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Levansucrase

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Last Updated: 20 November 2020

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General | Latest Info

Levansucrase

Databases
BRENDABRENDA entry
ExPASyNiceZyme view
Gene OntologyAmiGO / QuickGO
IntEnzIntEnz view
KEGGKEGG entry
MetaCycmetabolic pathway
PDB structuresRCSB PDB PDBe PDBsum
PRIAMprofile
Identifiers
CAS number9030-17-5
EC number2.4.1.10

Modify positions in B. Megaterium levansucrase library. Select positions for mutagenesis in B. Megaterium levansucrase are displayed as outlined white sticks. Catalytic amino acids and residue Y421, which has a pivotal role in hydrolysis and transfer, are show as white sticks. Corresponding residues in levansucrase from Gram-negative Gluconacetobacter diazotrophicus are depict in cyan. Possible hydrogen bond interactions with other residues and with sucrose are shown as dotted lines. Sucrose was align from the crystal structure of B. Subtilis levansucrase. Residues are found at analogous positions in inulosucrase from L. Johnsonii and in levansucrases from B. Megaterium, B. Subtilis and G. Diazotrophicus. Equivalent positions to Y421 are not shown on the right panel for the sake of clarity. Numbering corresponding to B. Megaterium levansucrase is displayed in black, and for other enzymes according to their color code. HPAEC-PAC analysis of product specificity of B. Megaterium levansucrase variants. FOS profile of variants R370H and K373R; FOS produced by variants R370Q and R370Q / K373R / F419W; and FOS synthesize by variant K373H. Products of wild-type levansucrase are shown as black dotted lines. Reactions were performed in Sorensen buffer using 2 U mL 1 of each enzyme and 0. 5 m sucrose. Products were analyzed after 24 h reaction, at comparable sucrose conversions for all variants. This figure is composed of cropped chromatograms focus on oligosaccharide products of variants; full-length chromatograms are included in Supplementary Information, Figs S5 and S6.

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INTRODUCTION

Fructans constitute primary carbohydrate reserve in 15 % of higher plants comprising approximately 40000 species and appear in a wide range of bacterial and fungal species. In plants, fructans are known to be involved in stabilization of cellular membranes, conferring protection from water stress caused by drought or low temperatures. Physiological roles of fructans in bacteria have been less described, and it has been postulated that fructan biosynthesis may be involved in a variety of processes, such as survival of bacteria in soil, phytopathogenesis and symbiosis. Of emerging importance is the use of fructo-oligosaccharides as health-promoting pre-biotics, which act as food sources for beneficial bacteria, such as Bifidobacteria. Synthesis of fructans starts with transfructosylation reaction in which a sucrose molecule plays the role of fructosyl donor and with a second molecule of sucrose initial acceptor of fructosyl moiety. Following initiation, extension, type of linkage and branching of fructan chain varies according to enzyme and organism. Fructan synthesis in plants is carried out by action of two or more FTFs that are able to produce complex mixtures of fructans with a great variety of linkages of fructosides. In bacteria, fructan biosynthesis is somewhat simpler because only one multifunctional enzyme is involve. This enzyme is named levansucrase when product is fructan of type levan or inulosucrase when fructan is of type inulin. Levansucrase catalyse transfer of fructosyl residue, from sucrose, to a variety of acceptors including water, glucose, sucrose and fructan. Attack of sucrose can occur via O1 to form 1-kestose or via O6 to form-2 6-link fructo-oligosaccharide 6-kestose. These reactions all occur via the Ping Pong mechanism involving formation of covalent fructosyl-enzyme intermediate. Within the sequence-derive classification of GHs and transglycosidases, bacterial FTFs are classified in family GH68 and, recently, first three-dimensional structure of FTF was publish. FTFs have also been shown to be structurally similar to sucrose-6phosphate hydrolases, invertases, fructanases and eukaryotic FTFs found in family GH32. Together, GH68 and GH32 thus comprise-fructofuranosidase Clan GH-J. The recent crystal structure of Gram-positive bacterium Bacillus subtilis levansucrase and its complex with sucrose reveal structural determinants for substrate recognition and spatial disposition of three key catalytic acidic residues at active site. The structure displays a five-bladed-propeller topology that is shared with elucidated crystal structures of Thermotoga maritima invertase, cellvibrio japonicus-Larabinanase and Cichorium intybus fructan 1-exohydrolase IIa. Despite their high degree of structural similarities in disposition of catalytic residues at active site, GH43-Larabinanase operate with inversion of anomeric configuration of substrate, while levansucrases and invertases are retaining enzymes. Endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus secretes constitutively express levansucrase, which is responsible for utilization of natural substrate sucrose.

