Hydrolysis Of Inulin Research Articles

Inulinase production from a new inulinase producer, Penicillium oxalicum BGPUP-4

Abstract The present study was carried out for the isolation and characterization of an efficient inulinase producing fungal strain. Out of 31 fungal isolates, only 16 strains showed the hydrolytic zone surrounding the fungal growth on an inulin-rich medium. Screening of 16 inulolytic fungal strains was carried out based on activity of enzyme on inulin and sucrose and their I/S ratio. Fungal isolate BGPUP-4 shown maximum inulinase activity (11.06 IU/mL) after 5 days of cultivation and the corresponding I/S ratio was 2.64. This I/S ratio confirmed the inulinase nature of the enzyme. Thin layer chromatography and densitometer scanning of the hydrolysate were obtained after hydrolysis of inulin by inulinases from 5 fungal strains having high enzyme activity. Micromorphological and macromorphological characteristics of the fungal isolate BGPUP-4 exhibiting maximum enzyme activity, revealed it to be Penicillium oxalicum . Furthermore, 18S rRNA gene sequencing and phylogenetic analysis confirmed the identification of the fungal strain. Out of various carbon sources tested, lactose (2%) in combination with inulin (1.5%, as an inducer) showed a stimulatory effect on inulinase production (19.14 IU/mL). Agitation mode of cultivation in shake-flask fermentations, supported maximum inulinase production (38.52 IU/mL) after 5 days of incubation. This is the first report on inulinase production from Penicillium oxalicum .

Endoinulinase production by a new endoinulinase producer Aspergillus tritici BGPUP6 using a low cost substrate

Endoinulinase is an inulolytic enzyme which is used for the production of fructooligosaccharides from inulin. A new endoinulinase producing fungal strain BGPUP6 was identified as Aspergillus tritici on the basis of its molecular characterization. Response surface methodology was used to optimize the endoinulinase production at shake-flask level from Aspergillus tritici BGPUP6 using raw Asparagus inulin as carbon source. Four independent variables (raw inulin, 2–4%; peptone, 0.3–0.7%; (NH4)H2PO4, 0.3–0.7% and pH, 4.5–6.5) were selected for the study based on the CCRD model of RSM. The other media supplements (FeSO4·7H2O, 0.001%; MgSO4·7H2O, 0.05% and KCl, 0.02%) were kept constant in the production medium, throughout the study. Endoinulinase production and biomass yield were selected as dependent responses. The optimal combination of media ingredients obtained from the study was 3% raw inulin, 0.5% peptone, 0.5% (NH4)H2PO4 and pH 5.5. Using the optimized media constituents, maximum endoinulinase production (25.01 IU/mL) and biomass yield (0.514g dry weight/50 mL) obtained were in good agreement with the predicted values. Crude enzyme produced was also used for the hydrolysis of inulin. The hydrolysate showed the presence of a mixture of fructooligosaccharides with varied degree of polymerization. This is the first report on the production of an endoinulinase from Aspergillus tritici.

Evaluation of oligosaccharide profiles in selected cooked tubers and roots subjected to in vitro digestion

The influence of the preparation method and the effect of in vitro digestion on the fructo-oligosaccharides (FOS) and malto-oligosaccharides (MALTOS) of sweet potato (Ipomoea batatas Lam), mandioquinha (Arracacia xanthorrhiza), white yam (Dioscorea alata L.), cassava (Manihot esculenta Crantz), and taro (Colocasia esculenta) were evaluated. Tubers/roots were cooked in two ways: whole/unpeeled and peeled/in cubes. In vitro digestion was performed on whole/unpeeled cooked vegetables. An oligosaccharide analysis was carried out by HPAE-PAD. The largest amounts of oligosaccharides were found in the whole/unpeeled cooked tubers/roots. Sweet potato showed the highest content of FOS (29.08 ± 1.60 mg/100 g) and the highest concentrations of MALTOS were observed in white yam (113.70 ± 3.41 mg/100 g). In vitro digestion promoted an increase in maltotriose in all tubers/roots. Sweet potato FOS were not changed by digestion. However, in mandioquinha, white yam, and cassava, FOS levels were higher after digestion. The increase in MALTOS after in vitro digestion may be associated with both the hydrolysis of oligosaccharides with a higher degree of polymerization and starch. FOS contents increased after digestion possibly by hydrolysis of inulin or release from the food matrix. Sweet potato, cassava, and white yams can contribute to an increased intake of prebiotics.

