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The purpose of this research was to evaluate the suitability of whey as an effective medium for the coproduction of inulinase and invertase by an oleaginous yeast Galactomyces geotrichum and to investigate the effects of some additional carbon and nitrogen sources. The nutritional factors and composition of the medium have a great impact on the production pathways of microbial enzymes. To deepen the research, a Taguchi design was employed to quickly scan the best conditions. First, the cheese whey was partly deproteinized and investigated as the sole medium for the yeast. The next step was performed to study the effects of inulin, sucrose and lactose as carbon sources and ammonium sulfate, yeast extract and casein as nitrogen sources. All analyses (Taguchi and ANOVA) were performed using Minitab software. Whey-based medium without any additional carbon and nitrogen sources gave inulinase and invertase activities as 54.6 U/mL and 47.4 U/mL, respectively. Maximum inulinase activity was obtained as 77.9 U/mL using inulin as the carbon source without any nitrogen source. The highest I/S ratio was found as 2.08. On the other hand, the highest invertase activity was carried out as 50.85 U/mL in whey-based medium using lactose as carbon source without any additional nitrogen source. This is the first report about partly deproteinized whey-based medium utilization for simultaneous inulinase and invertase production by G. geotrichum TS-61. Moreover, the effects of carbon and nitrogen sources were investigated in detail. Whey is a sufficient medium for inulinase and invertase production Inulin is an excellent carbon source for enhanced inulinase activity Taguchi orthogonal array presents an effective and quick screening method for the fermentation process [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Co-immobilization of cells and enzymes is often essential for the cascade biocatalytic processes of industrial-scale feasibility but remains a vast challenge. Herein, we create a facile co-immobilization platform integrating enzymes and cells in covalent organic frameworks (COFs) to realize the highly efficient cascade of inulinase and E. coli for bioconversion of natural products. Enzymes can be uniformly immobilized in the COF armor, which coats on the cell surface to produce cascade biocatalysts with high efficiency, stability and recyclability. Furthermore, this one-pot in situ synthesis process facilitates a gram-scale fabrication of enzyme-cell biocatalysts, which can generate a continuous-flow device conversing inulin to D-allulose, achieving space-time yield of 161.28 g L−1 d−1 and high stability (remaining >90% initial catalytic efficiency after 7 days of continuous reaction). The created platform is applied for various cells (e.g., E. coli, Yeast) and enzymes, demonstrating excellent universality. This study paves a pathway to break the bottleneck of extra- and intracellular catalysis, creates a high-performance and customizable platform for enzyme-cell cascade biomanufacturing, and expands the scope of biocatalysis process intensification. Co-immobilization of cells and enzymes is essential to leverage the advantages of both cell-free enzymes and whole-cells for biomanufacturing. Here, the authors design covalent organic frameworks to co-immobilize inulinase and E. coli for the conversion of inulin to D-allulose with the yield of 161 g/L/day. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction. Chicory root (Cichorium intybus L.) contains valuable components such as inulin, inulides, bitter substances, pectin, and fibers and is a promising non-traditional raw material for the production of new beer varieties and reduction of their cost. Materials and methods. Dried and roasted chicory roots, lager and caramel barley malt, enzyme preparation inulinase, granulated hops of "Agnus" variety, yeast Saccharomyces cerevisiae (race RH) were used. Inulin content was determined by spectrophotometric method, amine nitrogen was measured by Pope and Stevenson method, content of reducing substances was estimated by Wilstetter-Schudl method. Standard methods accepted in brewing were used to determine other beer characteristics, sensory parameters were evaluated using profile methods. Results and discussion. The technology for light beer production was proposed, according to which light malt and dried crushed chicory roots taken in the amount of 4% of malt are used as raw materials, and at the stage of mashing the enzyme preparation inulinase with activity of 14 units/g was introduced into the mixture. For the hydrolysis of inulin at the mashing stage, an inulase pause was provided at a temperature of 55-56 ℃ for 20-30 minutes. The method allows increasing the content of reducing substances in the wort by 1.6%, the apparent and actual fermentation degree by 3.9%, the content of alcohol and carbon dioxide in the beer by 3.2 and 10%, respectively. The finished beer had increased foam resistance and higher foam height, while the introduction of chicory did not impart excessive and extraneous bitterness. The innovative technology of dark beer provides mixing of aqueous extract of roasted chicory with malt wort cooled to the temperature of 85-90 ℃. It was found that the optimal mode of the extraction process was the temperature 85-90 ℃, hydromodule 1:6, and duration 90 min. Beer with chicory content of 3% was the best in terms of physical, chemical, and sensory properties. The improved method allows to increase the content of reducing substances in wort by 1.5%, apparent and actual fermentation degree by 2.2 and 3%, respectively, to increase the content of alcohol in beer by 2.3%, and carbon dioxide by 3.2%. It was proved that partial replacement of malt by chicory allows to reduce consumption of bitter hops for light beers by 20% (from 14.8 to 12.0 g/dal) and for dark beers by 10% (from 10.3 to 9.3 g/dal). Conclusions. The addition of dried and fried chicory roots makes it possible to obtain new varieties of high-quality beer with their cost reduction. [ABSTRACT FROM AUTHOR]

Abstract 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.