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MATERIALS AND METHODS

Workflow for recovery of levansucrase containing supernatants and for subsequent levan production at different pH. PH values in brackets indicate determine final production pH after mixing of respectively use buffers. Three independently grow cell cultures were prepared and handled as depict. Obtain results about amounts and sizes of recovered levans as well as contain volumetric levansucrase activities were finally compared among each other produce levan amounts and corresponding volumetric overall, transfructosylation and hydrolysis activities determine After 24 H of levan production at different pH using three different main cultures. Mean values including standard deviations were calculated from three different production pH per release pH / use cell culture, respectively differential weight distributions of geometric radii of levans produced at different pH. Respective radii distributions of levans produce at pH of levansucrase release are depict in and in of three different production pH per release pH of levansucrase: pH 4. 3 4. 65 5. 0 5. 35 and 5. 7. Experimentally determined production pH values derived from respective buffer mixtures are depicted in brackets in Fig. 1. The Data exemplarily show for cell culture OD = 2. 58 of G. Albidus TMW 2. 1191 and were highly similar to OD 2. 2 and 2. 84, respectively produce levan amounts, C at pH of levansucrase release using four different sucrose concentrations and corresponding volumetric activities. B, D determine After 24 H of levan production using three different main cultures. Mean values including standard deviations were calculated from singly detemined values per cell culture and specific condition, respectively. Stars indicate significant differences between compared conditions differential weight distributions of geometric radii of levans produce at different pH and sucrose concentrations. Experimentally determined production pH values derived from respective buffer mixtures are depicted in brackets in Fig. 1. The Data exemplarily show for cell culture OD = 2. 77 of G. Albidus TMW 2. 1191 and were highly similar to OD 2. 63 and 3.

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RESULTS AND DISCUSSION

Levansucrase SacB shows optimal levanase activity in the pH range of 5 to 6, not surprisingly similar to the pH activity range reported for levansucrase activity with sucrose. Maximum levanase activity towards HMw and LMW levans was found in range of 20 to 40C. Initial reaction rates at different HMw and LMW levan concentrations were first measured in order to observe kinetic behavior and determine kinetic parameters that describe hydrolytic activity of SacB. It was found that SacB follows Michaelis-Menten type kinetics when levan of low molecular weight is used as substrate as shown in Fig 1. By non-linear regression analysis of data, Michaelis Menten affinity constant of K M = 63. 4 mM, measured as fructose equivalents in levan, and first order catalytic constant of K cat = 9. 3 s-1 were determine, with correlation coefficient R 2 = 0. 97. These results differ from those values reported by Rapoport and Dedonder who found affinity constant of 5 mM for levan of 8 kDa, by Yamamoto et al. Who report catalytic constant of 5. 7 x 10 3 s-1 at 30C for levan of 10. 9 ~34 kDa and from those of Chambert et al. Who find an apparent limiting rate constant of 47 s-1 at 22C for levans of 15 3 kDa. In these reports, levanase activity was measured either by the initial rate of reducing sugars release, or by reduction of levan through turbidimetry. As shown below, levan consists of wide distribution of molecules, traditionally characterized by their GPC average Mw. Therefore, these kinetic parameters are indicative only of enzyme properties acting on initial distribution of levan molecules use as substrate. In spite of this consideration, interesting comparison results from SacB kinetic Data on sucrose and levan: in terms of K cat values, SacB is 17. 7 times faster on sucrose than on levan, with affinity 7. 9 times higher for sucrose than for levan. In the same context, SacB hydrolyses sucrose 7. 65 times faster than Levan, with a higher affinity for sucrose than for Levan. Fig 2 shows initial SacB levanase activity with high molecular weight levan as substrate, here report as total fructose concentration in reaction. In this case, linear first order kinetic behavior is observed up to total fructose concentration of 48 mg / mL; this is equivalent to HMw levan concentration of 0. 02 mM, highest concentration of HMw levan than can be prepare. Nevertheless, if the rate of hydrolysis of both HMw and LMW polymers are compared at same enzyme concentration in the first order region, first order kinetic constant is 8. 8 times higher for levan of low molecular weight, as shown in Fig 2. This is not unexpected, considering not only differences in physicochemical properties between both levans but also the structure report for HMw levan, with a large amount of branching.

