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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., Competing Interests: The authors declare they have no conflict of interest and have read and approved the manuscript., (© 2022 The Authors.)

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2':2,1'-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2':2,1'-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172)., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous work on Lolium perenne showed that sucrose:sucrose fructosyltransferase (SST) activity did not increase concomitantly with fructan synthesis in regrowing leaves or in mature leaf blades of plants that have been subjected to carbohydrate-accumulating conditions. This was contrary to the pattern of SST activity in roots and stubble. To obtain further insight into the fructan synthesizing activities and to explain this discrepancy, total fructosyltransferase activity (FT) was assayed by increasing the sucrose and the soluble enzyme concentrations and was compared to sedimentable phlein sucrase activity (PS) throughout the regrowth period following defoliation in leaves, stubble and roots. Before analysis on 2-month-old plants, PS activity was characterized in dark-grown coleoptiles, using [U-14C]sucrose. PS activity had a pH optimum of 6.0 and produced 1-kestose in addition to high molecular weight fructans with a mean DP of 9. In 2-month-old plants, sedimentable PS and FT soluble reactions contained an initial sucrose concentration of 160 mM and 400 mM and proteins equivalent to 1.4 and 2.1 g f. wt of tissue, respectively. In stubble and roots, the FT preparation catalysed the synthesis of large fructans, and the overall pattern resembled the native fructans when separated by anion exchange HPLC. In regrowing leaves, the FT preparation produced low-DP fructans relatively more than in vivo but synthesized the high-DP fructan characteristic of the tissue. Moreover, FT activity did not remain at a low level like SST activity but increased from day 5 after defoliation when fructans began to accumulate. PS activity formed very few low- DP fructans and 1-kestose was the main product. Trisaccharides generated by PS activity represented 2-5% of the total trisaccharide synthesis. High-DP fructans were detectable only when the products of the reaction were concentrated 100 times. Results are discussed with respect to the relative contribution of FT and PS activities for the synthesis of 1-kestose and fructans in roots, stubble and leaves of Lolium perenne. [ABSTRACT FROM AUTHOR]

Abstract Background Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. Results Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. Conclusions We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.

Accumulation of water-soluble carbohydrates was studied in leaf tissues of 8-wk-old plants of perennial ryegrass (Lolium perenne L. var. Bravo). The roots and leaf bases were cooled to low temperatures to reduce sink activity while source activity was enhanced by continuous illumination of the shoots. This resulted in accumulation of fructans, first in sheaths, subsequently in expanding leaves, and finally in blades. Fructan concentration increased from 6 to 23 mg g-1 f. wt in sheaths, from 8 to 30 mg g-1 f. wt in expanding leaves and from 6 to only 17 mg g-1 f. wt in mature leaf blades. Increase in concentration of low-DP fructans preceded accumulation of high-DP fructans. Expanding leaves accumulated significantly more glucose and fructose than did mature leaf tissue. Leaf blades contained a higher concentration of sucrose than either leaf sheaths or expanding leaves. Expanding leaves exhibited the greatest activity of SST (0.77 nkat g-1 f. wt), the mature leaf blades the least (0.09 nkat g-1 f. wt), whilst that of mature leaf sheaths was intermediate (0.27 nkat g-1 f. wt). In each leaf tissue, invertase activity was higher than SST activity. Under conditions of fructan accumulation, SST activity increased threefold in mature leaf sheaths and 1.2-fold in expanding leaves. In mature leaf blades, however, the activity remained constant at a low level. Invertase activity never increased. Inhibitors of protein synthesis (cycloheximide) and of RNA metabolism (actinomycin D) prevented any increase of SST activity in leaf sheaths. The results are discussed in relation to fructan metabolism in leaf tissues of Lolium perenne. [ABSTRACT FROM AUTHOR]

