Sucrose levan

D-fructose, C HijOo. Crystallizes in large needles m.p. 102-104 C. The most eommon ketose sugar. Combined with glucose it occurs as sucrose and rafftnose mixed with glucose it is present in fruit juices, honey and other products inulin and levan are built of fructose residues only. In natural products it is always in the furanose form, but it crystallizes in the pyranose form. It is very soluble in... 

The above chemicals can be obtained by fermentation (qv) of other sugars. However, some compounds require sucrose as a unique feedstock. Examples are the polysaccharides dextran, alteman, andlevan, which are produced by specific strains of bacteria (48,54—56). Dextrans are used to make chromatographic separation media, and sulfated dextran derivatives are used as plasma extenders (41). Levans show promise as sweetness potentiators and, along with alteman, have potential as food thickeners and bulking agents in reduced-caloric foods (55,56) (see Carbohydrates). 

The Constitution of a Levan Produced from Sucrose by Pseudomonas mors-prunorum (Wormald), V. E. Gilbert and M. Stacey,/. Chem. Soc.. (1948) 1560-1561. 

Two extracellular D-fructans, (2- 6)-linked S-D-fructofuranan or levan and the less common corresponding (2 l)-linked polysaccharide, of the inulin type, are elaborated by different bacteria. These polysaccharides are formed from sucrose by the action of sucrose fructosyltransferases. Terminal )S-D-fructofuranosyl groups are present in some bacterial heteropolysacchar-... 

Bacillus licheniformis produces a water-insoluble levan that has potential application as a selective plugging agent in MEOR. The microorganisms grow on sucrose, glucose, and fructose but produce levan only on sucrose. Thus plugging may be selectively controlled in the reservoir by substrate manipulation. Oil reservoirs that have a temperature of less than 55° C, a pH between 6 and 9, a pressure less than 500 atm, and a salt concentration of 4% or less are potentially suitable [1480]. 

Early reports on levan are obscured by incomplete descriptions of impure products.2 96 Greig-Smith found that Bacillus levaniformans(1) produced levan from sucrose96" in suitable nutrient solutions, but not from D-glucose, D-fructose, lactose or maltose.966 He therefore assumed that levan could only be formed from the nascent D-fructose and D-glucose resulting from the inversion of sucrose. Hydrolysis of levan yielded D-fructose only, and analysis of levan agreed with the empirical formula (C HiriOi) it was noted that levan was closely related to inulin but was not identical with it. 

Harrison, Tarr and Hibbert96 investigated the production of levan from sucrose by the action of Bacillus subtilis Cohn and B. mesentericus Trevisan. Nutrient solutions containing 10% carbohydrate, 0.1% peptone, 0.2% disodium hydrogen phosphate and 0.5% potassium chloride were incubated at 37° for six days. Levan formation occurred only with sucrose and raffinose, and not with melezitose, lactose, maltose, D-xylose, D-glucose or D-fructose. It was therefore suggested that only those carbohydrates with a terminal D-fructofuranose residue were satisfactory substrates for levan formation. 

Hestrin, Avineri-Shapiro and Aschner9 report that levan formation by means of B. subtilis is greater if cultural products other than levan are continually removed by dilution or dialysis. The organism was inoculated into a phosphate-buffered solution contained in a cellophane bag suspended in a large volume of sucrose peptone solution. 

The structure of the levan synthesized by the action of B. subtilis on sucrose was determined by Hibbert and Brauns.89 Levan, in a yield of 60-65% calculated on the D-fructose part of the sucrose, was obtained by precipitation of the concentrated culture into methanol, and purified by reprecipitation and electrodialysis. Hydrolysis of purified levan with 0.5% aqueous oxalic acid for one hour at 100° gave a 99% yield of crystalline D-fructose. Triacetyllevan was prepared by treatment with acetic anhydride in pyridine, and deacetylation with alcoholic alkali yielded material identical with the original levan.940... 