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Table 2

Native and mutant enzymes were purified from culture supernatants as described by Arrieta et al. And quantify by Bradford procedure using BSA as standard. Reaction mixtures containing 200 mM sucrose in 0. 1 M sodium acetate, pH 5. 0, were incubate with 0. 3 units of purified enzyme at 30 C. After 15 min, reaction was stopped by heating in boiling-water bath for 5 min. Glucose release from sucrose hydrolysis was measured by glucose oxidase / peroxidase-couple colorimetric reaction. K cat was calculated using 60000 as M r of functional unit of levansucrase. K M for sucrose was determined within the substrate concentration range of 1-150 mM at 30 C and pH 5. 0 kinetic parameters were determined by non-linear regression analysis of substrate-velocity plots using the LEONORA program. One unit of enzyme activity is defined as the amount of enzyme releasing 1 mol of glucose / min based on initial-velocity measurements under the following conditions: 0. 2 M sucrose in 0. 1 M sodium acetate buffer, pH 5. 0, at 30 C. Values are meanss.

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Conclusions

The Association of gut microbiome composition with occurrence and development of both gastrointestinal and extragastrointestinal diseases in humans has been in the spotlight over the last decades. Special attention has been devoted to bidirectional communication between central and enteric nervous systems, SO-call gut-brain-axis 1, with several studies analyzing the manner in which gut microbiome influences such interactions 34. Because molecules with prebiotic activity strongly effect composition and number of colonic microbial populations, inclusion of prebiotics in dairy and other food formulations is gaining momentum. While the function of readily available inulin-type oligosaccharides has long been study, there are fewer reports involving 6 F-molecules either as mixture or as purified single oligosaccharides. In part, this is justified by difficulties Associate to isolation of this type of sugars in sufficient yields to support studies. With few exceptions 35 36, report levansucrases often produce wide FOS distributions 16 or polymers of several kDa 10. Since 6-kestose and other small molecules are acceptor substrates in formation of large levan polymers, these sugars usually do not accumulate over reaction course. We report here the construction of the library of Levansucrase from B. Megaterium, which was focused on synthesis of short-chain oligosaccharides. Research known for its role in modulating the size of 1 F-and 6 F-products was revisited to analyze the contribution of diverse amino acid side-chains to product specificity. In general, variants containing mutation R370Q produce preferentially 1-kestose and neokestose, while variants carrying mutation K373H or K373L generate 6-kestose and 6-nystose as main products. Base on combination of microbial fermentation of side-products and chromatographic steps, molecules 1-kestose, 6-kestose and 6-nystose were isolated with purity superior to 95 %. Although the strategy for production and purification of these sugars was not optimized in this work and requires further development, it proves how engineered levansucrases can be applied for gaining access to custom oligosaccharides.

