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Heterogeneous expression of enzymes allows large-scale production with reduced costs. Changes in glycosylation often occur due to changes in the expression host. In the study, the catalytic and biochemical properties of Aspergillus awamori exo-inulinase 1 are compared for A. awamori and Penicillium verruculosum expression hosts. The tertiary structure contains seven sites of N-glycosylation, with two of them located near the active center. If expressed in P. verruculosum, the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in k cat . These substrates with a short chain of 2-4 monosaccharide units were used to characterize the interaction of the substrate with the amino acid residues in the active center while preventing the interaction of the substrate with N-linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of N-linked glycans. The fluctuation of the residues N40 and Q57, which interact with the hydroxyl group O5 of the fructose unit in the -1 subsite of the active center, was increased by 1.6 times. The fluctuation of the residue W335, which interacts with the hydroxyl group O1 of the fructose unit together with the catalytic residue D41 and affects the torsion angle geometry of the substrate molecules, was increased by 1.5 times. The residue R188, which analogously to W335 affects the torsion angle geometry of the substrate molecules, was also among the affected residues with a 1.2-fold increase in the fluctuation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fructose is a carbohydrate with essential applications in the food industry, mainly due to its high sweetness and low cost. The present investigation focused on optimising fructose production from commercial inulin using the enzymatic immobilisation method and applying the response surface methodology in a 12-run central composite design. The independent variables evaluated were the pH (-) and temperature (°C). The substrate consisted of a commercial inulin solution at a concentration of 1 g/L, while the catalyst consisted of the enzyme inulinase from Aspergillus niger (EC 232-802-3), immobilised in 2% m / v sodium alginate. A stirred vessel reactor was used for 90 min at 120 rpm, and quantification of reducing sugars was determined using DNS colorimetric and UV-Vis spectrophotometric methods at a 540 nm wavelength. After applying the response surface methodology, it was determined that the catalytic activity using the immobilisation method allows for a maximum total productivity of 16.4 mg/h under pH and temperature of 3.9 and 37 °C, respectively, with an efficiency of 96.4%. The immobilised enzymes' reusability and stability compared to free enzymes were evaluated, obtaining activity up to the fifth reuse cycle and showing significant advantages over the free catalyst.

The food enzyme inulinase (1- β -d-fructan fructanohydrolase; EC 3.2.1.7) is produced with the non-genetically modified Aspergillus welwitschiae strain NZYM-KF by Novozymes A/S. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of fructo-polysaccharides for the production of fructo-oligosaccharides. Since residual amounts of total organic solids (TOS) are removed during the food manufacturing process, toxicological studies other than allergenicity were considered unnecessary and dietary exposure was not calculated. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with tomato allergens were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to tomato, cannot be excluded, but is expected not to exceed that of tomato. As the prevalence of allergic reactions to tomato is low, also the likelihood of such reactions to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use., Competing Interests: If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu., (© 2024 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

This study aimed to produce enzymes (beta (β)-mannanase using a recombinant Aspergillus sojae AsT3 and inulinase using Aspergillus niger A42) and oligosaccharides (mannooligosaccharides (MOS), fructooligosaccharides (FOS)) using coffee waste, ground coffee, and coffee extract by solid-state fermentation (SSF). Plackett-Burman Design (PBD) was used to create a design for enzyme production with four different parameters (temperature, pH, solid-to-liquid ratio (SLR), and mix with coffee wastes and ground coffee). The highest β-mannanase and inulinase activities were 71.17 and 564.07 U/mg of protein respectively. Statistical analysis showed that the temperature was statistically significant for the production of both enzymes (P < 0.05). The produced enzymes were utilized in French Pressed coffee extracts to produce oligosaccharides. As a result of the enzymatic hydrolyzation, the highest mannobiose, mannotriose, mannotetraose, and total MOS levels were 109.66, 101.11, 391.02, and 600.64 ppm, respectively. For the FOS production, the maximal 1,1,1-kestopentaose was 38.34 ppm. Consequently, this study demonstrates that a recombinant Aspergillus sojae AsT3 β-mannanase and Aspergillus niger A42 inulinase produced from coffee wastes and ground coffee can be used in coffee extracts to increase the amount of oligosaccharides in coffee extracts., Competing Interests: Declaration of competing interest The authors have declared no competing interest in this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Inulinases are enzymes of great interest in the food industry, especially due to their application in the synthesis of fructose and fructo-oligosaccharides. Moreover, some inulinases ( I ) also present invertase activity ( S ), making them useful for sucrose hydrolysis processes. In the present study, the production of inulinase by Aspergillus niger URM5741 was evaluated and optimized using two statistical approaches. First, the composition of the cultivation medium was determined through a simplex centroid mixture design, followed by the selection of optimal fermentation conditions using the Box-Behnken design. Based on these experimental designs, the maximum activities of inulinase (16.68 U mL -1 ) and invertase (27.80 U mL -1 ) were achieved using a mixture of wheat, soy, and oat brans (5 g), along with 2.5% inulin and 40% moisture. The inulinase exhibited optimum temperature and pH of 60 °C and 4.0, respectively, displayed a high affinity for both substrates, as evidenced by very-low Michaelis constant values (1.07-1.54 mM). A relative thermostability was observed at 55-60 °C as indicated by half-lives values ( I: 169.06-137.27 min; S: 173.29-141.52 min) and D -values ( I: 561.61-456.00 min; S: 575.65-470.11 min) which were further confirmed by the high activation energy (123.01 and 143.29 kJ mol -1 ). The enzyme demonstrated favorable results in terms of inulin and sucrose hydrolysis, being a maximum release of reducing sugars of 6.04 and 15.80 g L -1 , respectively. These results indicate that the sequential statistical approach proved to be beneficial to produce inulinase by A. niger URM5741, with the obtained enzyme considered promising for long-term industrial applications., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© King Abdulaziz City for Science and Technology 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

This study aimed to the production of inulinase and fructooligosaccharides (FOSs) from carob under the solid-state fermentation (SSF) conditions by using Plackett-Burman Design (PBD). Based on the results the maximum inulinase and specific inulinase activities were 249.98 U/mL and 318.29 U/mg protein, respectively. When the fructooligosaccharide (FOS) results were evaluated, the maximum values of 1,1,1-Kestopentaose, 1,1-Kestotetraose, and 1-Kestose were 182.01, 506.16, 132.16 ppm while the lowest and highest total FOS values were 179.35 and 516.66 ppm, respectively. On the other hand, it was observed that the maximum inulinase activity was found at the center points of the design. Therefore, validation fermentations were carried out at center point conditions. Subsequently, the yielded bulk enzyme extracts were partially purified using Spin-X UF membranes with 10, 30, and 50 kDa cut-off values. After purification, the maximum inulinase activity was 247.30 U/mg using a 50 kDa cut-off value. Followed by this process, the purified enzyme was used to produce FOSs and the results indicated that the maximum total FOS amount was 28,712.70 ppm. Consequently, this study successfully demonstrates that Aspergillus niger A42 inulinase produced from carob under the SSF conditions can be used in FOSs production., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Irfan Turhan reports financial support was provided by Akdeniz University. Irfan TURHAN reports a relationship with Akdeniz University that includes: employment., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The food enzyme inulinase (1-β-d-fructan fructanohydrolase; EC 3.2.1.7) is produced with the genetically modified Aspergillus oryzae strain MUCL 44346 by PURATOS NV. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the production of fructo-oligosaccharides (FOS) from inulin extracted from chicory roots. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.01 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 100 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 10,000. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found with tomato allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to tomato, cannot be excluded. However, the likelihood of allergic reactions is expected not to exceed the likelihood of allergic reactions to tomato. As the prevalence of allergic reactions to tomato is low, also the likelihood of such reactions to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

