Characterization of a processive inulosucrase from Lactobacillus mulieris for efficient biosynthesis of high-molecular-weight inulin.

Inulin has been determined to have many exceptional properties and functions and has been used in the food and pharmaceutical fields. Recently, microbial high-molecular-weight inulin synthesized from sucrose by inulosucrase attracted much attention. In this study, a novel inulosucrase from Lactobacillus mulieris was constructed, overexpressed, purified, and identified. The recombinant enzyme displayed the maximum activity at pH 6.0 and 55 °C, and it exhibited high thermostability below 45 °C. After optimizing the production conditions, the conversion rate from 100 g/L sucrose to inulin reached 31 %, meanwhile, the maximum molecular weight of produced inulin reached 3.21 × 106 g/mol. The truncated IS showed a "processive" transfructosylation process, only synthesizing a small number of short-chain oligosaccharides with polymerization degrees below 6, which was in favor of the accumulation of high-molecular-weight inulin. Given this, L. mulieris inulosucrase might be a good potential candidate for the industrial production of high-molecular-weight inulin.

Molecular and biochemical characteristics of inulosucrase InuBK from Alkalihalobacillus krulwichiae JCM 11691.

Information about the inulosucrase of nonlactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the Gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0-9.0 and 50-55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multiangle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3-27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.

Temperature-dependent inulin nanoparticles synthesized by Lactobacillus reuteri 121 inulosucrase and complex formation with flavonoids.

Inulin nanoparticles (INNPs) are a biocompatible material which has a potential application for enhancing solubility and preventing degradation of compounds. In this work, we demonstrated that INNPs could be synthesized from sucrose using inulosucrase from Lactobacillus reuteri 121. Noticeably, dynamic light scattering (DLS) analysis showed that the derived INNPs exhibited uniformity in size, which was easily controlled by the reaction temperature. The effect of enzyme and sucrose concentration, as well as reaction time, was explored. Moreover, the solubility of INNPs in various organic solvents was also investigated, and we found that the INNPs were freely regenerated in water even though they had precipitated by organic solvents. Essentially, we demonstrated that the derived INNPs could be applied for flavonoid encapsulation. The solubility and stability of quercetin and fisetin in the INNPs complexes was higher than those of free compounds. These results make the INNPs very promising for many applications.

Exploring the sequence variability of polymerization-involved residues in the production of levan- and inulin-type fructooligosaccharides with a levansucrase.

The connection between the gut microbiome composition and human health has long been recognized, such that the host-microbiome interplay is at present the subject of the so-called "precision medicine". Non-digestible fructooligosaccharides (FOS) can modulate the microbial composition and therefore their consumption occupies a central place in a strategy seeking to reverse microbiome-linked diseases. We created a small library of Bacillus megaterium levansucrase variants with focus on the synthesis of levan- and inulin-type FOS. Modifications were introduced at positions R370, K373 and F419, which are either part of the oligosaccharide elongation pathway or are located in the vicinity of residues that modulate polymerization. These amino acids were exchanged by residues of different characteristics, some of them being extremely low- or non-represented in enzymes of the levansucrase family (Glycoside Hydrolase 68, GH68). F419 seemed to play a minor role in FOS binding. However, changes at R370 abated the levansucrase capacity to synthesize levan-type oligosaccharides, with some mutations turning the product specificity towards neo-FOS and the inulin-like sugar 1-kestose. Although variants retaining the native R370 produced efficiently levan-type tri-, tetra- and pentasaccharides, their capacity to elongate these FOS was hampered by including the mutation K373H or K373L. Mutant K373H, for instance, generated 37- and 5.6-fold higher yields of 6-kestose and 6-nystose, respectively, than the wild-type enzyme, while maintaining a similar catalytic activity. The effect of mutations on the levansucrase product specificity is discussed.

Inulin and its enzymatic production by inulosucrase: Characteristics, structural features, molecular modifications and applications.

Inulin, a natural fructan, cannot be hydrolyzed by digestive enzymes in the human body and plays a role as a dietary fiber and prebiotic. Due to its versatile physicochemical properties and physiological functions, inulin has been widely applied in food, pharmaceuticals, and many other fields. The microorganism-derived inulin-forming enzyme inulosucrase (ISase) (EC: 2.1.4.9) can biosynthesize higher-molecular-weight inulin than plants using sucrose as the sole substrate, and the enzyme also shows transfructosylation activity toward other saccharide acceptors. In this article, the properties, functions, and applications of inulin are overviewed. The biosynthesis of inulin by ISase is addressed, including ISase characteristics, structural features, molecular modifications and applications.