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Background

Acetic acid bacteria are Gramnegative, aerobic rods, which belong to the class of Proteobacteria. They are currently subdivided into 17 genera, which constitute the acetous group of Acetobacteraceae. Genera with the highest numbers of described species are Acetobacter, Komagataeibacter, Gluconobacter, Gluconacetobacter and Asaia. The main characteristic of AAB is their oxidative metabolism, enabling them to oxidize diverse alcohols and sugars to corresponding acids via membranebound dehydrogenases, which are part of the respiratory chain and whose active centres are orient into periplasm. For most oxidation reactions, substrates are taken up into periplasm, oxidize and subsequently released into the environment resulting in acidification of extracellular space. Most AAB can, in this way, cope with high alcohol or sugar concentrations, as energy can be generated without uptake of respective osmolytes into cytoplasm. Because of their acid and alcohol tolerance, some AAB are commonly found coexisting with lactic acid bacteria and yeasts, for example, in traditionally fermented foods like kefir, kombucha, cocoa beans or coffee. Acetobacter and Komagataeibacter spp. They specialize in ethanol conversion to acetic acid via two successive oxidative steps and are thus used for vinegar manufacture. By contrast, Gluconobacter spp. Preferably oxidize glucose to gluconic acid and usually occurs in sugary environments. Most species of Gluconacetobacter are N 2 fixing, endophytic symbionts of plants like sugar cane and coffee, while Asaia spp. Are beverage spoilers and symbionts of malariatransmitting mosquitoes. Remaining AAB genera mostly comprise one single species being distinctly less frequently isolated from natural sources than strains of five main genera. These more rarely occurring species have been mostly isolated from sugary plants, AT which recently described genera Bombella and Parasaccharibacter appear to be symbiotically associated with honey bees. AAB is hence predominantly found on or in plants and their temporary visiting, sugarfeeding insects, which can be considered as conveyers of AAB. While conversion of monosaccharides by AAB is well understood, consumption of sucrose being the most abundant carbohydrate in photosynthetic plants has not been systematically described in AAB. In general, sucrose can be used by bacteria either in phosphorylated form by sucrose6phosphate hydrolases or in nonphosphorylated form by fructosidases or exofructanases, all of them belonging to the glycoside hydrolase 32 family defined in CAZy database. Sucrose is also a natural substrate of secreted fructansucrases and glucansucrases, responsible for synthesis of fructan and glucantype homopolymers respectively. While link polyfructan levan and link polyglucan dextran are components of biofilms, release monosaccharide is directly used for metabolic purposes. Besides their respective transfructosylation or transglucosylation, GH68 and GH70 enzymes also hydrolyze sucrose, as water often ACT as acceptor molecule. While dextransucrase is exclusively expressed by lactic acid bacteria, GH68 encoding genes are present among Grampositive / negative bacteria, archaea, fungi and plants. Diverse previous works reveal AAB from genera Gluconobacter, Gluconacetobacter, Komagataeibacter, Asaia, Neoasaia and Kozakia as producers of active levansucrases.

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3.4 Immobilization of levansucrase

The yield of lactosucrose is affected by factors such as type of enzymes, source of microorganisms, and form of enzymes. In recent years, fructofuranosidase, levansucrase, and galactosidase are mostly enzymes used in synthesis of lactosucrose. A comparison between our results and previous reported results is presented in Table 1. For lactosucrose synthesize by fructofuranosidase, Ikegaki & Park found that content of lactosucrose synthesize by crude fructofuranosidase from Bacillus sp. 417 was 54 G / L. Mikuni et al. Report synthesis of lactosucrose by immobilized fructofuranosidase from Arthrobacter sp. K1, and corresponding content was 120 G / L, which is lower than our result. For lactosucrose synthesize by levansucrase, emphasis was focused on use of genetic modified levansucrase. It seems that different genetic modification methods had different effects on production of lactosucrose. Li et al. Use recombinant levansucrase from Leuconostoc mesenteroides B512 FMC to synthesize lactosucrose, and the corresponding content was 224 mg / ml. However, Lu et al. Also employ recombinant levansucrase to synthesize lactosucrose, and the corresponding content was only 88 mg / ml. Xu et al. Report synthesis of lactosucrose using recombinant levansucrase from Brenneria goodwinii and obtained similar results. Moreover, highest yield of lactosucrose was reported by Han et al. Using levansucrase from Pseudomonas aurantiaca, but substrate concentration was very high. For lactosucrose synthesize by galactosidase, yield of lactosucrose was relatively low, For example, content of lactosucrose synthesize by galactosidase derived from Bacillus circulans was only 56 G / L. Overall, our research fulfil improvement in yield of lactosucrose, and immobilized bienzymes show great Industrial application potential in lactosucrose production in advantages of high stability and reusability.