The glycoside hydrolase family contains enzymes that break the glycosidic bonds of carbohydrates by hydrolysis. Inulinase is one of the most important industrial enzymes in the family of Glycoside Hydrolases 32 (GH32). In this study, to identify and classify bacterial inulinases initially, 16,002 protein sequences belonging to the GH32 family were obtained using various databases. The inulin-effective enzymes (endoinulinase and exoinulinase) were identified. Eight endoinulinases (EC 3.2.1.7) and 4318 exoinulinases (EC 3.2.1.80) were found. Then, the localization of endoinulinase and exoinulinase enzymes in the cell was predicted. Among them, two extracellular endoinulinases and 1232 extracellular exoinulinases were found. The biochemical properties of 363 enzymes of the genus Arthrobacter , Bacillus , and Streptomyces (most abundant) showed that exoinulinases have an acid isoelectric point up to the neutral range due to their amino acid length. That is, the smaller the protein (336 aa), the more acidic the pI (4.39), and the larger the protein (1207 aa), the pI is in the neutral range (8.84). Also, a negative gravitational index indicates the hydrophilicity of exoinulinases. Finally, considering the biochemical properties affecting protein stability and post-translational changes studies, one enzyme for endoinulinase and 40 enzymes with desirable characteristics were selected to identify their enzyme production sources. To screen and isolate enzyme-containing strains, now with the expansion of databases and the development of bioinformatics tools, it is possible to classify, review and analyze a lot of data related to different enzyme-producing strains. Although, in laboratory studies, a maximum of 20 to 30 strains can be examined. Therefore, when more strains are examined, finally, strains with more stable and efficient enzymes were selected and introduced for laboratory activities. The findings of this study can help researchers to select the appropriate gene source from introduced strains for cloning and expression heterologous inulinase, or to extract native inulinase from introduced strains., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.)

The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions. Site-directed mutagenesis was used to construct the mutants of the enzyme after carrying out the MD simulations between Inu-ISO3 and its substrates. Mutation Trp79:Ala resulted in the total loss of activity when fructans were used as substrates, while with sucrose, the activity decreased by 98 %. In contrast, the mutations Phe113:Ala and Gln236:Ala increased the invertase activity when sucrose was used as a substrate. Although these amino acids are not part of the conserved motifs where the catalytic triad is located, they are essential for the enzyme's activity. In silico and experimental approaches corroborate the relevance of these residues for substrate binding and their influence on enzymatic activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) have emerged as key contributors to digestive discomfort and intolerance to certain vegetables, fruits, and plant-based foods. Although strategies exist to minimize FODMAP consumption and exposure, exogenous enzyme supplementation targeting the fructan-type FODMAPs has been underexploited. The objective of this study was to test the hydrolytic efficacy of a food-grade, non-genetically engineered microbial inulinase preparation toward inulin-type fructans in the INFOGEST in vitro static simulation of gastrointestinal (GI) digestion. Purified inulin was shown to undergo acid-mediated hydrolysis at high gastric acidity as well as predominantly inulinase-mediated hydrolysis at lower gastric acidity. Inulinase dose-response simulations of inulin, garlic, and high-fructan meal digestion in the gastric phase suggest that as little as 50 inulinase units (INU) and up to 800 INU per serving promote fructan hydrolysis better than the control simulations without inulinase. Liquid chromatography-mass spectrometry (LC-MS) profiling of fructo-oligosaccharides (FOS) in the gastric digestas following inulinase treatment confirms the fructolytic activity of inulinase under simulated digestive conditions. Altogether, these in vitro digestion data support the use of microbial inulinase as an exogenous enzyme supplement for reducing dietary fructan-type FODMAP exposure., Competing Interests: JG, MH, JF, KT, and SG are employed by BIO-CAT, Inc. The authors declare that this study was funded by BIO-CAT, Inc. The funder had the following involvement in the study: experimental design, data collection and analysis, interpretation of data, decision to publish, and writing of this article., (Copyright © 2023 BIO-CAT, Inc.)

Research Background: Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production., Experimental Approach: Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production., Results and Conclusions: The most common species were Aspergillus tubingensis (51.2%), followed by A. niger (23.1%), A. welwitschiae (23.1%) and A. uvarum (2.6%). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production., Novelty and Scientific Contribution: The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally., Competing Interests: CONFLICT OF INTEREST The authors declare no conflict of interest.

The present work aimed to model Aspergillus niger inulinase fermentation performed in the medium using sigmoidal functions, validate the selected models using an independent set of the experimental values, and perform a sensitivity analysis of the selected models. Based on the results, the selected models were Stannard and Fitzhugh models for substrate consumption (R 2  = 0.9976 and 0.9974, respectively), Huang model for inulinase production (R 2  = 0.9967), Weibull model for invertase-type production (R 2  = 0.9963), and modified logistic model for invertase-type activity/inulinase activity ratio (R 2  = 0.9292) with high R 2 values (>0.90). Kinetics predicted by particularly selected models mentioned above fit well with the experimental kinetic results. Besides, validation of the selected models with an independent set of the experimental data indicated that they gave satisfying results with high R 2 values for consumption and production (R 2  > 0.90). Sensitivity analysis of the selected models showed that the yielded R 2 values (R 2  ≥ 0.9775) were in good agreement with those obtained from the selected models. Consequently, A. niger inulinase fermentation was successfully modeled and the selected models were successfully validated with an independent set of the observed data. Besides, the sensitivity analysis also verified the reliability of the selected models. Those models can serve as universal equations to describe the A. niger inulinase fermentation., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

The enriched nutritional and functional properties of inulinase with wide attention are considered commercial/industrial food enzymes. It can be produced by many microorganisms such as yeasts, fungi, and bacteria. Nocardiopsis is a genus under Actinomycetes, which has biotechnologically important microorganisms. This study aims to isolate and identify marine Actinomycetes Nocardiopsis species and to evaluate the antibacterial potential of the inulinase enzyme obtained from it. Marine actinobacteria ( Nocardiopsis sp.) were isolated from sediment samples on YM agar. The isolate was identified by biochemical analysis of cell walls (amino acid and sugar). Enzyme screening assay was performed with temperature and pH influence in the production inulinase enzyme production. Antibacterial activity and minimal inhibitory activity of inulinase enzyme were performed with Staphylococcus , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Antimicrobial testing revealed that with higher concentrations of inulinase enzyme, the zone of inhibition of bacterial growth increased, and the minimum inhibitory concentration of inulinase enzyme that prevented the growth of bacteria was close to the standard tetracycline. Inulinase enzyme obtained from Nocardiopsis species shows good antibacterial activity against Staphylococcus aureus , K. pneumoniae , and P. aeruginosa in comparison to the standard, tetracycline., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Advanced Pharmaceutical Technology & Research.)

