthetic materials (polyurethane foam, stainless steel sponge, vegetal fiber, pumice stones, zeolites, and foam glass) and to produce fructooligosaccharides (FOS) from sucrose (165 g/L) is described. Cells were immobilized in situ by absorption, through direct contact with the carrier particles at the beginning of fermentation. Vegetal fiber was the best immobilization carrier as A. japonicus grew well on it (1.25 g/g carrier), producing 116.3 g/L FOS (56.3 g/L 1-kestose, 46.9 g/L 1-nystose, and 13.1 g/L 1-β-fructofuranosyl nystose) with 69% yield (78% based only in the consumed sucrose amount), giving also elevated activity of the β-fructofuranosidase enzyme (42.9 U/mL). In addition, no loss of material integrity, over a 2 day-period, was found. The fungus also immobilized well on stainless steel sponge (1.13 g/g carrier), but in lesser extents on polyurethane foam, zeolites, and pumice stones (0.48, 0.19, and 0.13 g/g carrier, respectively), while on foam glass no cell adhesion was observed. When compared with the FOS and β-fructofuranosidase production by free A. japonicus, the results achieved using cells immobilized on vegetal fiber were closely similar. It was thus concluded that A. japonicus immobilized on vegetal fiber is a potential alternative for high production of FOS at industrial scale.     

High temperatures limit plant growth but hasten flowering in root chicory (Cichorium intybus) independently of vernalisation

An increase in mean and extreme summer temperatures is expected as a consequence of climate changes and this might have an impact on plant development in numerous species. Root chicory (Cichorium intybus L.) is a major crop in northern Europe, and it is cultivated as a source of inulin. This polysaccharide is stored in the tap root during the first growing season when the plant grows as a leafy rosette, whereas bolting and flowering occur in the second year after winter vernalisation. The impact of heat stress on plant phenology, water status, photosynthesis-related parameters, and inulin content was studied in the field and under controlled phytotron conditions. In the field, plants of the Crescendo cultivar were cultivated under a closed plastic-panelled greenhouse to investigate heat-stress conditions, while the control plants were shielded with a similar, but open, structure. In the phytotrons, the Crescendo and Fredonia cultivars were exposed to high temperatures (35 °C day/28 °C night) and compared to control conditions (17 °C) over 10 weeks. In the field, heat reduced the root weight, the inulin content of the root and its degree of polymerisation in non-bolting plants. Flowering was observed in 12% of the heat stressed plants during the first growing season in the field. In the phytotron, the heat stress increased the total number of leaves per plant, but reduced the mean leaf area. Photosynthesis efficiency was increased in these plants, whereas osmotic potential was decreased. High temperature was also found to induced flowering of up to 50% of these plants, especially for the Fredonia cultivar. In conclusion, high temperatures induced a reduction in the growth of root chicory, although photosynthesis is not affected. Flowering was also induced, which indicates that high temperatures can partly substitute for the vernalisation requirement for the flowering of root chicory.