Background: Luffa cylindrica has numerous domestic and industrial uses. For example, this natural fiber supports immobilization through the covalent bonding of an inulinase. In addition, fructose syrup from crude inulin obtained from prickly pear (Opuntia ficus-indica) can be obtained in a plug-flow mini reactor with this immobilized inulinase on L. cylindrica. Results: A central composite design of experiments was used to maximize the enzymatic fructose production from crude inulin obtained from Opuntia ficus-indica in a plug-flow mini reactor. The experiments explore temperature (between 45 and 55°C), pH (4.0-5.0), and feed flow (0.1-0.2 ml/min). After verifying the adequacy of the quadratic model for productivity, it was maximized to find the optimal condition. It was at 49.97°C, 4.6 and 0.20 ml/min for the temperature, pH, and flow, respectively. Under the optimal condition, the quadratic model suggested a productivity of 2.456 ± 0.015 mg/h. Three validation experiments confirmed the validity of the model. Conclusions: The results confirmed the suitability of L. cylindrica as support for the immobilization of inulinase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The objective of this study was to create a fermentation process for the production of inulinase, an important enzyme with numerous applications in the food and pharmaceutical industries, using low-cost agricultural waste as substrates for Rhizopus oryzae NRRL 3563. High titer inulinase production in chicory roots by Rhizopus oryzae in a submerged culture was accomplished using a statistical experimental design. A two-level Plackett–Burman design followed by a three-level Box–Behnken design producing a high inulinase titer of 1085.11 U/mL, 2.83-fold the maximum level, was obtained in the screening experiment. The optimal levels were as follows: chicory root, 10 g/L; NaNO3, 5 g/L; and KCl, 0.2 g/L. The produced inulinase enzyme was purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.63-fold purification with 60% activity recovery. The enzyme had a molecular weight of approximately 130 KDa. The purified enzyme showed optimum activity at 50 °C and pH 6.0. The pH stability range was three to six and the temperature stability was up 70 °C. The purified inulinase could hydrolyze inulin and sucrose, but not cellobiose or soluble starch. Km and Vmax for inulin were determined to be 0.8 mg/mL and 50,000 U/mg, respectively. The two-level Plackett–Burman design was applied followed by a Box–Behnken model for optimization of fermentation conditions. Accordingly, the optimal combination of fermentation was a reaction time of seven hours, a temperature of 60 °C, and an enzyme concentration of 40,000 U/mL, which resulted in a 58.07% saccharification yield. The characteristics of the enzyme and its kinetic parameters suggested that it was highly effective in the fermentation of inulin and inulin-containing substrates. Additionally, it raises the potential of using inulinase enzymes in pharmaceutical and food industries. [ABSTRACT FROM AUTHOR]

Inulinase is an enzyme that catalyzes inulin to d‐fructose. This enzyme can be extracted from plants, but it is difficult to obtain it in large quantities, so its production cost is high. Therefore, microbial inulinase has great potential for industrial needs. In the last decade, there have been very few reports on actinobacterial inulinases, especially on purification and characterization of inulinase process extraction. This study aims to select actinomycetes that possess high inulinase activity from the soil. To screen inulinase‐producing bacteria, modified Czapex‐Dox agar supplemented with 1% inulin powder was used. The most effective isolate was Streptomyces sp. EFBO8, morphological and genotypic identification methods, confirmed that the strain is Streptomyces anulatus and that its nucleotide sequence has been deposited in GenBank under accession number OQ073700. To optimize inulinase production, kinetics were performed by using S. anulatus strain, which proved to be most productive with a value of 24,024 EU/mL. The enzyme was purified from the culture filtrate by precipitation with ammonium sulfate (NH4)2SO4, followed by column chromatography Sephadex (G‐50) separation. Purified protein has a molecular mass of 3331.83 Da. [ABSTRACT FROM AUTHOR]