Endophytic bacteria refer to bacteria which promote plant growth via direct and indirect mechanisms. Three endophytic bacteria isolated from Jerusalem artichoke exhibited plant growth induction and inulin production. These bacteria had functions of fructan degradation and synthesis from inulinase and levansucrase, respectively. Rossellomorea aquimaris 3.13 and Priestia megaterium 3.5 obtained inulinase/levanase enzyme with inulin and levan as substrates; enzyme production showed the optimum conditions in 1% inulin medium of 35 °C, pH 7.0. Bacillus velezensis 5.18 and Priestia megaterium 3.5 had inulosucrase/levansucrase enzyme with sucrose as a major carbon source; the enzyme had optimum temperature and pH conditions of 30 °C and pH 7.0, respectively. A combination of carbon sources had effect on decreasing enzyme activity; in addition, co-inoculation of bacteria showed a slight difference in enzyme production compared with single inoculation. The inulosucrase/levansucrase was produced earlier in co-culture containing bacteria with inulinase activity. Plant fructan synthesis was involved in 1-SST and 1-FFT, while 1-FEH encoded inulin degradation; these genes were evaluated in Jerusalem artichoke inoculated with the endophytic bacteria to quantify gene expression level using qPCR. All genes expressed in low levels at early stage of growth, responding to all endophytic bacteria. Significantly, Bacillus velezensis 5.18 induced all genes of the plant at 65 days of inoculation; Rossellomorea aquimaris 3.13 induced 1-FFT while Priestia megaterium 3.5 induced 1-SST., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest in the publication., (© King Abdulaziz City for Science and Technology 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Inulinases are enzymes involved in the hydrolysis of inulin, which can be used in the food industry to produce high-fructose syrups and fructo-oligosaccharides. For this purpose, different Aspergillus strains and substrates were tested for inulinase production by solid-state fermentation, among which Aspergillus terreus URM4658 grown on wheat bran showed the highest activity (15.08 U mL -1 ). The inulinase produced by this strain exhibited optimum activity at 60 °C and pH 4.0. A detailed kinetic/thermodynamic study was performed on the inulin hydrolysis reaction and enzyme thermal inactivation. Inulinase was shown to have a high affinity for substrate evidenced by very-low Michaelis constant values (0.78-2.02 mM), which together with a low activation energy (19.59 kJ mol -1 ), indicates good enzyme catalytic potential. Moreover, its long half-life ( t 1/2 = 519.86 min) and very high D -value (1726.94 min) at 60 °C suggested great thermostability, which was confirmed by the thermodynamic parameters of its thermal denaturation, namely the activation energy of thermal denaturation ( E* d = 182.18 kJ mol -1 ) and Gibbs free energy (106.18 ≤ Δ G * d ≤ 111.56 kJ mol -1 URM4658 inulinase is a promising and efficient biocatalyst, which could be fruitfully exploited in long-term industrial applications.A. terreus URM4658 inulinase is a promising and efficient biocatalyst, which could be fruitfully exploited in long-term industrial applications.

Inulin is the rich water-soluble storage polysaccharide after starch in nature, and utilization of inulin through hydrolysis of exo-inulinases has attracted much attention. Thermo-halo-alcohol tolerance is essential for exo-inulinase applications, while no report reveals the molecular basis involved in halo-alcohol tolerance of exo-inulinases via experimental data. In this study, two loops of exo-inulinase InuAMN8, including the loop built with 360 GHVRLGPQP 368 linking domains of Glyco&#95;hydro&#95;32N and Glyco&#95;hydro&#95;32C and another loop built with 169 GGAG 172 in the catalytic domain, were deleted to generate mutants MutG360Δ9 and MutG169Δ4, respectively. After heterologous expression, purification, and dialysis, InuAMN8, MutG169Δ4, and MutG360Δ9 showed half-lives of 144, 151, and 7 min at 50°C, respectively. InuAMN8 and MutG169Δ4 were very stable, while MutG360Δ9 showed a half-life of approximately 60 min in 5.0% (w/v) NaCl, and they showed half-lives of approximately 60 min in 25.0, 25.0, and 5.0% (w/v) ethanol, respectively. Structural analysis indicated that two cation-π bonds, which contributed to thermal properties of InuAMN8 at high temperatures, broke in MutG360Δ9. Four basic amino acid residues were exposed to the structural surface of MutG360Δ9 and formed positive and neutral electrostatic potential that caused detrimental effects on halo-alcohol tolerance. The study may provide a better understanding of the loop-function relationships that are involved in thermo-halo-alcohol adaptation of enzymes in extreme environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cen, Zhang, He, Tang, Wu, Zhou and Huang.)

Inulin can be hydrolyzed by inulinases to yield inulo-oligosaccharides (IOSs), which have great application potential in the food and nutraceutical industries. However, conventional enzymatic production of IOSs is limited by long hydrolysis times and poor thermo-stability of inulinases. Here, the self-assembling protein scaffold EutM was engineered to co-immobilize exo-inulinase (EXINU) and endo-inulinase (ENINU) for synergistic hydrolysis of inulin to produce IOSs with 3 to 5 monosaccharide units (DP3-5 IOSs). The immobilization of EXINU/ENINU onto the EutM scaffold resulted in an increase of catalytic efficiency, a 65% increase of the V max of ENINU, as well as an increase of thermo-stability, with 4.26-fold higher residual activity of EXINU after 22 h-incubation at 50 °C. After optimization, two efficient production protocols were obtained, in which the yield and productivity of DP3-5 IOSs reached 80.38% and 70.86 g·(L·h) -1 , respectively, which were at a high level in similar studies. Overall, this study provides an attractive self-assembling protein platform for the co-immobilization of inulinases, as well as optimized bioprocesses with great promise for the industrial production of DP3-5 IOSs., (Copyright © 2022. Published by Elsevier B.V.)

Bran can enrich snacks with dietary fibre but contains fructans that trigger symptoms in people with irritable bowel syndrome (IBS). This study aimed to investigate the bioprocessing of wheat and amaranth bran for degrading fructans and its application (at 20% flour-based) in 3D-printed snacks. Bran was bioprocessed with Saccharomyces cerevisiae alone or combined with inulinase, Kluyveromyces marxianus , Limosilactobacillus fermentum, or commercial starter LV1 for 24 h. Fructans, fructose, glucose, and mannitol in the bran were analysed enzymatically. Dough rheology, snack printing precision, shrinkage in baking, texture, colour, and sensory attributes were determined. The fructan content of wheat bran was 2.64% dry weight, and in amaranth bran, it was 0.96% dry weight. Bioprocessing reduced fructan content (up to 93%) depending on the bran type and bioprocessing agent, while fructose and mannitol remained below the cut-off value for IBS patients. Bran bioprocessing increased the complex viscosity and yield stress of dough (by up to 43 and 183%, respectively) in addition to printing precision (by up to 13%), while it lessened shrinkage in baking (by 20-69%) and the hardness of the snacks (by 20%). The intensity of snack sensory attributes depended on the bran type and bioprocessing agent, but the liking ("neither like nor dislike") was similar between samples. In conclusion, snacks can be enriched with fibre while remaining low in fructans by applying bioprocessed wheat or amaranth bran and 3D printing.

