|Content:||5,000 Units at 40oC|
|Formulation:||In 3.2 M ammonium sulphate|
|Stability:||> 4 years at 4oC|
|Synonyms:||fructan beta-fructosidase; beta-D-fructan fructohydrolase, beta-fructofuranosidase; beta-D-fructofuranoside fructohydrolase|
|Concentration:||Supplied at ~ 2,000 U/mL (40oC, pH 4.5 on kestose)|
|Expression:||Recombinant from Aspergillus niger|
EC 18.104.22.168: Hydrolysis of terminal, non-reducing (2,1)- and (2,6)-linked β-D-fructofuranose residues in fructans.
EC 22.214.171.124: Hydrolysis of terminal, non-reducing β-D-fructofuranoside residues in β-D-fructofuranosides.
~ 2,000 U/mg (60oC, pH 4.5 on kestose);
~ 1,000 U/mg (40oC, pH 4.5 on kestose)
|Unit Definition:||One Unit of exo-inulinase activity is defined as the amount of enzyme required to release one µmole of β-D-fructose reducing-sugar equivalents per minute from kestose (5 mg/mL) in sodium acetate buffer (100 mM), at pH 4.5 at 40oC.|
|Application examples:||Applications established in food industry for fructose syrup production and in the diagnostics industry for the measurement of fructans and inulins.|
High purity recombinant exo-Inulinase (Aspergillus niger) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
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Measurement of total fructan in foods by enzymatic/spectrophotometric method: Collaborative study.
McCleary, B. V., Murphy, A. & Mugford, D. C. (2000). Journal of AOAC International, 83(2), 356-364.
An AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measuring oligofructans and fructan polysaccharide (inulins) in mixed materials and food products. The sample is extracted with hot water, and an aliquot is treated with a mixture of sucrase (a specific sucrose-degrading enzyme), α-amylase, pullulanase, and maltase to hydrolyze sucrose to glucose and fructose, and starch to glucose. These reducing sugars are then reduced to sugar alcohols by treatment with alkaline borohydride solution. The solution is neutralized, and excess borohydride is removed with dilute acetic acid. The fructan is hydrolyzed to fructose and glucose using a mixture of purified exo- and endo-inulinanases (fructanase mixture). The reducing sugars produced (fructose and glucose) are measured with a spectrophotometer after reaction with para-hydroxybenzoic acid hydrazide. The samples analyzed included pure fructan, chocolate, low-fat spread, milk powder, vitamin tablets, onion powder, Jerusalem artichoke flour, wheat stalks, and a sucrose/cellulose control flour. Repeatability relative standard deviations ranged from 2.3 to 7.3%; reproducibility relative standard deviations ranged from 5.0 to 10.8%.Hide Abstract
Measurement of inulin and oligofructan.
McCleary, B. V. & Blakeney, A. B. (1999). Cereal Foods World, 44, 398-406.
Fructans are defined as any compound in which one or more fructosyl-fructose linkages constitute a majority of the linkages (1). This refers to polymeric material as well as to oligomers as small as disaccharide inulobiose. Fructans are widely distributed in the plant kingdom. They are present in monocotyledons, dicotyledons, and green algae. Fructans differ in molecular structure and in molecular weight. They may be classified into three main types, the inulin type, the levan (previously called phlein) type, and the graminan type (2). The inulin group consists of material that has mostly of exclusively the (2-1) fructosly-fructose linkage. Levan is material that contains mostly or exclusively the (2-6) fructosyl-fructose linkage. The graminan (or branched) type has both (2-1) and (2-6) fructosly-fructose linkages in significant amounts (e.g. graminan from Gramineae). The distribution of fructans in nature, and the production of fructooligosaccharides, such as neosugar, using fructosyltransferase, has been reviewed in a monograph (3). In the context of this article and the analytical procedure described, the term fructan relates only to inulin and graminan. The current analytical procedure has not been evaluated on levan.Hide Abstract
Measurement of inulin and inulin-degrading enzymes.
McCleary, B. V. (1998). “Proceedings of the Seventh Seminar on Inulin”, (A. Fuchs and A. Van Laere, Eds.), European Fructan Association, pp. 36-45.
A non-instrumental method for the measurement of fructan is described. The method simplifies fructan analysis, is easy to perform, uses standard laboratory equipment, and is accurate, reproducible and specific. The procedure employs highly purified and specific enzymes to hydrolyse sucrose, starch and fructans (inulins and graminan).Hide Abstract
Fructans - Analytical approaches to a fibre that ferments.
Blakeney, A. B., McCleary, B. V. & Mugford, D. C. (1997). Chemistry in Australia, 17-19.
Fructans are defined as any compound where one or more fructosyl-fructose linkages constitute a majority of the linkages. This refers to polymeric material as well as oligomers as small as the diasaccharide inulobiose. Material included in this definition may or may not contain attached glucose. The terms oligomer and polymer are used by fructan researchers to distinguish between materials that can be specifically characterised and those that can not. Fructans are widely distributed in the plant kingdom. They are present in monocotyledons, dicotyledons and in green algae.Hide Abstract
Diurnal patterns of growth and transient reserves of sink and source tissues are affected by cold nights in barley.