Jerusalem artichoke (JA) is a nutritional vegetable for human diet depending on its natural structure, especially high inulin content and it is the second inulin source for commercial production in the world, after chicory. It was aimed to investigate the inulinase production capability of Galactomyces geotrichum TS61 (GenBank accession: MN749818) using JA as an economical and effective substrate comparing with the pure chicory inulin and to optimize the fermentation using Taguchi design of experiment (DOE) in this study. Besides, the effects of sucrose on inulinase production either combined with JA or in its absence were also studied. Taguchi L16 orthogonal array was employed for optimization. Both of inulinase activities obtained from JA and pure inulin gave the maximum result at the 10th experimental run as 40.21 U/mL and 57.35 U/mL, respectively. The optimum levels were detected for each factor as, 30 g/L JA, 30 g/L sucrose, pH 5.5, and four days for time. The predicted value was found as 41.63 U/mL that was similar to the obtained result as 41.17 U/mL. Finally, inulinase activity was increased approximately 8-folds after optimization. The sucrose-free medium had similar effects with higher concentrations of JA at long incubation time. This is the first investigation about inulinase production by G. geotrichum. Low-cost inulinase production was achieved using an economical substrate Sucrose effects were investigated in detail on inulinase production Use of Taguchi DOE supported effective enzyme production [ABSTRACT FROM AUTHOR]

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 (R2 = 0.9976 and 0.9974, respectively), Huang model for inulinase production (R2 = 0.9967), Weibull model for invertase‐type production (R2 = 0.9963), and modified logistic model for invertase‐type activity/inulinase activity ratio (R2 = 0.9292) with high R2 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 R2 values for consumption and production (R2 > 0.90). Sensitivity analysis of the selected models showed that the yielded R2 values (R2 ≥ 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. [ABSTRACT FROM AUTHOR]

In this study, thirty yeast strains isolated from the gut of coprophagous "Gymnopleurus sturmi" and twenty-four from the dung of ruminants were shown to be producers of cellulases. Cellulolytic yeast isolates could also produce other hydrolytic enzymes such as pectinase, lipase, β-glucosidase, catalase, inulinase, urease, gelatinase, and protease. The oroduction of amylase was present in only one isolate of dung of ruminants. On the other hand, the production of tannase was absent in these isolates. All the yeasts isolated from two sources could utilize various carbon sources, including sorbitol, sucrose, and raffinose, and withstand high concentrations of glucose (300 g/L), salt (100 g/L), and exogenous ethanol. They could grow in a wide pH range of 3 to 11. The growth was stable up to a temperature of 40 °C for isolates from the gut of coprophage and 37 °C for the yeast from the dung of ruminants. These activities and growing conditions were similar to the diet of coprophagous insects and the composition of ruminant manure, likely because the adaptation and distribution of these microorganisms depend on the phenology and trophic preferences of these insects. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (t1/2 = 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 ≤ ΔG*d ≤ 111.56 kJ mol−1). These results indicate that A. terreus URM4658 inulinase is a promising and efficient biocatalyst, which could be fruitfully exploited in long-term industrial applications. [ABSTRACT FROM AUTHOR]

The study aimed to isolate rare halophilic actinomycetes from hypersaline soils of Algerian inland Wetland Ecosystems "Sebkhas‐Chotts" located in arid and hot hyperarid lands with international importance under the Ramsar Convention and to explore their enzyme‐producing and antibacterial abilities. The halophilic actinomycetes were selectively isolated using agar‐rich media supplemented with 5, 10, and 15% (W/V) of total salts. Thirty‐one isolates were obtained and 16S rRNA gene sequencing analysis revealed the presence of members affiliated to rare halophilic actinobacterial genera (Actinopolyspora and Nocardiopsis) accounting for 74.19% (23 isolates out of 31) and 25.8% (8 isolates), respectively. Both phylotypes are alkalitolerant and halophilic thermotolerant actinomycetes displaying significant hydrolytic activities relative to (amylase, asparaginase, cellulase, esterase, glutaminase, inulinase, protease, pectinase, xylanase), and over 96% of tested isolates exhibited all common enzymes, mainly active at 10% of growing salt. In addition, high antibacterial activity was observed against Bacillus cereus, Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus. The findings showed that saline wetlands ecosystems represent a rich reservoir for the isolation of significant rare halophilic actinomycetes with potential adaptive features and valuable sources for novel bioactive metabolites and biocatalysts of biotechnological interest. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Three signal peptides from α-mating factor (α-MF), inulinase (INU) and native levansucrase (LS) were compared for secretion efficiency of Bacillus subtilis levansucrase SacB-T305A in Pichia pastoris GS115. The first complete secretion of bacterial levansucrase in yeasts under methanol induction was achieved while using α-MF signal. The secreted recombinant Lev(α-MF) proved to be glycosylated by combination of NanoLC-MS/MS and Endo H digestion. Interestingly, glycosylation not only improved significantly the polymerase thermostability, but also reversed the products profiles to favor synthesis of high molecular weight (HMW) levan which accounted for approximately 73 % to total levan-type polysaccharides. It indicated for the first time that the glycosylation of recombinant B. subtilis levansucrase affected significantly the products molecular weight distribution. It also provided a promising enzymatic way to effectively product HMW levan from sucrose resources. [Display omitted] • Complete secretion of Bacillus subtilis levansucrase in Pichia pastoris. • Recombinant levansucrase occupied vast majority of secretory proteins. • Half-life of polymerase at 40 °C increased to 2.4 fold. • Glycosylated levansucrase favor synthesis of high molecular weight (HMW) levan. • HMW levan (average 2000 kDa) accounted for approximately 73 % of products. [ABSTRACT FROM AUTHOR]