Highly efficient production of D-lactic acid from chicory-derived inulin by Lactobacillus bulgaricus.

Inulin is a readily available feedstock for cost-effective production of biochemicals. To date, several studies have explored the production of bioethanol, high-fructose syrup and fructooligosaccharide, but there are no studies regarding the production of D-lactic acid using inulin as a carbon source. In the present study, chicory-derived inulin was used for D-lactic acid biosynthesis by Lactobacillus bulgaricus CGMCC 1.6970. Compared with separate hydrolysis and fermentation processes, simultaneous saccharification and fermentation (SSF) has demonstrated the best performance of D-lactic acid production. Because it prevents fructose inhibition and promotes the complete hydrolysis of inulin, the highest D-lactic acid concentration (123.6±0.9g/L) with a yield of 97.9% was obtained from 120g/L inulin by SSF. Moreover, SSF by L. bulgaricus CGMCC 1.6970 offered another distinct advantage with respect to the higher optical purity of D-lactic acid (>99.9%) and reduced number of residual sugars. The excellent performance of D-lactic acid production from inulin by SSF represents a high-yield method for D-lactic acid production from non-food grains.

Immobilization of inulinase from Aspergillus niger on octadecyl substituted nanoporous silica: Inulin hydrolysis in a continuous mode operation

Abstract Inulinase hydrolyzes inulin and produce high fructose syrup in a single-step reaction. However, finding the best working conditions and the highest inulinase stability, especially in continuous mode operations are of critical value. Native (endo-inulinase (EC 3.2.1.7)) and chemically modified inulinases (endo-inulinase stabilized with pyridoxal 5′-phosphate/ascorbic acid in a semi-rational chemical modification approach) were immobilized on octadecyl substituted nanoporous silica (with the average pore size of 66 nm) by using dioxane to extend the hydrophobic interactions between inulinase refolding intermediates and the hydrophobic alkyl moieties of the support. Native and modified inulinases revealed their maximum surface hydrophobicity at 40% and 50% dioxane in water, respectively. Immobilized inulinases (native and modified) showed 7.89 and 3.73 fold increase in their half lives compared to the free native sample., higher reusability compared to free inulinases (native and modified) and also could retain their activities after 10 hydrolysis cycles in a continuous mode of experimental conditions.

Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60°C) was 5°C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

Hydrolysis kinetics of inulin by imidazole-based acidic ionic liquid in aqueous media and bioethanol fermentation

This article focused on the inulin-containing energy biomass, Jerusalem artichoke, to explore environment-friendly processes for bioethanol production. An imidazole-based acidic ionic liquid (VImaILs) was prepared as the catalyst of inulin hydrolysis in aqueous media. The hydrolysis kinetics was studied under different conditions. The kinetic parameters of hydrolysis by VImaILs and dilute sulfuric acid were estimated and compared. This work demonstrated that the hydrolysis rate of inulin into reducing sugars by VImaILs was obviously faster than that by the latter. The proposed kinetic model successfully predicted the inulin hydrolysis in wider ranges of experimental conditions. The hydrolysate was fermented into ethanol by Saccharomyces cerevisiae which activity was not inhibited by the VImaILs. The conversion efficiency of inulin-type sugars to ethanol was greater than 92.5% of the theoretical yield. And the ethanol production capacity reached 123.76g/(L). This system integrated the chemical and biological processes to prepare ethanol in an environment-friendly way.

NMR Study of the Hydrolysis and Dehydration of Inulin in Water: Comparison of the Catalytic Effect of Lewis Acid SnCl4 and Brønsted Acid HCl

Various NMR techniques were employed to study the catalytic performance of the Lewis acid SnCl4 and the Bronsted acid HCl in the conversion of inulin to value-added compounds by hydrolysis and subsequent dehydration. The hydrolysis of inulin was examined to reveal the catalytic abilities of SnCl4 besides its intrinsic acidity by in situ 1H and 13C NMR at 25 °C. The dehydration reaction of inulin with SnCl4 as catalyst was followed by high temperature in situ 1H NMR at 80 °C. The fructose moieties were dehydrated to 5-(hydroxymethly)furfural (5-HMF), but the glucose fragment of inulin was inactive for dehydration reaction under this condition. The formation of 5-HMF and its transformation into formic acid and levulinic acid through a rehydration reaction could be monitored by in situ NMR spectroscopy. Moreover, diffusion ordered spectroscopy NMR revealed that the Lewis acid ion, Sn4+ interacts with the inulin model compounds, i.e., sucrose and fructose. The synergistic effects of complexation and acidity f...