This paper presents a method for rapid quantification of sucrose and fructans by thin-layer chromatography (TLC) and densitometry. In the optimized protocol, glass-backed TLC plates were developed in a solution of propan-1-ol:ethyl acetate:water (45:35:20, v/v/v) for 3.5 h at 30°C. In this system, sucrose, the fructan trisaccharides: 1-kestose, 6G-kestose and 6-kestose, fructan tetrasaccharide and fructan pentasaccharide were separated. An internal standard of stachyose was useful for most of the applications examined. The fructans were detected by spraying with urea-phosphoric acid and heating at 110 °C for 6 min. The blue colour developed was stable for 18 h provided that plates were kept in the dark. Quantification was achieved by reflectance densitometry and comparison with standards. The standard curves for the oligosaccharides were not linear, owing to the spreading of spots on the TLC plates, but were highly repeatable. The TLC quantification method provided a more rapid analysis of sucrose and fructan oligosaccharides than was possible by high-performance liquid chromatography analysis because time-consuming pre-treatment of samples was not needed. The method will be useful for both enzyme and physiological studies. [ABSTRACT FROM AUTHOR]

This paper reports the conditions for the assay of sucrose-sucrose fructosyltransferase (SST); fructan-fructan fructosyltransferase (FFT), invertase and fructan hydrolase (FH) in extracts of leaves of Lolium rigidum Gaudin. Invertase had a temperature optimum between 30 and 35 °C and a pH optimum between 4.5 and 5.0 whereas the enzymes of fructan metabolism had temperature optima at 25 °C and pH optima at about 5.5. Analysis of the products of enzyme assays showed that crude SST activity produced mainly 1-kestose from 100 mM sucrose and when incubated with 500 mM sucrose, neokestose and possibly 6-kestose were also formed in low concentrations. FFT produced two tetrasaccharides and neokestose after incubation with 100 mM 1-kestose and FH produced fructose exclusively after incubation with high-molecular-weight fructan extracted from Lolium rigidum and Helianthus tuberosus L. and with a commercial preparation of bacterial levan. Enzyme activities were also measured during a phase of fructan accumulation. FH activity declined by 50% but SST and FFT activities increased about 2.5-fold during this period. The data supported the proposed synthetic pathway of fructan accumulation from sucrose via 1-kestose into high-molecular-weight fructan and indicated that fructan synthesis may occur at low substrate concentrations. Extracts from leaves which had not accumulated fructans contained measurable activities of SST and FFT. [ABSTRACT FROM AUTHOR]

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60. [ABSTRACT FROM AUTHOR]

• Regioselectivity of fructosyl transfer to puerarin varies between levansucrases. • Bs _SacB synthesizes puerarin-4'-O-β-D-fructofuranoside. • Gd _LsdA synthesizes β-D-fructofuranosyl-(2→6)-puerarin and linear oligofructosides. • Bs _SacB transforms β-D-fructofuranosyl-(2→6)-puerarin into polyfructosides. • Cascade Gd _LsdA/ Bs _SacB reactions yield polyfructosides directly from puerarin. Enzymatic glycosylation is an efficient way to increase the water solubility and the bioavailability of flavonoids. Levansucrases from Bacillus subtilis (Bs _SacB), Gluconacetobacter diazotrophicus (Gd _LsdA), Leuconostoc mesenteroides (Lm _LevS) and Zymomonas mobilis (Zm _LevU) were screened for puerarin (daidzein-8-C-glucoside) fructosylation. Gd _LsdA transferred the fructosyl unit of sucrose onto the glucosyl unit of the acceptor forming β- d -fructofuranosyl-(2→6)-puerarin (P1a), while Bs _SacB, Lm _LevS and Zm _LevU synthesized puerarin-4'- O -β-D-fructofuranoside (P1b) and traces of P1a. The Gd _LsdA product P1a was purified and assayed as precursor for the synthesis of puerarin polyfructosides (PPFs). Bs_ SacB elongated P1a more competently forming a linear series of water-soluble PPFs reaching at least 21 fructosyl units, as characterized by HPLC-UV-MS, HPSEC and MALDI-TOF-MS. Simultaneous or sequential Gd_ LsdA/ Bs_ SacB reactions yielded PPFs directly from puerarin with the acceptor conversion ranging 82–92 %. The bi-enzymatic cascade synthesis of PPFs in the same reactor avoided the isolation of the intermediate product P1a and it is appropriate for use at industrial scale. [ABSTRACT FROM AUTHOR]