Inulinase enzyme (EC 3.2.1.80) is an enzyme capable of hydrolyzing inulin and is used as a catalyst in the production of High Fructose Syrup (HFS) and fructooligosaccharides (FOS). Inulinase enzymes can be produced by yeasts found in waste such as kepok banana peels (Musa acuminata x balbisiana). The aims of this study were to obtain the inulinase enzymeproducing yeast from kepok banana peels and to determine the effect of commercial inulin concentration and incubation time on enzyme activity. This research was conducted experimentally using a factorial completely randomized design with factors being inulin concentration of 1% (K1), 3% (K2) and 5% (K3) and incubation time of 6th (W1), 12th (W2), 18th (W3), and 24th hours (W4). The results showed that there were 4 isolates of inulinolytic yeasts obtained from kepok banana peels, namely YP1, YP2, YP4, and YP6. However, the most potential isolate to be used in inulinase production was YP2 isolate because it had the highest inulinolytic ratio value of 1.96. The results of inulinase activity showed that only inulin substrate with concentration of 5% (K3) had a significant or significant effect on inulinase production. [ABSTRACT FROM AUTHOR]

Halophiles are excellent sources of enzymes which are not only salt stable but also perform catalysis under extreme reaction conditions. The habitat of salt mines and its microbial diversity have not been thoroughly explored. In the present study, seven halophilic inulinase-producing bacteria (H1N1–H1N7) were isolated from the Bahadarkhel salt mines of Pakistan, and were identified based on their morphological, biochemical and molecular features. The strains H1N3, H1N4, H1N6, H1N7 were capable of growing at 20% salt concentration and therefore were considered as extremely halotolerant. Inulinase activities of the isolates were detected in inulin-containing broth media, and were further confirmed on inulin-based agar plates. The strains HIN1, H1N3, H1N5, and H1N7 exhibited the maximum zone of clearance (≥15 mm) on inuline agar medium. The strain H1N1 showed the highest zone of clearance of 19 mm. Morphological, biochemical and molecular characterization showed that the inulinase producing isolates were related to Bacillus and Klebsiella genera. The molecular identification was based on 16S rRNA gene sequencing. The strain H1N2 was capable of producing the four tested halozymes including inulinase, amylase, protease, and lipase. The strains HIN3, HIN4, and H1N5 were capable of producing three halozymes, i.e., inulinase, amylase, and protease while the strains H1N6 and H1N7 produced inulinases, lipases and proteases. These salt tolerant bacterial strains exhibiting diverse enzyme characteristics and phylogeny could be highly useful for future practical applications. [ABSTRACT FROM AUTHOR]

Inulinases are an important class of enzymes used in many fields, especially in the food and pharmaceutical industries, to produce fructose syrups. Microbial inulinases are important in the hydrolysis of inulin to produce fructose syrup and FOS. These enzymes are produced by various strains of microorganisms, of which Aspergillus sp. and Kluyveromyces sp. are the most commonly used strains for inulinase production. The goal of the study was to biosynthesis inulinase using the Aspergillus terreus ICCF 262 strain, with inulin and orange peel powder as carbon and energy sources, the enzyme being isolated from the fermentation medium by fractional precipitation with ammonium sulfate followed by purification on DEAE-Cellulose using ion exchange chromatography. Within 7 days of cultivating the fungal strain on a mineral medium containing inulin and orange peel at a final concentration of 2% in the fermentation medium, yields of biotechnological interest were higher than those previously reported in the literature. Through the procedure of isolating and purifying inulinase from the fermentation medium results a specific activity of between 164.6 - 396.4 U / mg protein. [ABSTRACT FROM AUTHOR]

The objective of this study was to convert inulin to high output fructose with the commercial inulinase in the continuous ultrafiltration membrane bioreactor (UFMB). The effects of operational conditions were explored for the volumetric productivity. The model equations of kinetics and membrane fouling were also founded. The stable fructose production with the volumetric productivity was attained at the optimum condition of inulin concentration (100 g/L), E/S ratio (40 U/g) and residence time (20 min). The kinetics results showed that the experimental data obtained were fit to the model equations. Preliminary experimental results were attained the fundamental membrane fouling mechanism. These results demonstrated its feasibility and availability for efficient fructose production, which were beneficial to provide theoretical basis for further researches. The operational conditions were explored for the volumetric productivity. Model equations and parameters of kinetics were founded and obtained. The membrane fouling mechanism of UFMB system was founded. [ABSTRACT FROM AUTHOR]