Synthesis of fructooligosaccharides (FosA) and inulin (InuO) by GH68 fructosyltransferases from Bacillus agaradhaerens strain WDG185.

Fructooligosaccharides (FOS) and inulin, composed of ß-2-1 linked fructose units, have a broad range of industrial applications. They are known to have various beneficial health effects and therefore have broad application potential in nutrition. For (modified) inulin also for non-food purposes more applications are arising. Examples are carboxymethylated inulin as anti-scalant and carboymlated inulin as emulsifiers. Various plants synthesize FOS and/or inulin type of fructans. However, isolating of FOS and inulin from plants is challenging due to for instance varying chains length. There is an increasing demand for FOS and inulin oligosaccharides and alternative procedures for their synthesis are attractive. We identified and characterized two fructosyltransferases from Bacillus agaradhaerens WDG185. FosA, a ß-fructofuranosidase, synthesises short chain fructooligosaccharides (GF2-GF4) at high sucrose concentration, whereas InuO, an inulosucrase, synthesises a broad range of inulooligosaccharides (GF2-GF24) from sucrose, very similar to plant derived inulin. FosA and InuO showed activity over a broad pH range from 6 to 10 and optimal temperature at 60°C. Calcium ions and EDTA were found to have no effect on the activity of both enzymes. Kinetic analysis showed that only at relatively low substrate concentrations both enzymes showed Michaelis-Menten type of kinetics for total and transglycosylation activity. Both enzymes showed increased transglycosylation upon increasing substrate concentrations. These are the first examples of the molecular and biochemical characterization of a ß-fructofuranosidase (FosA) and an inulosucrase enzyme (InuO) and its product from a Bacillus agaradhaerens strain.

Biosynthesis of inulin from sucrose using inulosucrase from Lactobacillus gasseri DSM 20604.

Inulin is composed of fructose residues connected by ß-(2, 1) glycosidic linkages with many promising physiochemical and physiological properties. In this study, an inulin-producing inulosucrase gene from Lactobacillus gasseri DSM 20604 was cloned, expressed and purified. SDS-PAGE and gel filtration found that the recombinant inulosucrase is a monomeric protein with a molecular weight of 63KDa. The optimal pH for its sucrose hydrolysis and transfructosylation activities was pH 5.5. The optimal temperatures were measured to be 45, 25, and 35°C for sucrose hydrolysis, transfructosylation, and total activity, respectively. Biosynthesis studies showed that the optimal enzyme dosage was 4.5U/g sucrose. Higher sucrose concentrations immensely contributed to inulin biosynthesis; the inulin yield reached its maximum after 1.5h of reaction. Structural analyses of the polysaccharide produced by the recombinant enzyme from sucrose revealed that it is an inulin-type fructan with a molecular weight of 5.858×106Da.

Effect of nitrogen fertilisation on the overall quality of minimally processed globe artichoke heads.

BACKGROUND: Although nitrogen (N) fertilisation is essential for promoting crop yield, it may also affect the produce quality. Here, the influence of three N fertiliser rates (0 kg ha-1 as a control, 200 kg ha-1 and 400 kg ha-1 referred to as N0 , N200 and N400, respectively) on the overall quality of minimally processed globe artichoke heads was investigated during refrigerated storage for 12 days. RESULTS: Throughout the storage time, N fertilised samples had higher inulin contents than those unfertilised. In addition, the respiratory quotient of N200 and N400 samples was 2-fold and 2.5-fold lower than N0 ones, whose values were close to the normal range for vegetables. All the samples reported good microbiological standards, although N200 and N400 achieved lower mesophilic and psychotropic counts than N0 throughout the storage time. After 8 and 12 days of refrigerated storage, the N200 samples showed the highest scores of positive sensory descriptors. CONCLUSION: A fertiliser level of 200 kg N ha-1 is suitable for obtaining minimally processed globe artichoke heads with good nutritional, sensory and microbiological quality, characterised by low endogenous oxidase activities. Proper packaging systems and procedures are, however, crucial for extending the product shelf-life and, thus, promoting its exportation on a wider scale. © 2016 Society of Chemical Industry.

Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum.

BACKGROUND: IslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme. RESULTS: Screening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS. CONCLUSIONS: Domain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.

Inulin-type fructans: a review on different aspects of biochemical and pharmaceutical technology.