Development of heterogeneous preparation with inulinase for tubular reactor systems

We developed highly stable heterogeneous preparations of inulinase immobilized on the ion-exchange matrixes and show that such preparations effectively split the chemically pure inulin and the inulin contained in plant extracts.The optimal conditions of hydrolysis are created for the migration of inulin solution from top to bottom (the downsteam flow) through the column filled by immobilized compounds extracted from Kluyveromyces marxianus and Helianthus tuberosus with the flow rate of 3mL/min. For the bottom-up migration of the inulin solution (i.e. the upstream flow) through the column the flow rate of 5mL/min was found to be the most optimal for the hydrolysis.When the extract of H. tuberosus was allowed to pass through the column filled by the immobilized plant inulinase from top to bottom with the flow rate of 5–10mL/min, the conditions for hydrolysis appear to be the most optimal. The conditions for hydrolysis were also found to be favourable for the bottom-up moving of the H. tuberosus extract through the column for the flow rate of 3–5mL/min.For the yeasts inulinase immobilized on VION KN-1 the most optimal flow rates of the H. tuberosus extract in the reactor were found to be similar for the following rates of inulin flow, viz. 3mL/min for the downstream flow and 5mL/min for the upstream flow.A theoretical description of the process of the inulin hydrolysis has been proposed and experimentally verified, which allows predicting the time of the full hydrolysis at different operating conditions with an error not exceeding 20%. It could be applied for the design of continuous reactors.

Efficient Secretory Overexpression of Endoinulinase in Escherichia coli and the Production of Inulooligosaccharides.

Endoinulinase production was achieved by heteroexpression of endoinulinase-encoding gene from Aspergillus ficuum which is an eukaryotic organism in Escherichia coli BL21 (DE3). Further analysis demonstrated that the native signal peptide existed in inu2 gene lowered the enzyme expression level. To realize extracellular accumulation of target protein and improve its expression level, native signal peptide was substituted with pelB, ompC, and pelB fusing with the native signal peptides; then, the effects on endoinulinase production were investigated. As a result, E. coli A606-3, with replacement of pelB as its signal peptide, showed the highest endoinulinase enzyme activity (75.22U/mg). Also, it suggested that eukaryotic signal peptides have an inhibition on enzyme expression in prokaryotic organism. Moreover, the condition for inulooligosaccharide (IOS) production from inulin was optimized, and an IOS yield of 94.41% was achieved under the condition of 15% (w/v) inulin, purified endoinulinase dosage of 5U/g inulin, 55°C, and pH 4.6 for 24h. The major products of hydrolysis of inulin were identified as DP3 to DP7.

Efficient Conversion of Inulin to Inulooligosaccharides through Endoinulinase from Aspergillus niger.

Inulooligosaccharides (IOS) represent an important class of oligosaccharides at industrial scale. An efficient conversion of inulin to IOS through endoinulinase from Aspergillus niger is presented. A 1482 bp codon optimized gene fragment encoding endoinulinase from A. niger DSM 2466 was cloned into pPIC9K vector and was transformed into Pichia pastoris KM71. Maximum activity of the recombinant endoinulinase, 858 U/mL, was obtained at 120 h of the high cell density fermentation process. The optimal conditions for inulin hydrolysis using the recombinant endoinulinase were investigated. IOS were harvested with a high concentration of 365.1 g/L and high yield up to 91.3%. IOS with different degrees of polymerization (DP, mainly DP 3-6) were distributed in the final reaction products.