Kluyveromyces marxianus is the most suitable fungus for inulinase industrial production. However, the underlying transcriptional activation mechanism of the inulinase gene (INU1) is hitherto unclear. Here, we undertook genetic and biochemical analyses to elucidate that a glycolysis regulator KmGcr1p with unique Q‐rich repeats is the key transcriptional activator of INU1. We determined that INU1 and glycolytic genes share similar transcriptional activation patterns and that inulinase activity is induced by fermentable carbon sources including the hydrolysis products of inulin (fructose and glucose), which suggests a novel model of product feedback activation. Furthermore, all four CT‐boxes in the INU1 promoter are important for KmGcr1p DNA‐binding in vitro, but the most downstream CT‐box 1 primarily confers upstream activating sequence activity in vivo. More intriguingly, the use of artificial and natural GCR1 mutants suggests that the Q‐rich repeats act as a functional module to maintain KmGcr1p transcriptional activity by contributing to its solubility and DNA‐binding affinity. Altogether, this study uncovers a novel transcriptional activation mechanism for the inulinase gene, that is different from the previous understanding for filamentous fungi, but might have universal significance among inulinase‐producing yeasts, thereby leading to a better understanding of the regulation mechanism of yeast inulinase genes. [ABSTRACT FROM AUTHOR]

Inulinase was immobilised by entrapment method in polyacrylamide/polyethylene glycol composite and evaluated for its efficiency for short-chain fructooligosaccharides (3–6 degrees of polymerisation) production in batch hydrolysis system. Aqueous two-phase polymerisation technique was used to synthesise the composite, where aqueous polyethylene glycol 1000 containing the enzyme was used as dispersant with ammonium persulfate as initiator. The characteristics of free and immobilised inulinase were investigated and compared, and the results showed shift of pH and temperature optimum and change in stability caused by the immobilisation material. The immobilised preparation retained 50% of its initial activity after 20 successive batch cycles of 1 h each. The conversion degree of highly polymerised inulin to fructooligosaccharides (3–6 degrees of polymerisation) was 36% when using 30% PAA/PEG, w/v. ATPS extracted inulinase was effectively immobilised in PAA/PEG composite. Immobilised enzyme showed good pH and thermal stability. Immobilised catalyst presents good retention of activity after 20 reuses. The entrapped enzyme is effective in producing FOS with the DP from 3 to 6 with proved prebiotic activities. The optimum conditions for batch operating mode were: 2% (w/v) inulin, 30% (w/v) PAA/PEG composite, pH = 4.4 and T = 40ºC. [ABSTRACT FROM AUTHOR]

Background: Engineering thermal adaptations of enzymes is a popular field of study. Enzymes active at low temperature have been used in many industries; however, reports seldom describe improvements in enzyme activity at low temperatures using protein engineering. Results: Multiple amino acid sequence alignment of glycoside hydrolase (GH) family 32 showed an unconserved region located in the catalytic pocket. The exo-inulinase InuAGN25 showed the highest frequency of charged amino acid residues (47.4%) in this region among these GH 32 members. Notably, five consecutive charged amino acid residues (137 EEDRK141) were modeled as a loop fragment in this region of InuAGN25. Deletion of the loop fragment broke two salt bridges, one cation-p interaction, and the a-h elix-loop-310-helix structure at the N-terminal tail. The mutant exo-inulinase RfsMutE137D5 without the loop fragment was expressed in Escherichia coli, digested using human rhinovirus 3C protease for removal of the fused sequence at the N-terminus, and purified using immobilized metal affinity chromatography. Compared to the wild-type enzyme, the optimum temperature and t1/2 at 50°C of purified RfsMutE137D5 decreased by 10°C and 31.7 min, respectively, and the activities at 20°C and 30°C increased by 11% and 18%, respectively. Conclusions: In this study, we engineered the loop to obtain the mutant exo-inulinase that showed an improved performance at low temperatures. These findings suggest that the loop may be a useful target in formulating rational designs for engineering thermal adaptations of GH 32 exo-inulinases. [ABSTRACT FROM AUTHOR]

One of the major issues for the microbial production of polyhydroxyalkanoates (PHA) is to secure renewable, non-food biomass feedstocks to feed the fermentation process. Inulin, a polydisperse fructan that accumulates as reserve polysaccharide in the roots of several low-requirement crops, has the potential to face this challenge. In this work, a "substrate facilitator" microbial consortium was designed to address PHA production using inulin as feedstock. A microbial collection of Bacillus species was screened for efficient inulinase producer and the genome of the selected strain, RHF15, identified as Bacillus gibsonii , was analysed unravelling its wide catabolic potential. RHF15 was co-cultured with Cupriavidus necator , an established PHA producer, lacking the ability to metabolize inulin. A Central Composite Rotary Design (CCRD) was applied to optimise PHA synthesis from inulin by the designed artificial microbial consortium, assessing the impact of species inoculum ratio and inulin and N-source concentrations. In the optimized conditions, a maximum of 1.9 g L−1 of Polyhydroxybutyrate (PHB), corresponding to ~80% (g polymer /g CDW) polymer content was achieved. The investigated approach represents an effective process optimization method, potentially applicable to the production of PHA from other complex C- sources. [Display omitted] • C. necator is an established PHA producer lacking the ability to metabolize inulin. • B. gibsonii RHF15 was discovered as a new inulinase producer. • Co-culturing the two bacterial strains on inulin lead to effective PHB accumulation. • Central Composite Rotary Design allowed optimization of consortium performances. [ABSTRACT FROM AUTHOR]