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.)

In this presented paper, concanavalin A-modified cysteine-functionalized Fe 3 O 4 /Ag core/shell magnetic nanoparticles were synthesized and used as a support material for inulinase enzyme, which has been intensively used for the preparation of high-fructose syrup by hydrolyzing inulin. Inulinase adsorption capacity of Con A-functionalized Ag-coated magnetic nanoparticles was optimized by changing medium pH, temperature, and initial inulinase concentration, and maximum inulinase adsorption capacity was found to be 655.32 mg/g nanoparticle by using 1.00 mg/mL of inulinase solution in pH 3.0 buffer system at 25 °C. Finally, efficient inulin degradation capacity of the inulinase immobilized magnetic nanoparticles was demonstrated by TLC studies and released fructose amount was determined as 0.533 mg/mL only within the 5 min of hydrolysis. This newly developed hydrolysis strategy holds considerable promise to produce high-fructose syrup in many industries.

Inulin is a kind of polysaccharide that can be obtained various biomass. Inulooligosaccharides (IOS), a kind of oligosaccharides that can be obtained from inulin by enzymatic hydrolysis using inulinases, have been regarded as the functional food ingredients. Commercially available inulinases produced by natural Aspergillus niger contained both endo- and exo-inulinase activities. For IOS production from inulin, it is desirable to use only endo-inulinase as exo-inulinase would produce mainly the monosacchairde fructose from inulin. In the present study, a simple inulin-mediated ethanol precipitation method was developed to separate endo- and exo-inulinases present in natural inulinases. IOS production from inulin using the enriched endo-inulinase was then optimized in process conditions including pH and temperature, achieving a high yield of ∼94%. The resultant IOS products had a degree of polymerization ranging from 2 to 7. The study demonstrated a novel method for obtaining partially purified or enriched endo-inulinase for IOS production from inulin in an efficient process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Zhang, Wang and Yang.)

Background: Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis., Results: Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s -1 and 568.93 L mmol -1 s -1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s -1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol -1 s -1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference., Conclusions: The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

During preservation, Jerusalem artichoke (JA) tubers are subjected to deterioration by mold fungi under storage, which signifies a serious problem. A new blue mold ( Penicillium polonium ) was recorded for the first time on JA tubers. Penicillium mold was isolated, identified (morphologically, and molecularly), and deposited in GenBank; (MW041259). The fungus has a multi-lytic capacity, facilitated by various enzymes capable of severely destroying the tuber components. An economic oil-based procedure was applied for preserving and retaining the nutritive value of JA tubers under storage conditions. Caraway and clove essential oils, at a concentration of 2%, were selected based on their strong antifungal actions. JA tubers were treated with individual oils under storage, kept between peat moss layers, and stored at room temperature. Tubers treated with both oils exhibited lower blue mold severity, sprouting and weight loss, and higher levels of carbohydrates, inulin, and protein contents accompanied by increased levels of defense-related phytochemicals (total phenols, peroxidase, and polyphenol oxidase). Caraway was superior, but the results endorse the use of both essential oils for the preservation of JA tubers at room temperature, as an economic and eco-safe storage technique against the new blue mold.

The aim of study was to optimize fermentation parameters for inulinase production from Rhizopus oryzae by a statistical approach and to carry out purification of inulinase. Five isolated fungal strains were screen out inulin degradation by using Lugol's iodine solution. R. oryzae exhibited maximum zone of clearance around the colony and was used as an inulinase producer. The effect of carbon sources (inulin, glucose, maltose, sucrose, lactose, onion peel, stevia root, wheat bran) as medium component and fermentation parameters (temperature (25-45 °C), initial pH (4-7), time (3-7 days)) on inulinase production was investigated by Plackett-Burman Design. Wheat Bran (WB) , temperature, pH, and incubation time were found to be significant for the production of inulinase ( P  < 0.05). Furthermore, Box-Behnken Design was employed to optimize fermentation conditions. The maximum experimental results for inulinase activity and specific activity were 348.36 EU/mL and 3621.78 EU/mg, respectively. The results were obtained at 5 days of incubation time, 35 °C of incubation temperature, initial pH of 5.5, and 2% (w/v) WB. Also, inulinase was purified by using ammonium sulfate precipitation, gel filtration chromatography with 2.19-fold and its molecular weight was found as 89.12 kDa. The optimal pH and temperature of the purified enzyme were 4.0 and 60 °C, respectively. Furthermore, the purified enzyme showed excellent stability at 60 °C. In conclusion, the present study offers cost-effective method to produce inulinase from Rhizopus oryzae . Also, it can be suggested that the purified inulinase has strong potential for usage in production of fructose syrup and other industrial areas., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© Association of Food Scientists & Technologists (India) 2020.)

Freshly harvested Jerusalem artichoke tubers contain inulinase, an enzyme that requires inactivation, because of its ability to hydrolysis inulin into fructose, which can be consumed by microorganism during marination. As the traditional pickling process takes 6 months, and involves the addition of a large amount of salt (18-20%), this production strategy is uneconomical and increases the nitrite intake. Additionally, miscellaneous bacteria produced during pickling affect the product taste. In this study, the enzyme inactivation effects of NaCl, NaHCO 3 , and ultrasound were evaluated. NaHCO 3 treatment results in the highest degree of enzyme inactivation; however, the quality and flavor of the obtained Jerusalem artichoke pickles were not ideal. The Jerusalem artichoke pickles in which the enzymes were inactivated using a combination of NaCl and ultrasound exhibited better flavor than those exposed to NaHCO 3 ; further, this combination reduced the inulinase activity of the Jerusalem artichokes to 2.50 U/mL, and maintained the inulin content at 61.22%. The strains LS3 and YS2, identified as Enterococcus faecalis and the salt-tolerant yeast Meyerozyma guilliermondii , respectively, were the dominant microorganisms isolated from the pickle juice. Jerusalem artichokes with inactivated inulinase were pickled with microbial powder, separated, purified, and dried to remove the natural Jerusalem artichoke sauce. This process shortened the fermentation cycle and improved product quality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Liu, Ji, Deng, Feng, Shi and Gao.)