Synergistic effect between the recombinant exo-inulinase and endo-inulinase on inulin hydrolysis

An exo-inulinase gene from Kluyveromyces marxianus and an endo-inulinase gene from Aspergillus niger were cloned and expressed in Yarrowia lipolytica, respectively. Then, the purified recombinant exo-inulinase (rEXINU) and endo-inulinase (rENINU), either individual or combination with another, were used for hydrolysis of inulin and inulin in tuber meal of Jerusalem artichoke. After the analysis of the dynamic hydrolysis process, it was found that the rEXINU and the rENINU acted synergistically with each other, and the maximum degree of synergistic effect on the level of fructose formation (DSEFru) could be obtained with a molar ratio of the rEXINU to the rENINU at 1:1. Furthermore, the investigation of the hydrolysis product in the synergistic hydrolysis process showed that a wide range of oligosaccharides were first generated and then converted to fructose. Based on the enzyme kinetics, substrate specificity and product inhibition of the rEXINU and the rENINU, a synergism mechanism of the them for the inulin hydrolysis was proposed. During the process of the synergistic action, the rENINU preferentially degraded the inulin with high DP, but was inhibited by the produced oligosaccharides. Then the released oligosaccharides which were the optimum substrates of the rEXIUN were rapidly hydrolyzed by the enzyme to produce fructose and glucose. Meanwhile, this process relieved the inhibition of the produced oligosaccharides to the rENINU. Besides, in the synergistic hydrolysis of the inulin in tuber meal of Jerusalem artichoke, the depolymerization of the rENINU could result in the increase of the subtrate solubility to improve the hydrolysis efficiency. Therefore, this kind of the synergistic effect between the exo-inulinase and the endo-inulinase may provide a potential driving force in the industrial application of inulin and inulin-containing materials.

Biotechnological potential of microbial inulinases: Recent perspective

ABSTRACTAmong microbial enzymes, inulinases or fructo-furanosylhydrolases have received considerable attention in the past decade, and as a result, a variety of applications based on enzymatic hydrolysis of inulin have been documented. Inulinases are employed for generation of fructose and inulo-oligosaccharides (IOS) in a single-step reaction with specificity. The high fructose syrup can be biotransformed into value-added products such as ethanol, single cell protein, while IOS are indicated in nutraceutical industry as prebiotic. Myriad microorganisms produce inulinases, and a number of exo- and endo-inulinases have been characterized and expressed in heterologous hosts. Initially, predominated by Aspergilli, Penicillia, and some yeasts (Kluyveromyces spp.), the list of prominent inulinase producers has gradually expanded and now includes extremophilic prokaryotes and marine-derived microorganisms producing robust inulinases. The present paper summarizes important developments about microbial inulinases and their applications made in the last decade.

Modelación matemática del efecto de la temperatura en la actividad y la estabilidad térmica de la inulinasa de Kluyveromyces marxianus NRRL Y-7571

A mathematical model was used in this study to describe the effect of temperature on the activity and thermal stability (non-reactive conditions) of the inulinase of Kluyveromyces marxianus NRRL Y-7571. The model was taken from the literature, which was developed using the Arrhenius equation and experimental data. A first-order kinetic equation was takenfor enzyme inactivation. The model parameters were determined by linear and nonlinear regression, reporting confidence intervals. The experimental data showed that the maximum activity acting on sucrose and inulin was reached at 55 °C and that these activities were highly sensitive to higher temperatures. Furthermore, inulolytica activity was more thermally stable than the invertase activity in the range from 48 to 60 °C. Using the model, the process temperature for the hydrolysis of sucrose and inulin was determined at the intersection of the curves of the relative activity and relative half-life, resulting 49 °C for both processes. Although this model was used with reference to the sucrose and inulin hydrolysis, the approach is a useful tool that can be applied to other enzymatic processes for determining the operating temperature, which is ultimately determined by an economic evaluation.

Biocatalytic conversion of inulin and sucrose into short chain oligosaccharides for potential pharmaceutical applications