The levan synthesized by B. subtilis from raffinose was shown by Mitchell and Hibbert100 to be identical in structure with that obtained from sucrose. 

Challinor, Haworth and Hirst101 determined the chemical structure of the levan produced by the action of B. mesentericus on sucrose. Methylated levan appeared homogeneous when fractionally precipitated from mixed solvents. Fractional distillation of the hydrolytic products of methylated levan yielded tetramethyl-D-fructofuranose in an amount corresponding to a levan chain length of from ten to twelve fructofuranose units, joined as previously940 shown through the 2- and 6-positions. 

Other levans produced by widely different organisms all have similar structures, e. g., the levans1020 produced from sucrose by B. megatherium, Phytomonas pruni and P. prunicola, and those1020 produced from sucrose... 

Hestrin and Avineri-Shapiro98 have recently suggested a mechanism for levan production from sucrose and raffinose by levansucrase. The enzyme was used in the form of the autolyzate from A. levanicum (rendered sterile with chloroform and thymol9). This autolyzate was incubated for twenty-four hours at 37° with four volumes of 3% sucrose solution and one volume of phosphate buffer (pH 5.0). 

It was found98 that the amount of sucrose consumed could be accounted for entirely as levan and reducing sugars. Fructose formed part of the reducing sugars produced, and there was no appreciable interconversion of aldose and ketose. When raffinose was used as substrate, the products were levan, melibiose and D-fructose (but not sucrose and D-galactose). The following equation expresses fully the net result of the action of the autolyzate ... 

The specificity of levansucrase98 is dependent not only on the d-fructoside but also on the aldoside residue of the substrate. Neither inulin nor methyl D-fructofuranoside was hydrolyzed by levansucrase, and even when these two substrates were hydrolyzed by inulase (prepared from inulin-fermenting Torula yeast) or by yeast invertase respectively, no levan formation occurred with levansucrase. However, neither methyl D-fructofuranoside nor inulin inhibited levan formation from sucrose by levansucrase. No levan was formed from potassium D-glucose... 

Indirect evidence of the reversal of enzymic synthesis of levan has recently been obtained.10Ba Enzyme preparations from B. subtilis were shown to contain an enzyme capable of hydrolyzing levan (in addition to the levan-synthesizing enzyme) in a system composed of D-glucose, levan, yeast invertase (to hydrolyze sucrose formed) and the B. subtilis enzyme preparation. 

Hehre, Genghof and Neill106 c have shown that levans isolated from sucrose cultures of Streptococcus salivarius (and of a spore-forming bacterium) exhibit precipitation and complement fixation when tested against antisera produced by immunization with bacteria obtained from sucrose cultures of either species. 

It is of interest that similar enzymes, capable of exchanging glycosidic linkages, appear to be involved in the synthesis of some polysaccharides. Thus the polysaccharide dextran is formed from sucrose by enzyme preparations from Leuconostoc46 while the polysaccharide levan is produced from sucrose or raffinose by enzymes of other bacteria.49 The reactions may be written as follows ... 

Dijkhuizen and coworkers identified and characterized a Lactobacillus levansucrase (systematic name sucrose [6)-P-D-fructofuranosyl-(2 ]n a-D-glucopyranoside 6-P-D-fructosyltransferase EC 2.4.1.10) from L. reuteri strain 121, which could produce a high molecular weight levan polysaccharide from fructose [207]. 

In 1993, suicide by an E. colt containing the npt -sacFL-B suicide cassette (nptll gene encodes kanamycin resistance) was reported (Recorbet et al., 1993). In the absence of sucrose the sacK gene does not induce expression of the sacB gene. To induce cell death, sucrose was added to the soil in which the organism was released. This causes the cells to produce levansucrase (a sacB product), which in turn causes the periplasm of the cells to fill with levan. This leads to cell death by lysis. Sucrose... 

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