The objectives of this study were to purify Aspergillus niger inulinase produced from sugar-beet molasses in the shaking incubator (100 mL) and stirred-tank bioreactors (5-L and 30-L) by using some downstream processes and to determine enzyme kinetics and characterization. The results showed that the best centrifuge-time combination was 16,873 ×g-5 min with the purification coefficient of 1.4. Besides, with the ultrafiltration process, the inulinase activities yielded using the shaking incubator, pH-controlled/uncontrolled small-scale bioreactors, and large-scale bioreactor were increased from 1101.3, 2079.2, 1561.3, and 787.5 U/mL to 12,065.2, 21,789.0, 11,296.9, and 2948.1 U/mL with purification coefficients of 5.33, 1.38, 1.46, and 1.67, respectively. Additionally, for the inulinase from shaking incubator and pH-uncontrolled bioreactor, the values of K m for inulin and sucrose were 17.8 and 49.4 mg/mL and 28.8 and 25.9 mg/mL, respectively. As the enzyme amount added to the substrate increased, the activity also increased. Mn 2+ is the activator of the enzyme, and Cu 2+ and Ag + are inhibitors of the enzyme. The molecular weight of inulinase has been determined to be between 60 and 70 kDa. Consequently, this study ensures valuable and significant information about the purification and characterization of inulinase for industrial implementations., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Exo-inulinases are versatile enzymes that have gained attention in recent years due to their ability to hydrolyze linear and branched polyfructose chains found in inulines. Agavin, a branched inulin, is found in Agave plant, the raw matter to produce tequila. Our group has isolated several microbial strains from agave bagasse, an agro-industrial residue from tequila production that increases yearly. Strain ISO3, identified as Kluyveromyces marxianus, showed a remarkable activity towards agavin, and from its fermentation liquor an inulinolytic enzyme (Inu-ISO3) was purified. The isolated enzyme is a glycosylated dimeric protein with a molecular mass of ~256 kDa, as determined by DLS and SEC. The enzyme has an isoelectric pH of 4.6 and has both inulinase and invertase activities with an I/S ratio (ratio of activity with agavin to activity with sucrose) of 1.39. The enzyme has temperature and pH optima of 50 °C and 5.5, respectively, and follows hyperbolic kinetics with agavin (k cat of 339 ± 27 s -1 and K M of 11.8 ± 1.5 mM). The remarkable activity of Inu-ISO3 on linear and branched inulin spotlights this enzyme as a potential player in the treatment of agricultural residua for the generation of added-value products., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Utilization of agricultural wastes as cheap natural resources for production of bioactive products is currently attracting global attention. For this purpose, this study focused on isolation of Aspergillus wewitschiae MN056175 as promising producer of inulinase, then investigating physiochemical, kinetics and thermodynamics of the obtained inulinase, and its ability to extract bioactive fructo-oligosaccharides (FOS) from Cynara scolymus leaves (artichoke leaves, AL). A. wewitschiae MN056175 inulinase gave the maximum activity at temperature 60 °C and inulin concentration 1%. The kinetics including K m and V max were determined to be 105.26 mg·ml -1 and 83.33 μmol·ml -1 ·min -1 , respectively. The thermodynamics including, E a (activation energy) and E d (activation energy for denaturation) were determined to be 21.82 and 73.21 kJ·mol -1 , K d , T 1/2 , D-value, ΔH°, ΔG° and ΔS° at 40, 50 and 60 °C which indicated the stability of A. wewitschiae MN056175 inulinase. Moreover, this inulinase was capable of hydrolyzing Cynara scolymus leaves into reducing sugar and 15 FOS with different DP, total carbohydrate, and protein content under different conditions designed by central composite design (CCD). The 15 AL FOS showed different high antioxidant and prebiotic activities. Central FOS with probiotic bacteria exhibited significant antimicrobial activity against tested gram positive bacteria in a way higher than those recorded against gram negative bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052 bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72 h. In a 5-l fed-batch fermentation, as high as 148.9 g/L of inulin was directly used within 120 h, and only 1.2 g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4 g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9 g/L was obtained.

Objective: The fermentation medium contains many complex components (vitamins, minerals, etc.) for better growth of the microorganisms. The increasing purity and number of these components used in the medium seriously affect the cost of the microbial process. This study aimed to further optimize the concentration of the components used in the medium (yeast extract and peptone) for inulinase fabrication by Aspergillus niger from sugar-beet molasses in shake flask fermentation by using Central Composite Design (CCD) and to kinetically identify the fermentation., Results: The results indicated that the optimal medium composition consisted of only 4.2% (w/v) yeast extract. By using the fermentation environment, the inulinase generation, inulinase/sucrase ratio, maximum inulinase generation rate, maximum sugar depletion rate, and substrate utilization yield were determined as 1294.5 U/mL, 1.2, 159.6 U/mL/day, 7.4 g/L/day, and 98.1%, respectively. The kinetic analysis of the fungal development (logistic model) indicated that a specific development rate and initial biomass concentration were 0.89/day and 1.79 g/L, respectively. Inulinase and sucrase productions are mixed-development associated since the α value ≠ 0 (8.46 and 4.31 U/mgX) and the β value ≠ 0 (5.15 and 4.83 U/mgX day), respectively (Luedeking-Piret model). Besides, the maintenance value (Z) (0.009 gS/gX day) was lower than γ value (1.044 gS/gX), showing that A. niger commonly uses the substrates for enzyme fabrication and fungal development (modified Luedeking-Piret model)., Conclusions: The enzyme activity was increased by optimizing the concentration of the components used. It was demonstrated that the proposed kinetic models can victoriously define fungal development, enzyme fabrication, and sugar depletion.

Animals are thought to use only glucose polymers (glycogen) as energy reserve, whereas both glucose (starch) and fructose polymers (fructans) are used by microbes and plants. Here, it is reported that the gall midge Mayetiola destructor, and likely other herbivorous animal species, gained the ability to utilize dietary fructans directly as storage polysaccharides by a single horizontal gene transfer (HGT) of bacterial levanase/inulinase gene followed by gene expansion and differentiation. Multiple genes encoding levanases/inulinases have their origin in a single HGT event from a bacterium and they show high expression levels and enzymatic activities in different tissues of the gall midge, including nondigestive fat bodies and eggs, both of which contained significant amounts of fructans. This study provides evidence that animals can also use fructans as energy reserve by incorporating bacterial genes in their genomes., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The goals of this study were to optimize the medium formulation for enhanced production of Aspergillus niger inulinase using Plackett-Burman Design (PBD) and to model the fermentation in optimal medium formulation. Results indicated that (NH 4 ) 2 SO 4 (negative effect), yeast extract and peptone (positive effect) were determined as significant factors affecting the inulinase production. Different media including Medium A (non-enriched), Medium B (contains both negative and positive factors) and Medium C (contains only positive factors) were formed and inulinase fermentations were performed. Findings showed that the best nutritional formulation was Medium C, which yielded to be 1011.02 U/mL, 834.28 U/mL, 1.22, 4383.44 U/mg, 4186 U/mg, 158.49 U/mL/day, 128.60 U/mL/day and 94.54% of P Inulinase , S Inulinase , I/S ratio, S Inulinase , S Sucrase , Q Inulinase , Q Sucrase and SUY, respectively. Additionally, fungal growth, enzyme or protein production and substrate consumption were modeled using the logistic model, Luedeking-Piret model, and modified Luedeking-Piret model, respectively, and found that enzyme or protein production was non-growth associated. Besides, maintenance value (Z) was lower than γ value, indicating that A. niger mainly utilizes the sugars for enzyme production and fungal growth. Consequently, optimum medium composition was successfully determined by PBD and also the kinetic models fitted the experimental data very well with high regression coefficient.