The production of fructooligosaccharides (FOS) and inulooligosaccharides (IOS) of varying degrees of polymerization (DP) was optimized by orthogonal experimental designs using central composite design (CCD) and response surface methodology (RSM). The FOS produced were quantified and maximal GF2, GF3 and GF4 were 211.09 mM, 156.06 mM and 43.99mM respectively at pH 5.6 and 60 °C. Maximal IOS produced were quantified and F3, F4 and F5 were 70.3 mM,38.8 mM and 12.43 mM respectively at pH 6.0 and 60 °C. The detection and quantification of oligosaccharides of specific chain length from inulin hydrolysis and sucrose elongation were carried out using high performance liquid chromatography with refractive index (HPLC-RI) detection, thin layer chromatography (TLC) and mass spectrometry with electrospray ionization (MS-ESI). FOS and IOS were ionized and detected in the positive ion mode using 1 mM LiCl, [M+Li]+. The FOS produced by the action of fructosyltransferases were monosaccharides (m/z=187.98), disaccharides (m/z=349.19), 1-kestose (GF2; m/z=511.25), nystose (GF3; m/z=673.27) and fructofuranosyl nystose (GF4; m/z=835.45). IOS produced were identified as fructose (F; m/z=187.01), inulobiose (F2; m/z=349.22) inulotrioses (F3; m/z=511.23), inulotetraoses (F4; m/z=673.29) and inulopentaoses (F5; m/z=835.22).

Mesoporous Sulfonated Carbon Materials Prepared by Spray Pyrolysis

AbstractA one‐step approach was developed for the production of mesoporous sulfonated carbon materials by means of an aerosol synthesis. Nebulizing a clear aqueous solution of sucrose and sulfuric acid through a heated oven leads to subsequent dehydration, carbonization and sulfonation of the carbohydrate structure, in less than two seconds residence time. Acid site concentrations ranging from 0.1 to 0.6 mmol g−1 can be obtained. Porosity can easily be introduced via salt templating, and can be adjusted by varying the loading and type of salt used. The highest surface area was obtained with Li2SO4, giving a BET surface area of 506 m2 g−1 and a mesopore size distribution between 2 and 8 nm. Fructose dehydration and inulin hydrolysis showed that the porous materials synthesized by salt templating are more active than the bulk ones, especially for inulin hydrolysis, for which the initial activity is enhanced by a factor of seven, making these materials competitive with the most active commercial resins.

Butanol production from inulin-rich chicory and Taraxacum kok-saghyz extracts: Determination of sugar utilization profile of Clostridium saccharobutylicum P262

Developing alternative applications for wastes stemming from the processing of industrially applicable crops adds value to industrial crops and limits agricultural land use. Kazak dandelion (Taraxacum kok-saghyz; TKS) and Chicory are rich in inulin. In this study, unhydrolyzed inulin-rich chicory and TKS extracts were assessed as substrates for acetone–butanol–ethanol (ABE) production, using different solventogenic Clostridium species/strains. Extracts from chicory and TKS were rich in inulin (∼42 and 36g/L respectively). In addition to inulin, the chicory extract also contained glucose (3.5g/L), sucrose (6.2g/L), fructose (19.5g/L), and kestose (6.0g/L), while the TKS extract contained fructose (28.3g/L), kestose (2.7g/L) and an unidentified sugar (2.0g/L). Among the species/strains tested, Clostridium saccharobutylicum P262 demonstrated superior ability to utilize inulin, with at least 86% and 153% higher cell growth and ABE concentration, respectively, on inulin relative to the other species/strains. With pure commercial inulin, TKS and chicory extracts, C. saccharobutylicum P262 utilized 29.0, 25.2 and 40.5g/L total sugars respectively, and produced 9.7, 8.5 and 12.5g/L ABE, respectively. However, when fructose, the major product of inulin hydrolysis was used as the sole substrate, cell growth and ABE production by C. saccharobutylicum P262 were at least 74% and 83% lower, respectively, relative to the other species tested. These results demonstrate the suitability of C. saccharobutylicum P262 for butanol production from unhydrolyzed plant inulin and also identify limited fructose utilization as a potential metabolic engineering target for improving the fermentability of unhydrolyzed inulin-rich plant extracts to butanol.

Rapid and efficient bioconversion of chicory inulin to fructose by immobilized thermostable inulinase from Aspergillus tubingensis CR16