[Display omitted] • Solid culture boosts inulinase and invertase production with no catabolic repression. • Inulinase is the preponderant enzyme under solid and submerged cultures. • Aspergillus brasiliensis growth is higher in solid than in submerged culture. Extracellular inulinase and invertase production by Aspergillus brasiliensis was evaluated in solid-state fermentation (SSF) and submerged fermentation (SmF) with a high sucrose concentration. The parameters estimated from online CO 2 analysis revealed a lower Lag phase in SSF compared to SmF; the specific CO 2 production rate (μ CO2) decreased in both systems as the sucrose concentration increased. The highest maximum CO 2 production rate (10.45 ± 0.72 mg/h mL) and total CO 2 production (138.53 ± 6.48 mg/mL) were obtained in SSF with a sucrose concentration of 210 g/L. Inulinase and invertase production (1.13 ± 0.02 and 5.84 ± 0.12 U/mL, respectively) in SmF was independent of the initial sucrose concentration; meanwhile, both activities increased in SSF as the sucrose concentration increased, reaching maximum values of 5.88 ± 0.78 and 41.69 ± 3.60 U/mL, respectively, at the highest sucrose concentration (210 g/L). The zymographic analysis showed one band of inulinase and two bands of invertases involved in sucrose hydrolysis. [ABSTRACT FROM AUTHOR]

The main objectives of this study were to investigate the effect of phenol on the production of Aspergillus niger A42 (ATCC 204447) inulinase and to model the best fermentation in the medium with inhibitor. When the phenol concentrations added into the fermentation medium increased from 0 to 5 g/L, the inulinase and invertase‐type activities and the maximum inulinase and invertase‐type production rates were reduced from 203.0 to 12.6 U/ml and from 156.9 to 3.1 U/ml, and from 32.6 to 2.9 U–1 · ml–1 · day–1 and from 32.6 to 1.1 U–1 · ml–1 · day–1. Additionally, the modified Gompertz, modified logistic, and Baranyi and Huang models successfully predicted the experimental data of inulinase production, invertase‐type production, and substrate consumption, respectively. Consequently, renewable resources can be used in inulinase productions without detoxification when the hydrolysate contains low concentrations of phenol. Furthermore, the models can serve as universal equations in modeling the enzyme fermentation in shake flask fermentation. Practical applications: Inulinase is used in various fields such as the production of prebiotic products fructooligosaccharides (FOS) and inulooligosaccharides (IOS), bioethanol, and fructose syrup. In addition to pure carbon sources, industrial wastes such as molasses or renewable resources are used as carbon sources. To use renewable resources, various pretreatments are applied before the upstream process. During pretreatment, various inhibitors such as phenols that are mostly formed by the degradation of lignin adversely affected fermentation. Phenolic compounds are removed from the fermentation medium by detoxification processes. The detoxification process is a very costly and time‐consuming, although it is very important for the efficiency of the fermentation. For this, the determination of the tolerance ranges of the microorganism to the phenol compounds is essential. Knowing the tolerance of Aspergillus niger used in this study to the phenol compound will benefit the industry in the use of dilute, direct or detoxifying hydrolysates. [ABSTRACT FROM AUTHOR]

This study aimed to investigate new bacterial sources with the ability to produce inulinase and to optimize fermentation conditions for inulinase production. The inulinase production was carried out using newly isolated Bacillus licheniformis and Bacillus velezensis among identified sixteen bacterial strains. The four essential variables incubation time (1-3 days), temperature (25-40°C), pH (5-7), and Wheat Bran (WB) level (1-5%) determined by the Plackett–Burman design (PB) were selected for optimization studies with Box-Benhken Design (BBD). These parameters were found to be very effective on inulinase production. The maximum inulinase activities for B. licheniformis and B. velezensis were 401.18 EU/mL and 344.61 EU/mL. Both inulinases sustained 50% of their initial activity at 30°C for 9 days. The results point out that bacteria are an important source as inulinase producer. The new isolates can be used in production of inulinase for industrial processes. [ABSTRACT FROM AUTHOR]

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. • β-mannanase and inulinase fermentations were carried out using SSF method. • The maximum mannanase and inulinase activities were found to be 71,174 U/mg and 564,065 U/mg of protein respectively. • In order to produce mannooligosaccharides and fructooligosaccharides, the enzymatic hydrolysis was carried out. • Enzymes application was increased the oligosaccharide activity of the coffee extract. [ABSTRACT FROM AUTHOR]