Inulinases are used for the production of high-fructose syrup and fructooligosaccharides, and are widely utilized in food and pharmaceutical industries. In this study, different carbon sources were screened for inulinase production by Aspergillus niger in shake flask fermentation. Optimum working conditions of the enzyme were determined. Additionally, some properties of produced enzyme were determined [activation (E a )/inactivation (E ia ) energies, Q 10 value, inactivation rate constant (k d ), half-life (t 1/2 ), D value, Z value, enthalpy (ΔH), free energy (ΔG), and entropy (ΔS)]. Results showed that sugar beet molasses (SBM) was the best in the production of inulinase, which gave 383.73 U/mL activity at 30 °C, 200 rpm and initial pH 5.0 for 10 days with 2% (v/v) of the prepared spore solution. Optimum working conditions were 4.8 pH, 60 °C, and 10 min, which yielded 604.23 U/mL, 1.09 inulinase/sucrase ratio, and 2924.39 U/mg. Additionally, E a and E ia of inulinase reaction were 37.30 and 112.86 kJ/mol, respectively. Beyond 60 °C, Q 10 values of inulinase dropped below one. At 70 and 80 °C, t 1/2 of inulinase was 33.6 and 7.2 min; therefore, inulinase is unstable at high temperatures, respectively. Additionally, t 1/2 , D, ΔH, ΔG values of inulinase decreased with the increase in temperature. Z values of inulinase were 7.21 °C. Negative values of ΔS showed that enzymes underwent a significant process of aggregation during denaturation. Consequently, SBM is a promising carbon source for inulinase production by A. niger. Also, this is the first report on the determination of some properties of A. niger A42 (ATCC 204,447) inulinase.

We develop a technique for the sorption of inulinase from Kluyveromyces marxianus on the KU-2 matrix cation-exchanger. The most appropriate conditions for immobilization are: 25 °C, pH 4.5, incubation time 1.5 h, protein content during immobilization 10 mg/g of carrier. At higher (20-100 mg/g) concentrations, inulinase forms local high-concentration domains on the ion-exchanger surface, the enzyme is adsorbed on the protein, not on the carrier. 62% of the initial catalytic activity of inulinase is immobilized on KU-2 by the adsorption method. Upon the enzyme immobilization on KU-2, the number of unordered structures in the protein is reduced by ~10%, that points to the compaction of the molecule. Hydrogen bonds are formed only between the sulfo groups of carrier and the protein molecule, without affecting other structures of the cation exchanger. The highest activity of the inulinase immobilized on KU-2 was observed at 70 °C (cf. 50 °C for the native enzyme). Heating of the solution for 60 min in the temperature range of 50-80 °C failed to inactivate completely the immobilized enzyme; the complete loss of its ability to hydrolyze inulin was achieved only after more than 1 h incubation at 90 °C., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bacterial inulinases are the key enzymes in the enzymatic hydrolysis of inulin and production of fructooligosaccharides (FOSs) and high fructose syrup (HFS). An extremophilic inulinase was purified from Alkalibacillus filiformis using 80% ethanol precipitation, ultrafiltration, and Q-Sepharose anion exchange chromatography. The purified inulinase was highly active in a wide range of pH, temperature, chemical reagents, and NaCl concentrations. The enzyme immobilization on cobalt ferrite magnetic nanoparticles (CoFe 2 O 4 MNPs) was carried out by carrier binding method with covalent linkage and showed improved stability and reusability within a broad temperature and pH range, compared with the free enzyme. Using free and immobilized inulinases from A. filiformis, 122 g L -1 and 160 g L -1 fructose with 61% and 80% conversion, respectively, were obtained, with inulin as the substrate. The enzymatic properties, such as notable stability under extreme conditions, make the inulinase from A. filiformis a promising candidate for related biotechnological applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This paper describes the development of a simple method for mixed non-covalent and covalent bonding of partially purified inulinase on functionalized multiwall carbon nanotubes ( f- MWCNTs) with polypyrrole (PPy). The pyrrole (Py) was electrochemically polymerized on MWCNTs in order to fabricate MWCNTs/PPy nanocomposite. Two multiple forms of enzyme were bound to N-H functional groups from PPy and -COO - from activated MWCNTs to yield a stable MWCNTs/PPy/PEG immobilized preparation with increased thermal stability. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used to confirm functionalization of nanoparticles and immobilization of the enzyme. The immobilization yield of 85% and optimal enzyme load of 345  μ g protein onto MWCNTs was obtained. The optimum reaction conditions and kinetic parameters were established using the UV-Vis analytical assay. The best functional performance for prepared heterogeneous catalyst has been observed at pH 3.6 and 10, and at the temperatures of 60 and 80ºC. The half-life ( t ) of the immobilized inulinase at 60 and 80ºC was found to be 231 and 99 min, respectively. The reusability of the immobilized formulation was evaluated based on a method in which the enzyme retained 50% of its initial activity, which occurred after the eighteenth operation cycle.1/2 ) of the immobilized inulinase at 60 and 80ºC was found to be 231 and 99 min, respectively. The reusability of the immobilized formulation was evaluated based on a method in which the enzyme retained 50% of its initial activity, which occurred after the eighteenth operation cycle., (© 2019 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Air flow rate and agitation speed for inulinase production by Kluyveromyces marxianus were optimized based on metabolic heat release profiles. Shear stress and oxygen transfer (k L a) values were compared to assess the effects of aeration and agitation. At agitation rates of ≤ 100 rpm, the oxygen mass transfer rates were small and eventually led to less inulinase production, but at agitation rates > 150 rpm, loss of biomass resulted in less inulinase activity. Bio-reaction calorimeter (BioRc1e) experiment with aeration rates ≤ 0.5 lpm showed low k L a while at 1.5 lpm frothing of reactor contents caused loss of biomass and inulinase activity. The optimum conditions for aeration and agitation rate for K. marxianus in BioRc1e were 1 lpm and 150 rpm. Heat yield values obtained for the substrate, product and biomass reinstated the ongoing metabolic process. The heat release pattern could be a promising tool for optimization of bioprocess and in situ monitoring, with a possibility of interventions during the biotransformation process. At optimized aeration and agitation conditions, a two-fold increase in inulinase activity could be noticed.