Abstract Background Fructose, a monosaccharide, has gained wide applications in food, pharmaceutical and medical industries because of its favourable properties and health benefits. Biocatalytic production of fructose from inulin employing inulinase is the most promising alternative for fructose production. For commercial production, use of immobilized inulinase is advantageous as it offers reutilization of enzyme and increase in stability. In order to meet the demand of concentrated fructose syrup, inulin hydrolysis at high substrate loading is essential. Results Inulinase was immobilized on chitosan particles and employed for fructose production by inulin hydrolysis. Fourier transform infrared spectroscopy (FTIR) analysis confirmed linkage of inulinase with chitosan particles. Immobilized biocatalyst displayed significant increase in thermostability at 60 and 65 °C. Statistical model was proposed with an objective of optimizing enzymatic inulin hydrolytic process. At high substrate loading (17.5 % inulin), using 9.9 U/g immobilized inulinase at 60 °C in 12 h, maximum sugar yield was 171.1 ± 0.3 mg/ml and productivity was 14.25 g/l/h. Immobilized enzyme was reused for ten cycles. Raw inulin from chicory and asparagus was extracted and supplied in 17.5 % for enzymatic hydrolysis as a replacement of pure inulin. More than 70 % chicory inulin and 85 % asparagus inulin were hydrolyzed under optimized parameters at 60 °C. Results of high performance liquid chromatography confirmed the release of fructose after inulin hydrolysis. Conclusions The present findings prove potentiality of immobilized thermostable inulinase from Aspergillus tubingensis CR16 for efficient production of fructose syrup. Successful immobilization of inulinase on chitosan increased its stability and provided the benefit of enzyme reutilization. Box-Behnken design gave a significant model for inulin hydrolysis. Extraction of raw inulin from chicory and asparagus and their enzymatic hydrolysis using immobilized inulinase suggested that it can be a remarkable cost-effective process for large-scale fructose production.

An efficient method for the immobilization of inulinase using new types of polymers containing epoxy groups.

New glycidyl methacrylate copolymers containing different numbers of epoxy groups were synthesized and used to develop effective procedures for inulinase immobilization. The beneficial characteristics of the carriers included a high degree of crosslinking, stability at ambient temperature, an appropriate surface, and the presence of reactive epoxy groups. Some factors affecting the efficiency of immobilization of crude inulinase, including the kind and amount of carrier, the number of epoxy groups, as well as buffer pH and buffer concentration were examined. The yield of immobilization of this enzyme on the investigated type of microspheres was higher than on the commercial carrier, Eupergit® C. After immobilization, the optimum temperature for inulinase activity shifted from 55 to 45 °C, whereas the optimum pH = 5 remained unchanged. The basic parameters of inulin hydrolysis were examined, and the possibility of applying the obtained biocatalyst in continuous conditions was tested. Inulin at a concentration of 0.5 % (w/v) was almost completely hydrolyzed to fructose (in a yield of 98 %) at a flow rate of 0.1 mL/min. A tenfold increase in the speed of flow resulted in an increase in the yield of oligosaccharides (DP2-DP6) up to ~41 % in the overall hydrolysate, as analysed by HPLC-RID and LC-ESI/MS. These results indicate that two forms of inulinase, an exo- and an endo-acting enzyme, were immobilized on our carrier. The enzyme showed good operational stability in a packed column over 28 days. There were no significant decreases in the efficiency of continuous hydrolysis during this time (about 17.4 % in comparison to its initial value).

Characterization of a thermostable inulin fructotransferase from Clostridium clostridioforme AGR2157 that produces difructose dianhydride I from inulin

A gene encoding a difructose dianhydride I (DFA I)-forming inulin fructotransferase (IFTase) from Clostridium clostridioforme AGR2157 was cloned and extracellularly expressed in Escherichia coli. SDS-PAGE and gel filtration analysis of the purified enzyme showed molecular mass of 43 and 128kDa, respectively, indicating that the recombinant enzyme was a trimer with three identical subunits. The enzyme displayed the highest activity at pH 5.5 and 50°C with the specific activity of 2.076Umg−1, and it was stable under heat treatment of up to 80°C for 4h. In comparison with other reported IFTases (DFA I-forming), the recombinant IFTase from C. clostridioforme demonstrated the best thermostability. Km and Vmax were calculated to be 0.42mM and 2.69mMmin−1, respectively. The main product of the hydrolysis of inulin by purified enzyme was identified as DFA I according to nuclear magnetic resonance analysis, and the minor products were suggested to be sucrose (GF), 1-kestose (GF2), nystose (GF3), and fructofuranosyl nystose (GF4). The smallest fructo-oligosaccharide substrate was determined to be GF3. When 10, 50, and 100gL−1 of inulin were catalyzed by the purified enzyme, the maximal yields of DFA I were approximately 85%, 73%, and 71%, respectively.