This paper presents the results of research of biochemical and physicochemical properties of extracellular inulinase synthesized by the new citric acid producer Aspergillus niger-1 (12000). A method for the isolation of extracellular homogeneous inulinase from the culture broth has been developed. It was revealed that this exoinulinase, possessing at the same time invertase activity (I/S=1.14), showed inulolytic activity and stability in a wide range of pH (3.0-7.0) and temperature (20-50 oC). The metal ions MnCl2, NaCl, MgSO4, CoCl2 partially increase, and FeCl3, ZnSO4, AgNO3 suppress or completely inhibit the catalytic activity of exoinulinase. The molecular weight is 63 kDa which is typical for inulinases produced by the strains belonging to the genus Asprgillus. Fructose and glucose, which are the end products of enzymatic hydrolysis of inulin, indicate the exo-type action of the studied exoinulinase, as well as the possibility of its application in biotechnology for the production of various products from inulin. The revealed properties of this exoinulinase are also important for the optimization of the processes of citric acid synthesis by Aspergillus niger-1 from inulin under varying conditions of the fermentation. [ABSTRACT FROM AUTHOR]

Introduction: Fructose is a carbohydrate found in fruits, vegetables, and honey. It is a glucose monosaccharide isomer that has important applications in food industry. Because that is important to investigate its obtaining mechanisms. Present work shows inulinase in sodium alginate physical retention for inulin conversion to fructose process through hydrolysis, establishing inulin as a potential resource for obtaining it, since it is present in a crops that are produced in an agro-industrial way wide variety. Objective: To determine hydrolysis pH and temperature conditions, using immobilized inulinase in sodium alginate. Materials and methods: Enzymatic reaction was carried out in a stirred glass reactor, provided with temperature control for three hours at 120 rpm. For reducing sugars quantification, DNS method and Uv-Vis spectrophotometry were applied at a wavelength of 540 nm. A central composite design was used for hydrolysis conditions analysis. Results and Discussion: Statistical analysis established as favorable conditions for obtaining fructose a temperature of 40 ° C and a pH of 6, reaching productivity of up to 12.27 mg/h, which is consistent with scientific literature. Conclusions: Temperature and pH factors influence enzymatic conversion process and consequently had an effect on total fructose productivity (mg/h). [ABSTRACT FROM AUTHOR]

The authors used mesoporous silica microspheres as a support for the immobilization of inulinase from Aspergillus brasiliensis MTCC 1344 by the process of cross‐linking. Under optimized operating conditions of pH 6.0, particle/enzyme ratio of 2.0:1.0 and glutaraldehyde concentration of 7 mM, a maximum immobilization yield of 90.7% was obtained after a cross‐linking time of 12.25 h. Subsequently, the cross‐linked inulinase was utilized for the hydrolysis of 5% inulin, and a maximum fructose concentration of 31.7 g/L was achieved under the optimum conditions of pH 6.0 and temperature 60°C in 3 h. Furthermore, on performing reusability studies during inulin hydrolysis, it was observed that the immobilized inulinase could be reused up to 10 subsequent cycles of hydrolysis, thus providing a facile and commercially attractive process of high‐fructose syrup production. [ABSTRACT FROM AUTHOR]

Sixty Klebsiella pneumoniae isolates which isolated from urine and sputum were screened and found that 11 isolates from urine and 9 isolates from sputum had an ability to produce inulinase and Klebsiella pneumoniaeU7 revealed maximum level in inulinase productivity. Inulinase was partially purified by using 70% saturation of ammonium sulfate. Fructo-oligosaccharide (FOS) was produced by reacting the partially purified inulinase with inulin as its substrate and the produced FOS was characterizing by using UV-Spectrophotometer and HPLC analysis in comparison with FOS standard. The product FOS have an immunomodulatory effect on immunity in mice by elevated of total white blood cells count and cytokine (IL10 and INF-γ) in comparing with the control. [ABSTRACT FROM AUTHOR]

The aim of the study was to carry out high-activity and low-cost inulinase production from Rhizopus oryzae HBF351, a new fungal strain, in solid phase fermentation. For this, it was firstly aimed to determine the best substrate that supports high enzyme production and to optimize some parameters that affect the fermentation condition by a statistical approach. Five different substrates (wheat bran, oat bran, onion peel, apple pulp and lettuce root powder) were investigated to find the best substrat supporting maximum enzyme productions. Box–Behnken design (BBD) was employed to optimize fermentation conditions (initial pH (4-7), incubation temperature (25°C-45°C), initial moisture level (50-100%) and incubation time(3-7 days)) for the production of inulinase. In the study, wheat bran was the best substrate that supports enzyme activity. pH, incubation temperature, moisture level and incubation time have been shown to be highly effective parameters on enzyme production (p <0.0001). In the study, the fitness of the model was evaluated using R2 (regression coefficient) (0.99) and a good correlation were obtained between experimental and predicted results. Optimum conditions providing maximum inulinase activity (385.1 IU gds-1) were found as pH 5.18, 33.4 °C, 72.1 % moisture level and 4.7 days. As a result, this study provides a cost-effective method to produce inulinase from Rhizopus oryzae. Also, it can be suggested that the produced inulinase has strong potential for usage in the production of fructose syrup and other industrial areas due to its high activity. [ABSTRACT FROM AUTHOR]