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2.15.3.1.2 Fructansucrases

Fructansucrases encompass both Levansucrases and inulosucrases. Levansucrases synthesize levans and inulosucrases synthesize inulins. Fructansucrases have been identified in S. Salivarius, 63 S. Mutans, 64 Le. Citreum, 65 Le. Mesenteroides, 66 La. Reuteri, 67 68 and La. Sanfranciscensis. 69 Levansucrases are widely distributed in bacteria, for instance in Gram-positive bacteria Paenibacillus polymyxa, 70 Bacillus amyloliquefaciens, 71 B. Stearothermophilus, 72 and B. Subtilis, 73 and in Gram-negative bacteria Erwinia amylovora, 74 Gluconacetobacter diazotrophicus, 75 and Zymomonas mobilis. 76 in general, fructansucrases from LAB are larger than their counterparts from non-LAB. Also, fructan biosynthesis is known to occur in plants and fungi, involving a set of enzymes that are evolutionarily related to invertases and are hence different from their bacterial counterparts. Fructansucrases catalyze two different reactions, depending on the nature of the acceptor molecule: hydrolysis of sucrose and fructosyl transfer, which can be divided into polymerization and tri-or tetrasaccharide biosynthesis. In each case, the priming reaction of fructansucrase is coupling of fructose moiety of sucrose molecule to another nonreducing-fructose with free primary alcohol group at position C-2 in acceptor molecule. As sucrose is used as acceptor in this initial priming reaction, bacterial fructans contain nonreducing glucose unit at the end of the fructan chain. In subsequent steps, fructansucrase elongate primer. Hence, levans and inulin-type fructans are synthesize according to the following reactions:

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2 EXPERIMENTAL

Previous studies reveal that spherical high molecular weight levan molecules produced by G. Albidus TMW 2. 1191 are functionally diverse regarding their hydrocolloid and rheological properties depending on their molecular size. Moreover, production pH during batch fermentation is crucial for size distributions of these levans as also shown in the present work. However, little is known about influencing factors of levansucrase release as well as of levansucrase activity, both of which are crucial for efficiency of the complex production process of polydisperse levan taking place under continuously changing conditions. By application of the development buffer System it was confirmed that levansucrase is constitutively expressed by G. Albidus TMW 2. 1191 as report for some dextransucrases secreted by water kefir LAB. Higher volumetric levansucrase activities were detected in buffer supernatants at higher release pH. Higher productivity towards levan formation at higher release pH could be due to comparatively higher levansucrase amounts released at higher pH. This view is supported by the fact that use of the same crude enzyme preparation for levan production yields comparable levan amounts. Moreover, higher volumetric activities were determined in buffers incubate with higher cell densities, indicating that more levansucrase was released by a higher number of metabolic active cells. However, continuous increase in productivity with rising release pH could not be verified in the second experimental series focusing on the impact of sucrose concentration on levan formation, in which significant increases in levan amounts and volumetric overall activities could solely be observed between pH 4. 3 / 4. 65 or rather pH 4. 65 / 5. 0. Hence, in addition to environmental pH, cell densities and / or growth phase of levan producing cultures influence levan formation, as higher cell densities were applied in the second experimental series. Growth phase-dependent expression of levansucrases was also reported for enteric bacterium Rahnella aquatilis, which it has to be considered that expression and secretion / release of sucrases are in fact independent processes. For instance, water kefir isolates Lactobacillus hordei TMW 1. 1822 releases its sucrose-converting dextransucrases in similar amounts into the environment in dependence on sucrose, but independently of applied environmental release pH while accumulating dextransucrases within cell independent of sucrose. In the case of Lactobacillus hordei TMW 1. In 1822, it was further observed that release pH affects mean activity / productivity of dextransucrase at different pH. This probably results from concomitantly increased stability of dextransucrase towards its denaturation at non-optimum pH, if it had been initially recover actively at its approximate optimum pH. A similar feature is unlikely for levansucrase release by G. Albidus TMW 2. 1191, as it appears to be comparably active and productive towards high Molecular weight levan production over a broad pH range. This suggests that this type of levansucrase is structurally adapted to changes in extracellular pH, which naturally result from gluconic acid production by G. Albidus TMW 2.