The main objective of the present work was to modify multiwalled carbon nanotubes (MWCNTs) using 3-aminopropyl-triethoxysilane (APTES) to generate amino-terminated surfaces for inulinase immobilization, which can be further used for fructose production. CCRD of response surface methodology was used for optimization of inulinase immobilization on MWCNTs. At optimized parameters (APTES concentration 4%; sonication time 4 h; enzyme coupling time 1.5 h and enzyme load 15 IU), maximal inulinase activity and immobilization yield was 60.7% and 74.4%, respectively. Immobilized inulinase showed same pH optima of free enzyme, while an elevation in temperature optima to 60 °C was observed after its immobilization. Immobilized inulinase also shown enhancement in pH stability and thermostability. Overall, 4.54-fold rise in half-life of inulinase was detected after immobilization at 60 °C. K m and V max of inulinase decreased after immobilization. Immobilized inulinase preserved 28% of its residual activity after 10 consecutive batch cycles of inulin hydrolysis for the production of fructose., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aguamiel is a natural sap produced by some species of agave plants, such as Agave salmiana, A. atrovirens , or A. angustifolia . It is a product with a high concentration of fructose, glucose or sucrose, although its composition may vary depending on the season in which it is produced, and may also contain agave fructans (or agavins) or fructooligosaccharides (FOS). It has been reported that FOS can be produced by enzymes that act on sucrose or inulin, transfructosylating or hydrolyzing these materials, respectively. Due to the sugar content in aguamiel, the application of an enzymatic complex produced by Aspergillus oryzae DIA MF was carried out. This complex was characterized by 1-D electrophoresis SDS-PAGE, and its transfructosylation and hydrolysis activities were determined by HPLC. In order to determine the conditions at which the concentration of FOS in this beverage increased, kinetics were carried out at different temperatures (30, 50, and 70°C) and times (0, 1, 2, 3, 4, 5, 10, and 15 h). Finally, the antioxidant and prebiotic activities were evaluated. FOS concentration in aguamiel was increased from 1.61 ± 0.08 to 31.01 ± 3.42 g/ L after 10 h reaction at 30°C applying 10% enzymatic fraction-substrate (v/v). Antioxidant activity was highly increased (34.81-116.46 mg/eq Trolox in DPPH assay and 42.65 to 298.86 mg/eq Trolox in FRAP assay) and growth of probiotic bacteria was higher in aguamiel after the enzymatic treatment. In conclusion, after the application of the enzymatic treatment, aguamiel was enriched with FOS which improved antioxidant and prebiotic properties, so it can be used as a functional food.

To date, the high cost of enzyme production, lack of enzyme reusability and operational stability are the main limitations of the enzyme's application in industry. In this work, inulinase was covalently immobilized on the surface of glutathione-coated gold magnetic nanoparticles (GSH-AuMNPs). The synthesized NPs were fully characterized. The effects of different restriction factors such as substrate concentration, temperature, and pH on the performance and stability of the enzyme were examined. The maximum activity and immobilization yield were estimated 83% and 93%, respectively. The immobilized inulinase showed maximum activity at pH 4.5 and 60 °C. The kinetic parameters of the immobilized enzyme were not changed significantly after the immobilization process. The reusability assessment indicated that approximately 78% of the initial activity of immobilized inulinase remained after ten times recycling. The storage stability of inulinase was improved by the immobilization process. The inulin hydrolysates were checked by HPLC and the end products only contained two components, 98% of fructose and up to 2% of glucose in both free enzyme and immobilized enzyme systems. This study introduced a simple, effective and inexpensive immobilization process, which is applicable in different biomedical, biotechnological and food industries., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, various levels of ultra-high pressure (UHP) were combined with the enzymatic synthesis of the fructooligosaccharide (FOS) using Pectinex Ultra SP-L and inulinase. The combination enhanced the FOS yields up to 2.5- and 1.5-fold, respectively, compared to atmospheric condition (0.1 MPa). However, the enzymatic reaction was dependent on the levels of pressure, the reaction times, and the initial sucrose concentrations. The combined UHP and inulinase showed that the maximum FOS yield (71.81%) was obtained under UHP at 200 MPa for 20 min with 300 g/L of initial sucrose as a substrate, while the FOS yield (57.13%) using Pectinex Ultra SP-L was obtained under UHP at 300 MPa for 15 min with 600 g/L of initial sucrose as a substrate. The FOS composition produced by Pectinex Ultra SP-L under the UHP was 1-kestose (GF 2 ), nystose (GF 3 ), and 1F-fructofuranosylnystose (GF 4 ), whereas the FOS produced by inulinase composed of only GF 2 and GF 3 . The combined UHP is a useful tool in the industrial application for FOS production. Highlights UHP activated the activity of Pectinex Ultra SP-L yet inactivated inulinase Pressure level, time, and sucrose concentration significantly affect FOS yields under UHP UHP enhanced FOS production with time-saving benefits within 15-20 min.

There is evidence that a diet low in Fermentable Oligo-, Di-, Monosaccharides And Polyols (FODMAPs) alleviates symptoms in approx. 70% of the patients suffering from irritable bowel syndrome. Through fructans, wheat containing products are a major source of FODMAPs in the western diet. Although fructans are partially degraded during dough fermentation by Saccharomyces cerevisiae invertase, wheat bread contains notable fructan levels. In this study, it was shown that Kluyveromyces marxianus strain CBS6014 can degrade more than 90% of the fructans initially present in wheat whole meal during bread making, which can be attributed to its high inulinase activity. As K. marxianus CBS6014 was not able to consume maltose during fermentation, alternative sugars (sucrose) or glucose releasing enzymes (amyloglucosidase) had to be included in the bread making recipe to ensure sufficient production of CO 2 and high bread quality. Five volatile flavor compounds were produced in significantly different levels when K. marxianus CBS6014 was used as starter culture compared with the conventional S. cerevisiae bakery strain. These differences were, however, not detected when sensory analysis of the crumb was performed. This study demonstrates the potential of inulinase-producing K. marxianus strains for the production of (whole meal) breads low in FODMAPs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Inulinase are industrial food enzymes which have gained much attention in recent scenario. In this study, Inulinase producing eight bacterial colonies were isolated and screened from three different plant root tubers soil sample. Among 8 inulinase producing colonies, the higher yielding colony was selected with 25.10 U/mL for further studies. The best inulinase producing colony was identified by partial 16S rRNA gene sequence as Bacillus sp. The crude inulinase was purified by using ammonium sulphate precipitation, dialysis and ion exchange chromatography on DEAE - sephacel and obtained 1.9 purification fold with total activity 293 U. The purified enzyme was subjected to characterization studies and it was found to be stable at 30-60 °C and optimum temperature was at 55 °C. The enzyme was stable at pH 3.0-7.0 and optimum pH was at 6.5. The K m and V max value for inulinase was found to be 0.117 mg/mL and 4.45 μmol min mg -1 respectively, demonstrate its greater affinity. Hence, this enzyme can be widely used for the production of fructose, and fructooligosaccharides, which are important ingredients in food and pharmaceutical industry.

In order to find an alternative for commercial inulinase, a strain XL01 identified as Penicillium sp. was screened for inulinase production. The broth after cultivated was centrifuged, filtered, and used as crude enzyme for the following saccharification. At pH 5.0 and 50 °C, the crude enzyme released 84.9 g/L fructose and 20.7 g/L glucose from 120 g/L inulin in 72 h. In addition, simultaneous saccharification and fermentation of chicory flour for D-lactic acid production was carried out using the self-produced crude inulinase and Lactobacillus bulgaricus CGMCC 1.6970. A high D-lactic acid titer and productivity of 122.0 g/L and 1.69 g/(L h) was achieved from 120 g/L chicory flour in 72 h. The simplicity for inulinase production and the high efficiency for D-lactic acid fermentation provide a perspective and profitable industrial biotechnology for utilization of the inulin-rich biomass.