Inulin is a natural storage polysaccharide with a large variety of food and pharmaceutical applications. It is widely distributed in plants, being present as storage carbohydrate in more than 30,000 vegetable products. Due to their wide distribution in nature and significant role in industry, the extraction, isolation and characterization of inulin-type fructans are gaining attention in recent years. Inulin sources have recently received increasing interest as they are a renewable raw material for the production of bioethanol, fructose syrup, single-cell protein and single cell oil, obtainment of fructooligosaccharides and other useful products. This review focuses on the state-of-the-art of biochemical and pharmaceutical technology of inulin-type fructans.

Biodisponibilidade de minerais em ratos alimentados com frutanos do tipo inulina / Mineral bioavailability in rats fed inulin-type fructans

Neste estudo foi avaliado o efeito de frutanos do tipo inulina (FTI) na biodisponibilidade de minerais em ratos, com particular enfoque para o Ca e o Fe. Foram realizados dois ensaios: no primeiro utilizou-se um modelo fatorial 2×2×2, com dois níveis de Fe (0 e 35 mg/kg), Ca (0 e 5 g/kg) e FTI (0 e 100 g/kg) nas rações; no segundo, utilizou-se um modelo de repleção de Hb em ratos anêmicos. As fontes de FTI foram a Raftilose P95 (ensaios 1 e 2), fonte purificada, e a farinha de yacón (FY; ensaio 2); os sais de Fe utilizados foram o Fe4(P2O7)3 (ensaios 1 e 2) e o FeSO4 microencapsulado com alginato (ensaio 2). No primeiro ensaio foram avaliados parâmetros bioquímicos de Fe, concentração de minerais nos ossos, fígado e baço, e parâmetros de resistência e histomorfometria óssea. Após 33 d, não foram observados efeitos na retenção óssea de Ca, porém observou-se maior volume trabecular ósseo e parâmetros histomorfométricos de formação óssea nos animais deficientes em Fe alimentados com os FTI. No segundo ensaio foram determinados parâmetros hematológicos, de biodisponibilidade de Fe, e parâmetros intestinais (balanço mineral; pH, ácidos graxos de cadeia curta e espécies de Fe no conteúdo intestinal). A FY recuperou os animais da deficiência de Fe mais rapidamente do que a Raftilose. Esse efeito ocorreu em função de alterações intestinais provocadas pelo aumento da fermentação desses carboidratos, que afetou a solubilidade do Fe da dieta. Concluiu-se que os FTI aumentaram a biodisponibilidade de minerais, possivelmente por influência da concentração do mineral e pelas interações entre os minerais presentes na ração, pelo tempo de ensaio, pela matriz alimentar, e pelo modelo experimental utilizado.

This study evaluated the effect of inulin-type fructans (ITF) on mineral bioavailability in rats, focusing mainly on Ca and Fe. Two experiments were carried out: the first one considered a 2×2×2 factorial model using two levels of Fe (0 and 35 mg/kg), Ca (0 and 5 g/kg) and ITF (0 and 100 g/kg) in the diets; the second experiment considered a Hb repletion model in anemic rats. Raftilose P95 (experiments 1 and 2; purified source) and yacon flour (YF; experiment 2) were used as ITF sources; the Fe salts used were Fe4(P2O7)3 (experiments 1 and 2) and microencapsulated FeSO4 with alginate (experiment 2). In the first experiment, Fe biochemical parameters, mineral concentration in the bones, liver and spleen, bone strength and histomorphometrical parameters were evaluated. After 33 d, no effects on bone retention were observed, but a larger bone volume and larger bone-formation parameters were observed on Fe-deficient animals fed with ITF. In the second experiment, hematological, Fe bioavailability and intestinal parameters (mineral balance, pH, short-chain fatty acids and Fe species in the intestinal content) were determined. Fe-deficient animals fed with YF recovered more rapidly than those fed with Raftilose. This effect took place as a function of intestinal alterations caused by the increased fermentation of these carbohydrates, which affected dietary Fe solubility. It can be concluded that ITF increased the bioavailability of minerals, possibly due to the influence of mineral concentration and interactions between minerals in the diet, experiment duration, food matrix and experimental model used.

Biotechnological potential of inulin for bioprocesses.

Inulin consists of linear chains of ß-2,1-linked D-fructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end. In this review article, inulin and its applications in bioprocesses are overviewed. The tubers of many plants, such as Jerusalem artichoke, chicory, dahlia, and yacon contain a large amount of inulin. Inulin can be actively hydrolyzed by microbial inulinases to produce fructose, glucose and inulooligosaccharides (IOS). The fructose and glucose formed can be further transformed into ethanol, single-cell protein, single cell oil and other useful products by different microorganisms. IOS formed have many functions. Therefore, inulin can be widely used in food, feed, pharmaceutical, chemical and biofuels industries.