There is a great interest in prebiotics consumptions eminence by their health promoting properties and economic market value. Filamentous fungi have been implicated as the key producers of fructosyltransferase (Ftase) and inulinase for synthesis of fructooligosaccharides (FOSs), which is used as a bioactive ingredient in functional foods. In this study, sixty-one indigenous coprophilous fungal strains were isolated, purified to monoculture and investigated for their potential use in biotransformation of sucrose and inulin into fructooligosaccharides (FOSs) and inulooligosaccharides (IOSs) by producing Ftase and inulinase enzymes, respectively. The molecular identification by 18S rDNA sequencing and morpho-taxonomic keys revealed that axenic fungal strains belonged to the genera Aspergillus, Neocosmospora , Trichoderma, Mucor and Fusarium. The crude enzyme extract from the isolates showed hydrolysis zones of 15–30 mm on the 2,3,5-triphenyltetrazolium chloride (TTC) and Lugol's iodine solution assay plates confirming the Ftase and inulinase activities, respectively. The submerged culture filtrates of eight fungal isolates showed high Ftase activity while six different fungal isolates exhibited high inulinase activity. The reaction products analysed with thin-layer chromatography and high-performance liquid chromatography coupled with refractive index detection indicated the presence of FOSs and IOSs. The strains isolated in this study have potential biotechnological implications to produce FOSs and IOSs. Image 1 • Here, sixty-one indigenous coprophilous fungal strains were isolated and purified. • The isolates were purified to monoculture and identified by 18S rDNA sequencing. • Identified as genera Neocosmospora , Trichoderma, Aspergillus, Mucor and Fusarium. • Fungi were able to produce Ftase and inulinase for synthesis of oligosaccharides. • TLC and HPLC analysis revealed isolates produced short-chain FOSs GF 3 , GF 4 and GF 5. [ABSTRACT FROM AUTHOR]

The market for prebiotics is gaining strength and is growing greatly in recent years since the consumers demand are changing and are highly influenced by the increasing consumption of healthier products. The fructosyltransferase (FTase) mainly produced by filamentous fungi is responsible for the synthesis of fructooligosaccharides which are widely used as a bioactive ingredient in functional foods. The objectives of this work were to select the best Aspergillus strains fructosyltransferase producer, purify FTase using ion-exchange chromatography coupled to FPLC under Superdex-G75 and to produce fructooligosaccharides. Six strains of Aspergillus sp. were studied, and A. tamarii Kita was highlighted because of the greater fructosyltransferase activity. At the production stage, the maximum activity of crude extract (1629.03 U/gds) was obtained under the following conditions: wheat bran 3.0g, sucrose concentration at 20%, 106 spore/mL, 70% moisture, 12h in the presence of light, at 30 °C for 48h. Superdex-G75 was able to purify the enzyme with high activity, and after SDS-PAGE under zymography conditions, one band of protein was observed at 89.7 kDa. The specific activity of the final purified material was 112,629.03 U/gds with purification ratio of 49 times and yield of 36%. FTase presented optimum temperature at 60 °C and pH 5.0, respectively. Together, these results indicated that the FTase of Aspergillus tamarii Kita is an excellent candidate for the industrial production of FOS. • Extracellular fructosyltransferase (FTase) (~89.7 kDa) from Aspergillus tamarii Kita was purified and characterized. • The purification of FTase was performed at room temperature on a fast protein liquid chromatography system. • The specific activity of the FTase was 112,629.03 U/gds with purification ratio of 49 times and yield of 36%. • FTase purified by A. tamarii presented optimum temperature at 60°C and pH 5.0, respectively. • FTase executed by A. tamarii generate economical production of fructooligosaccharides. [ABSTRACT FROM AUTHOR]