Introduction

Levansucrases are bacterial extracellular enzymes that convert sucrose into-2 6-link fructooligosaccharides of varied chain length and high-molecular weight level These enzymes are present in many plant-relate bacteria such as Pseudomonas syringae, Gluconacetobacter diazotrophicus, Zymomonas mobilis and Erwinia amylovora, but also in Bacillus subtilis, B. Megaterium and several lactic acid bacteria such as Lactobacillus sanfranciscensis, L. Reuteri and Leuconostoc mesenteroides. FOS, which are derived from plant storage polysaccharide inulin, are already widely recognized as prebiotics. They are industrially produced from plant sources and used in various food-and health-related products. On contrary, other types of FOS are not commercially available and therefore their biological effects are scarcely study. Still, few papers, for example, report that-2 6-link FOS are selectively ferment by bifidobacteria showing even stronger prebiotic effects than their-2 1 link counterparts. Neokestose, fructosylglucosylfructoside produced from sucrose by fungus Xanthophyllomyces dendrorhous, also shows bifidogenic effect on human gut microbiota. Notably, recent paper by Marsh and coworkers states that water kefir grains originating from different regions of the world contain Z. Mobilis as main bacterial component. As Z. Mobilis possesses levansucrase, water kefir, popular healthy drink produced by fermentation of sucrose-containing water with water kefir grains as starter, most likely contains levan and FOS. The prebiotic effect of polymeric fructans on lactobacilli and bifidobacteria is most probably assisted by other bacteria in the gut that degrade these large molecules to oligomers. For further study of physiological effects of-2 6-link FOS and levan, biotechnologically feasible production systems applying wild-type enzymes or select mutant variants should be establish. We have clone and heterologously express three genomic levansucrase genes lsc1, lsc2 and LSC3 from plant pathogen, Pseudomonas syringae pv. Tomato. Respective proteins have highly similar sequences and general catalytic properties. We have shown that purified LSC3 Protein has very high catalytic constant. Higher K cat has been recorded only for levansucrase of B. Megaterium whereas levansucrases of G. Diazotrophicus and Z. Mobilis have eight and 18 times lower K cat values than LSC3, respectively. LSC3 is a very efficient polymerizer, producing two types of fructans from sucrose: high-molecular weight levan and short-chain FOS. The spectrum of FOS produced by LSC3 is highly similar to that of prebiotic inulin-type FOS mixture as verified using different analysis methods: thin layer chromatography, nanoelectrospray ionization mass spectrometry and high-performance liquid chromatography. Importantly, LSC3 transfructosylated eleven out of twelve nonconventional acceptor substrates tested by us. Among them, sorbitol, xylobiose, galacturonic acid, mannitol, xylitol and methyl-glucopyranoside were shown to serve as fructosyl acceptors for levansucrases for the first time. In search, isolation and characterization of levansucrase mutants, we have elaborate and applied several high-throughput methods. Firstly, for selection of random mutants of LSC3 Protein, we introduce microplate-base assay of levansucrase activity on permeabilized cells of levansucrase-expressing E. Coli as catalyst. This method was further applied for preliminary study of site-directedly mutate LSC3 variants. As the majority of levansucrase activity was detected in cytoplasmic fraction of levansucrase-expressing E.

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Levan and Fructooligosaccharide

Zymomonas accumulate levan and some fructooligosaccharide as by-products during fermentation of sucrose. This characteristic has enabled development of an effective one-step enzymatic process for synthesizing levan. By mixing solution of sucrose with extracellular levansucrase derived from Z. Mobilis, excellent quality, uniquely colloidal levan can be produce. Levans have a wide range of potential applications, including as food thickeners and as glazing agents in cosmetics. Interestingly, it was also recently reported that Z. Mobilis levans exhibit antitumor activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levansucrase also catalyze fructosyl transfer from sucrose to various mono-and di saccharides to form hetero-fructooligosaccharides, which have attracted special attention due to their many physiological and physical uses. When Zymomonas extracellular levansucrase was been used as biocatalyst for FOS production in sugar syrup, yield of FOS was 24-32 %, and was comprised of a mixture of 1-kestose, 6-kestose, neokestose, and nystose.

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Sources

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