Background: Taking into account its thermotolerance, high growth rate, and broad substrate spectrum, Kluyveromyces marxianus can be considered an ideal consolidated bioprocessing (CBP). A major obstacle to ethanol production using K. marxianus is the low production of lignocellulolytic enzymes, which reduces the cellulose hydrolysis and ethanol production. Thus, further improvement of enzyme expression and secretion is essential., Results: To improve the expression of lignocellulolytic enzymes, the inulinase promoter and signal sequence from K. marxianus was optimized through mutagenesis. A T(-361)A mutation inside the promoter, a deletion of AT-rich region inside 5'UTR (UTR∆A), and a P10L substitution in the signal sequence increased the secretory expression of the feruloyl esterase Est1E by up to sixfold. T(-361)A and UTR∆A increased the mRNA expression, while the P10L substitution extended the hydrophobic core of signal sequence and promoted secretion of mature protein. P10L and T(-361)A mutations increased secretory expressions of other types of lignocellulolytic enzymes by up to threefold, including endo-1,4-β-glucanase RuCelA, endo-1,4-β-endoxylanase Xyn-CDBFV, and endo-1,4-β-mannanase MAN330. During the fed-batch fermentation of strains carrying optimized modules, the peak activities of RuCelA, Xyn-CDBFV, MAN330, and Est1E reached 24 U/mL, 25,600 U/mL, 10,200 U/mL, and 1220 U/mL, respectively. Importantly, higher yield of enzymes by optimized promoter and signal sequence were achieved in all tested carbon sources, including the major end products of lignocellulose saccharification and fermentation, with growth on xylose resulting in the highest production., Conclusions: The engineered promoter and signal sequence presented increased secretory expressions of different lignocellulolytic enzymes in K. marxianus by means of various carbon resources. Activities of lignocellulolytic enzymes in fed-batch fermentation were the highest activities reported for K. marxianus so far. Our engineered modules are valuable in producing lignocellulolytic enzymes by K. marxianus and in constructing efficient CBP strains for cellulosic ethanol production.

The enzymatic hydrolysis of poly- and oligosaccharides from plants seems like an advantageous approach for sugars production. Two inulinases producing fructose from plant oligosaccharides were isolated from yeast Kluyveromyces marxianus and plant Helianthus tuberosus. Both enzymes were immobilized on polymeric carriers by using the static adsorption approach. We could save 80.4% of the initial catalytic activity of plant inulinase immobilized on KU-2 cation-exchange resin and 75.5% of yeast enzyme activity adsorbed on AV-17-2P anion-exchange resin. After immobilization, the Km values increased 1.5 and 6 times for enzymes from K. marxianus and H. tuberosus, respectively. The optimal temperatures for catalysis of both enzymes were increased from 48-50 °C up to 70 °C. The activities of both immobilized enzymes remained unchanged after the 10 cycles of 20-min hydrolysis reaction at 70 °C model batch reactor. Sorbents, native and immobilized enzymes did not exhibit any mutagenic or cytotoxic activity., (Copyright © 2018. Published by Elsevier B.V.)

The consumption of natural and low calorie sugars has increased enormously from the past few decades. To fulfil the demands, the production of healthy sweeteners as an alternative to sucrose has recently received considerable interest. Fructose is the most health beneficial and safest sugar amongst them. It is generally recognised as safe (GRAS) and has become an important food ingredient due its sweetening and various health promising functional properties. Commercially, high fructose syrup is prepared from starch by multienzymatic process. Single-step enzymatic hydrolysis of inulin using inulinase has emerged as an alternate to the conventional approach to reduce complexity, time and cost. The present review, outlines the enzymatic strategies used for the preparation of high fructose syrup from inulin/inulin-rich plant materials in batch and continuous systems, and its conclusions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Response surface methodology has been implemented for the utilization of corn bran for inulinase production by Penicillium oxalicum . CCRD of RSM with 15 runs was practiced to optimize three independent variables: moisture (70-90%), incubation time (4-8 days) and pH (5-8). However, other media constituents viz. inulin (1%), NaNO 3 (0.2%), NH 4 H 2 PO 4 (0.2%), KH 2 PO 4 (0.2%), MgSO 4 ·7H 2 O (0.05%) and FeSO 4 ·7H 2 O (0.001%) were kept constant during solid state fermentations. Solid state fermentations were carried out at 30 °C at flask-level. A substantial inulinase production (77.95 IU/gds) was obtained under the optimized conditions i.e., moisture (80%), incubation time (6.0 days) and pH (6.5). Multiple correlation coefficient ' R ' for inulinase production was 1.00, which justifies good agreement between experimental and predicted values. Besides, ' 2 ' value close to one, also authenticates the validity of the model. The experimentation carried out at laboratory scale shown corn bran a good substrate for inulinase production by R 2 ' value close to one, also authenticates the validity of the model. The experimentation carried out at laboratory scale shown corn bran a good substrate for inulinase production by P. oxalicum .

Tithonia rotundifolia is an easily available and abundant inulin rich weed reported to be competitive and allelopathic. This weed inulin is hydrolyzed by inulinase into fructose. Response surface methodology was employed to optimize culture conditions for the inulinase production from Arthrobacter mysorens strain no.1 isolated from rhizospheric area of Tithonia weed. Initially, Plackett- Burman design was used for screening 11 nutritional parameters for inulinase production including inulin containing weeds as cost effective substrate. The experiment shows that amongst the 11 parameters studied, K 2 HPO 4 , Inulin, Agave sisalana extract and Tithonia rotundifolia were the most significant variables for inulinase production. Quantitative effects of these 4 factors were further investigated using Box Behnken design. The medium having 0.27% K 2 HPO 4 , 2.54% Inulin, 6.57% Agave sisalana extract and 7.27% Tithonia rotundifolia extract were found to be optimum for maximum inulinase production. The optimization strategies used showed 2.12 fold increase in inulinase yield (1669.45 EU/ml) compared to non-optimized medium (787 EU/ml). Fructose produced by the action of inulinase was further confirmed by spectrophotometer, osazone, HPTLC and FTIR methods. Thus Tithonia rotundifolia can be used as an eco-friendly, economically feasible and promising alternative substrate for commercial inulinase production yielding fructose from Arthrobacter mysorens strain no.1., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The Jerusalem artichoke is a perennial plant that belongs to the sunflower family. As a non-grain crop, Jerusalem artichoke possesses a number of desirable characteristics that make it a valuable feedstock for biorefinery, such as inulin content, rapid growth, strong adaptability, and high yields. This review provides a comprehensive introduction to renewable Jerusalem artichoke-based biomass resources and recent advances in bio-based product conversion. Furthermore, we discuss the latest in the development of inulinase-producing microorganisms and enhanced inulin hydrolysis capacity of microbes by genetic engineering, which lead to a more cost-effective Jerusalem artichoke biorefinery. The review is aimed at promoting Jerusalem artichoke industry and new prospects for higher value-added production.