Barros, K. A., Esteves‐Ferreira, A. A., Inaba, M., Meally, H., Finnan, J., Barth, S., Davis, S. J. & Sulpice, R. (2020). Plant, Cell & Environment, 43(6), 1404-1420.
Barley is described to mostly use sucrose for night carbon requirements. To understand how the transient carbon is accumulated and utilized in response to cold, barley plants were grown in a combination of cold days and/or nights. Both daytime and night cold reduced growth. Sucrose was the main carbohydrate supplying growth at night, representing 50–60% of the carbon consumed. Under warm days and nights, starch was the second contributor with 26% and malate the third with 15%. Under cold nights, the contribution of starch was severely reduced, due to an inhibition of its synthesis, including under warm days, and malate was the second contributor to C requirements with 24-28% of the total amount of carbon consumed. We propose that malate plays a critical role as an alternative carbon source to sucrose and starch in barley. Hexoses, malate, and sucrose mobilization and starch accumulation were affected in barley elf3 clock mutants, suggesting a clock regulation of their metabolism, without affecting growth and photosynthesis however. Altogether, our data suggest that the mobilization of sucrose and malate and/or barley growth machinery are sensitive to cold.Hide Abstract
Immobilized Inulinase for the Continuous Conversion of Inulin in the Fluidized-Bed Reactor.
Hang, H., Cheng, X., Yan, F., Wang, C. & Sun, K. (2020). Catalysis Letters, 150, 1849-1855.
The feasibility of operation on a fluidized bed reactor (FBR) with inulinase imbedded in the gelatin alginate microspheres was investigated in order to improve the higher inulin conversion yields and enzyme stability than that of the previously obtained with the packed bed reactors. The operation processes were based on statistical analyses, the operational conditions of immobilized inulinase in a FBR system have been determined for immobilized enzyme load of 18 g, substrate concentration of 80 g/L, expansion ratio of 1.4 and substrate flow rate at 0.5 mL/min. According to the above-mentioned research parameters, the continuous fructose preparation with FBR system was sustainable for 10 days (240 h) and gained the productivity of 86.4 g/Ld. Compared with the previous results of the packed-bed reactor, the immobilized inulinase in the FBR system was applied in the inulin conversion, which appeared more effective. This study suggested that a system for the continuous and efficient enzymatic conversion of inulin in the FBR was founded, which could be potentially applicable for the scale-up production.Hide Abstract
Nijman, R., Liu, Y., Bunyatratchata, A., Smilowitz, J. T., Stahl, B. & Barile, D. (2018). Journal of Agricultural and Food Chemistry, 66(26), 6851–6859.
Oligosaccharides are known to affect the health of infants. The analysis of these complex molecules in (human) milk samples requires state-of-the-art techniques. This study analyzed the composition and concentration of oligosaccharides in early (day 3) and mature (day 42) human milk as well as in five different infant formula brands. The oligosaccharide content decreased in human milk from 9.15 ± 0.25 g/L at day 3 to 6.38 ± 0.29 g/L at day 42 of lactation. All formulas resulted to be fortified with galacto-oligosaccharides, with one also fortified with polydextrose and another with long-chain fructo-oligosaccharides. About 130 unique oligosaccharide structures were identified in the human milk samples, whereas infant formula contained less diversity of structures. The comparisons indicated that composition and abundance of oligosaccharides unique to human milk are not yet reproduced in infant formulas. The analytical workflow developed is suitable for the determination of prebiotic oligosaccharides in foods that contain diverse carbohydrate structures.Hide Abstract
Huazano-García, A. & López, M. G. (2017). Applied Biochemistry and Biotechnology, 1-10.
Recently, agavins (branched neo-fructans) of short degree of polymerization have shown beneficial effects on the health of both healthy and overweight individuals. Therefore, the aim of the present work was to investigate the potential use of Agave angustifolia agavins on the generation of branched fructooligosacharides (a-FOS). A. angustifolia agavins were hydrolyzed using exo-, endo-inulinase, and a mixture of both (25 and 75%, respectively). Exo- and the inulinase mixture degraded quickly the agavins in relation to endo-inulinase treatment. Only endo-inulinase and the inulinase mixture generated a-FOS formation. Endo-inulinase degraded 31% of agavins, yielding approximately 20% of a-FOS after 48 h, whereas the inulinase mixture hydrolyzed 33% of agavins in just 90 min, but only yielded 10% of a-FOS. These results suggest that agave plants could be an abundant raw material for a-FOS production, which might have a huge prebiotic potential as new branched fructooligosaccharides with many applications in the alimentary and pharmaceutical industry.Hide Abstract
Hang, H., Wang, C., Cheng, Y., Li, N. & Song, L. (2017). Applied Biochemistry and Biotechnology, 1-18.
A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40°C for the free enzyme and 5.0-5.5 and 45-50°C for the immobilized enzyme. The kinetic parameters of Vmax and Km were 5.24 µmol/min and 57.6 mg/mL for the free inulinase and 4.32 µmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45°C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.Hide Abstract
Anwar, M. A., Kralj, S., van der Maarel, M. J. E. C. & Dijkhuizen, L. (2008). Applied and Environmental Microbiology, 74(11), 3426-3433.
Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with β(2-6) and β(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, 13C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by 13C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55°C, with a drastic decrease at 60°C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.Hide Abstract