BACKGROUND: This study developed a feasible catalytic method for d‐allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. RESULTS: d‐Allulose 3‐epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo‐inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L−1 Mn2+, the CSCA‐linkerE‐DAE fusion enzyme obtained through high cell‐density cultivation displayed a maximal exo‐inulinase activity of 21.8 U mg−1 and resulted in a yield of 6.3 g L−1d‐allulose and 39.2 g L−1d‐fructose using 60 g L−1 inulin as the raw material. Catechol‐modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA‐immobilized CSCA‐linkerE‐DAE (8 U g−1) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L−1d‐allulose and 32.7 g L−1 D‐fructose from 60 g L−1 inulin. CONCLUSION: This study shed light on a feasible and cost‐effective approach for the production of syrup containing d‐allulose and D‐fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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. Mn2+ is the activator of the enzyme, and Cu2+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Novel inulinase producer of K. pneumoniae for 2,3-butanediol production was isolated. • The enzyme was an intracellular inulinase with an optimal temperature of 30 °C. • Concentration of 2,3-butanediol and acetoin reached 80.4 g/L in batch fermentation. • A productivity of 2.23 g/(L⋅h) and yield of 0.426 g/g sugar were obtained. Inulin could be converted to bio-based chemicals by an inulinase producer without external inulinase, and the production of 2,3-butanediol was less than 50 g/L. In this work, a novel inulinase producer of Klebsiella pneumoniae H3 was isolated, and inulinase catalytic properties as well as 2,3-butanediol fermentation were investigated. The enzyme was an intracellular inulinase with an optimal pH of 6 ∼ 7 and a temperature of 30 °C. The use of inulin by H3 was dependent on the degree of polymerization (DP), and the average DP of inulin in fermentation broth increased from 2.82 to 8.08 in 24-h culture of batch fermentation. Acidic pretreatment was developed to increase inulin utilization by adjusting medium pH to 3.0 prior to sterilization. In batch fermentation with optimized medium and fermentation conditions, the concentration of target product (2,3-butanediol and acetoin) was 80.4 g/L with a productivity of 2.23 g/(L⋅h), and a yield of 0.426 g/g inulin. [ABSTRACT FROM AUTHOR]

Inulinase is an industrially applicable enzyme which is used widely in the production of ultra high fructose syrups and fructooligosaccharides. Production of inulinase enzyme in a cost effective manner is the major challenge faced. In the present research study, Trichoderma asperellum RSBR08 which has the capability to produce high exo-inulinase enzyme is produced by using the solid state fermentation. Process parameters for production of exo-inulinase by using solid state fermentation were optimized. Results showed high exo-inulinase production in garlic as substrate (199.2±6.3 U/gds), 45% moisture percentage (228.4±3.4), 26oC temperature (219.1±5.8) and 5.0 pH (213.6±4.5). Effect of different metals viz. Mg2+,Zn2+, K+,Ca2+, Na+,Mn2+ and Hg2+ were studied on the production of exo-inulinase enzyme in which Ca2+ induced the enzyme production (196.3±5.6) whereas Hg2+ has inhibited the inulinase production (37.0±3.3). Based on the above results, the solid substrate fermentation parameters optimized can be used industrially. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The feasibility of operation on a fluidized bed reactor (FBR) with inulinase imbedded in the gelatin alginate microspheres was investigated in order to improve the higher inulin conversion yields and enzyme stability than that of the previously obtained with the packed bed reactors. The operation processes were based on statistical analyses, the operational conditions of immobilized inulinase in a FBR system have been determined for immobilized enzyme load of 18 g, substrate concentration of 80 g/L, expansion ratio of 1.4 and substrate flow rate at 0.5 mL/min. According to the above-mentioned research parameters, the continuous fructose preparation with FBR system was sustainable for 10 days (240 h) and gained the productivity of 86.4 g/Ld. Compared with the previous results of the packed-bed reactor, the immobilized inulinase in the FBR system was applied in the inulin conversion, which appeared more effective. This study suggested that a system for the continuous and efficient enzymatic conversion of inulin in the FBR was founded, which could be potentially applicable for the scale-up production. The diagram of fluidized-bed reactor with immobilized inulinase for continuous inulin conversion [ABSTRACT FROM AUTHOR]