Desenvolvimento de margarina probiótica e simbiótica: viabilidade do probiótico no produto e resistência in vitro / Development of probiotic and synbiotic margarine: viability of probiotic in the product and in vitro resistance

O presente trabalho teve como objetivo verificar a viabilidade da cepa probiótica Bifidobacterium animalis subsp. lactis Bb-12 incorporado em margarina, suplementada com inulina, concentrado protéico de soro (WPC) e concentrado de caseína (CMP), bem como avaliar as características do produto e a resistência do probiótico às condições simuladas do trato gastrintestinal humano. Foram produzidos 7 diferentes tipos de margarinas de mesa (60% de lipídios: 60 % de óleo de palma + 40% de óleo de canola), empregando-se um modelo de mistura, onde inulina, WPC e CMP foram as variáveis estudadas. Uma formulação controle foi produzida (M8), sem adição desses ingredientes. A utilização da mistura do óleo de palma com óleo de canola favoreceu nutricionalmente as formulações, fornecendo produtos contendo ácidos graxos essenciais em sua composição e ausência de ácidos graxos trans. As formulações M1 a M7, exceto a formulação M2 após o 21º dia de armazenamento, apresentaram populações satisfatórias de Bb-12 para um alimento probiótico, com populações acima de 6 log UFC/g durante 35 dias de armazenamento. Margarinas suplementadas com inulina apresentaram populações satisfatórias durante todo o armazenamento, atingindo populações de 8,01 log UFC/g ao 35º dia (M1). Além disso, M3 e M6, revelaram populações de Bb-12 de 6,87 log UFC/g e 7,27 log UFC/g (dia 35), respectivamente. Por outro lado, M8 não foi caracterizada como margarina probiótica, uma vez que apresentou populações abaixo de 6 log UFC/g, já ao 1º dia de armazenamento. Embora WPC seja utilizado em pesquisas para aumentar a viabilidade de probióticos em alimentos, a suplementação de margarina com WPC sem inulina ou CMP não resultou em populações satisfatórias de Bb-12, apresentando decréscimo de 7,82 (dia 1) para 4,64 log UFC/g (M2, dia 35) (p<0,05). Durante todo o ensaio de resistência in vitro, Bb-12 apresentou sobrevivência significativamente superior (p<0,05) em M1 e revelou populações acima de 6 log UFC/g após 6h de...

This study aimed to determine the viability of probiotic Bifidobacterium animalis subsp. lactis Bb-12 incorporated in margarine, with inulin, whey protein concentrate (WPC) and caseinomacropeptide (CMP) supplementation. In addition, the in vitro resistance of Bb-12 incorporated in margarine and related properties were evaluated. Seven margarine-making trials (60% of fat: 60% of palm oil +40% canola oil) were produced, using a mixture model, where inulin, WPC and CMP were the variables studied. Also, a control formulation without these ingredients was manufactured. The use of blending palm oil with canola oil improved the margarine formulations nutritionally, providing products containing essential fatty acids in its composition and absence of trans fatty acids. The formulations M1 to M7, except M2 after 21 days of storage, revealed satisfactory Bb-12 populations for a probiotic food, with counts above 6 log CFU/g during 35 days of storage at 5±1ºC. Margarines supplemented with inulin presented suitable Bb-12 populations throughout the whole storage period, reaching up to 8 log CFU/g by the end of storage (M1). Also, M3 and M6, revealed Bb-12 populations of 6.87 log CFU/g and of 7.27 log CFU/g (day 35), respectively. In contrast, M8 was not characterized as probiotic margarine, since it showed Bb-12 populations below 6 log CFU/g on day 1. Even though whey protein is largely employed in probiotic foods, margarine supplementation with WPC without inulin or CMP did not lead to Bb-12 satisfactory populations, decreasing from 7.82 (day 1) to 4.64 log CFU/g (M2, day 35) (p<0.05). During the whole in vitro assays, Bb-12 survived significantly better (p<0.05) in M1 and revealed populations above 6 log CFU/g after 6h even after 28 days. M2 populations decreased drastically during the in vitro assays for all storage period tested (reduction of 5 log CFU/g after 2h of in vitro assays on day 7 and populations of 2.8 log CFU/g after 6h). For the other formulations, Bb-12...

The probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme.

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, (13)C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by (13)C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55 degrees C, with a drastic decrease at 60 degrees C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.

Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.).

Fructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status.

Physicochemical characterization and biological effects of inulin enzymatically synthesized from sucrose.