Fructooligosaccharides (FOS) have widely used for the manufacture of low-calorie and functional foods, because they can inhibit intestinal pathogenic microorganism growth and increase the absorption of Ca2+ and Mg2+. In this study, the novel fructosyltransferase (FTase) from Aspergillus oryzae strain S719 was successfully purified and characterized. The specific activity of the final purified material was 4200 mg−1 with purification ratio of 66 times and yield of 26%. The molecular weight of FTase of A. oryzae S719 was around 95 kDa by SDS-PAGE, which was identified as a type of FTase by Mass Spectrometry (MS). The purified FTase had optimum temperature and pH of 55 °C and 6.0, respectively. The FTase showed to be stable with more than 80% of its original activity at room temperature after 12 h and maintaining activity above 90% at pH 4.0–11.0. The Km and kcat values of the FTase were 310 mmol L−1 and 2.0 × 103 min−1, respectively. The FTase was activated by 5 mmol L−1 Mg2+ and 10 mmol L−1 Na+ (relative activity of 116 and 114%, respectively), indicating that the enzyme was Mg2+ and Na+ dependent. About 64% of FOS was obtained by the purified FTase under 500 g L−1 sucrose within 4 h of reaction time, which was the shortest reaction time to be reported regarding the purified enzyme production of FOS. Together, these results indicated that the FTase of A. oryzae S719 is an excellent candidate for the industrial production of FOS. [ABSTRACT FROM AUTHOR]

Given its potential role in the synthesis of novel prebiotics and applications in the pharmaceutical industry, a strong interest has developed in the enzyme levansucrase (LSC, EC 2.4.1.10). LSC catalyzes both the hydrolysis of sucrose (or sucroselike substrates) and the transfructosylation of a wide range of acceptors. LSC from the Gram-negative bacterium Erwinia tasmaniensis (EtLSC) is an interesting biocatalyst due to its high-yield production of fructooligosaccharides (FOSs). In order to learn more about the process of chain elongation, we obtained the crystal structure of EtLSC in complex with levanbiose (LBS). LBS is an FOS intermediate formed during the synthesis of longer-chain FOSs and levan. Analysis of the LBS binding pocket revealed that its structure was conserved in several related species. The binding pocket discovered in this crystal structure is an ideal target for future mutagenesis studies in order to understand its biological relevance and to engineer LSCs into tailored products. [ABSTRACT FROM AUTHOR]

Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5–4×105 times reduction of total sucrase activity. [Copyright &y& Elsevier]

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.

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 (A T ) was maximized by the experimental design in function of the reaction pHs and temperatures. The A T 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.

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.

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 6 G -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., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

An extracellular fructosyltransferase (Ftase) enzyme with a molar mass of ≈70 kDa from a newly isolated indigenous coprophilous fungus Aspergillus niger sp. XOBP48 is purified to homogeneity and characterized in this study. The enzyme was purified to 4.66-fold with a total yield of 15.53% and specific activity of 1219.17 U mg -1 of protein after a three-step procedure involving (NH 4 ) 2 SO 4 fractionation, dialysis and anion exchange chromatography. Ftase showed optimum activity at pH 6.0 and temperature 50 °C. Ftase exhibited over 80% residual activity at pH range of 4.0-10.0 and ≈90% residual activity at temperature range of 40-60 °C for 6 h. Metal ion inhibitors Hg 2+ and Ag + significantly inhibited Ftase activity at 1 mmol concentration. Ftase showed K m , v max and k cat values of 79.51 mmol, 45.04 µmol min -1 and 31.5 min -1 , respectively, with a catalytic efficiency ( k for the substrate sucrose. HPLC-RI experiments identified the end products of fructosyltransferase activity as monomeric glucose, 1-kestose (GF cat / K m ) of 396 µmol -1  min -1 for the substrate sucrose. HPLC-RI experiments identified the end products of fructosyltransferase activity as monomeric glucose, 1-kestose (GF 2 ), and 1,1-kestotetraose (GF 3 ). This study evaluates the feasibility of using this purified extracellular Ftase for the enzymatic synthesis of biofunctional fructooligosaccharides., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© King Abdulaziz City for Science and Technology 2020.)

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 2 3 -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., (Copyright © 2020 Elsevier B.V. All rights reserved.)