We first developed the method to produce inulin from sucrose using an enzyme from Bacillus sp. 217C-1. Synthesized inulin consists of a linear polymer having beta(2-1) linkages of d-fructose with one terminal glucose. The synthesized inulin has similar properties (pH and thermal stability, Maillard reaction, and in vitro fermentation) to plant-derived inulin. The marked difference is the polydispersity of the inulin chain length. Synthesized inulin with a narrow degree range of fructose polymerization shows better solubility in water than plant-derived inulin. Synthesized inulin (5%, w/w) treatment for 12 weeks reduced the elevation in body weight and serum and liver lipids in rats fed high fat- and high sucrose-supplemented diets, and blood glucose in rats fed a standard diet. Synthesized inulin (15%, w/w) significantly suppressed the elevation in blood glucose of human healthy subjects after dextrin loading. These results suggest that daily intake of synthesized inulin modulates carbohydrate and lipid metabolism.

Properties of fructan:fructan 1-fructosyltransferases from chicory and globe thistle, two Asteracean plants storing greatly different types of inulin.

Remarkably, within the Asteraceae, a species-specific fructan pattern can be observed. Some species such as artichoke (Cynara scolymus) and globe thistle (Echinops ritro) store fructans with a considerably higher degree of polymerization than the one observed in chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus). Fructan:fructan 1-fructosyltransferase (1-FFT) is the enzyme responsible for chain elongation of inulin-type fructans. 1-FFTs were purified from chicory and globe thistle. A comparison revealed that chicory 1-FFT has a high affinity for sucrose (Suc), fructose (Fru), and 1-kestose as acceptor substrate. This makes redistribution of Fru moieties from large to small fructans very likely during the period of active fructan synthesis in the root when import and concentration of Suc can be expected to be high. In globe thistle, this problem is avoided by the very low affinity of 1-FFT for Suc, Fru, and 1-kestose and the higher affinity for inulin as acceptor substrate. Therefore, the 1-kestose formed by Suc:Suc 1-fructosyltransferase is preferentially used for elongation of inulin molecules, explaining why inulins with a much higher degree of polymerization accumulate in roots of globe thistle. Inulin patterns obtained in vitro from 1-kestose and the purified 1-FFTs from both species closely resemble the in vivo inulin patterns. Therefore, we conclude that the species-specific fructan pattern within the Asteraceae can be explained by the different characteristics of their respective 1-FFTs. Although 1-FFT and bacterial levansucrases clearly differ in their ability to use Suc as a donor substrate, a kinetic analysis suggests that 1-FFT also works via a ping-pong mechanism.

A novel enzyme of Bacillus sp. 217C-11 that produces inulin from sucrose.

We found a bacterium that converts sucrose to a useful material, using about 6,000 samples of bacteria isolated from soil. This bacterium, Bacillus sp. 217C-11, was identified according to Bergey's manual, and produced a highly efficient enzyme that converted sucrose into inulin. So, the enzyme was purified to homogeneity through five chromatographic steps, to identify its enzymatic properties. The molecular mass of the enzyme was estimated to be 45,000, and this enzyme was a monomer protein (by SDS-PAGE). The optimum pH and temperature of this enzyme were 7-8 and 45-50 degrees C, respectively. The enzyme reacted only with sucrose, but did not with other disaccharides, fructooligosaccharides and inulin. This paper will show that our enzyme is a novel one, which is different from the other well-known enzymes concerned in inulin production.

Molecular characterization of inulosucrase from Leuconostoc citreum: a fructosyltransferase within a glucosyltransferase.

The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.

Transgenic potato (Solanum tuberosum) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke (Cynara scolymus) roots.

The ability to synthesize high molecular weight inulin was transferred to potato plants via constitutive expression of the 1-SST (sucrose:sucrose 1-fructosyltransferase) and the 1-FFT (fructan: fructan 1-fructosyltransferase) genes of globe artichoke (Cynara scolymus). The fructan pattern of tubers from transgenic potato plants represents the full spectrum of inulin molecules present in artichoke roots as shown by high-performance anion exchange chromatography, as well as size exclusion chromatography. These results demonstrate in planta that the enzymes sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1-fructosyltransferase are sufficient to synthesize inulin molecules of all chain lengths naturally occurring in a given plant species. Inulin made up 5% of the dry weight of transgenic tubers, and a low level of fructan production also was observed in fully expanded leaves. Although inulin accumulation did not influence the sucrose concentration in leaves or tubers, a reduction in starch content occurred in transgenic tubers, indicating that inulin synthesis did not increase the storage capacity of the tubers.