FR2617502A1 - Enzymatic preparation of D-1,5-anhydrofructose - Google Patents

Abstract

Process for the enzymatic preparation of D-1,5-anhydrofructose from a carbohydrate; an aqueous solution of an alpha -4-glucane, or of a degradation product of such a glucane, is incubated with an enzyme produced by a macroascomycetes or discomycetes fungi, the D-1,5-anhydrofructose formed then being separated from the solution and purified.

Description

 The present invention relates to a new process for the preparation of 1,5-anhydro-D-fructose. It includes, as a new product, this purified anhydro-fructose, as well as its application to the production of an antibiotic.

 Up to now, anhydro-1,5D-fructose has had no industrial interest. While we studied the enzymatic oxidation, using certain Pseudomonas, of 1,5-anhydro-D-glucitol in the corresponding fructose, this one was not even isolated and was characterized only by its 2,4-phenylhydrazone dinitro (NARM4URA et al.

J. Biochem, 1986, 99, 609-613). Besides, glucitol being a deoxy-1 form of glucopyranose could not constitute an economic raw material, for the production of anhydro-1,5D-fructose. On the other hand, a chemical process is known for preparing 1,5-anhydro-ketohexoses, the class to which anhydro-fructose belongs; it consists of the hydroxylaminolysis of 2-hydroxy glycal esters, followed by deacylation and deoximation (LICHTEN
HALER et al. Tetrahedron Letters, 1980, 21, 1429-1432).

However, deacylation leads to complications and the authors were unable to obtain anhydro-1,5D-fructose other than in the form of its oxime. This known process would therefore be difficult to apply to industrial production.

est obtenuepar la culture aérobie des Micromycètes, Microthecium ou Melanospora, sur une solution de fécule de pomme de terre etc qlucose ; l'action de ces champignons est exceptionnelle, puisque cent autres Micromycètes ne l effectuent pas.However, since the discovery, by the authors of the present invention, of the role of precursor that anhydro-1,5D-fructose plays in the production of the antibiotic microthecin (publication of Japanese patent application No. 79,122796) , it has become very interesting to be able to obtain this fructose economically. According to the Japanese process, microthecin

is obtained by aerobic culture of Micromycetes, Microthecium or Melanospora, on a solution of potato starch etc qlucose; the action of these fungi is exceptional, since a hundred other Micromycetes do not perform it.

 The present invention starts from the unexpected facts that microthecin can be obtained more advantageously, if anhydro-1,5D-fructose is first prepared (hereinafter designated by the abbreviation AF) and then transformed into microthecin , and that this conversion can be carried out with the help of certain Macro-Ascomycetes and Discomycetes.

 The process according to the invention consists in subjecting an aqueous solution of an alpha 1-4 glucan, or of a product of its degradation, to the action of an enzyme produced by a Macro-Ascomycete or Discomycete fungus.

The enzyme can in particular come from a fungus from one of the following two groups a) species up to the microthecin stage
Discina perlata
Discina parma
Gyromitra gigas
Gyromitra infula
Mitrophora hybrida
Morchella conica
Morchella costata
Morchella elata
Morchella hortensis
Morchella rotunda
Morchella vulgaris
Peziza aurantia
Peziza badia
Sarcosphaera eximia b) species stopping at the AF stage
Disciotis venosa
Gyromitra esculenta
Helvella crispa
Helvella lacunosa
Leptopodia elastica
Certain Morchella strains
Verpa digitaliformis
In the case of enzymes from group (a), the reaction should be stopped at the AF stage, which can be obtained by the action of EDTA; (ethylene diamine tetracetic acid).

 The glucans i A-1-4, serving as raw material, that is to say substrate for the enzymatic reaction according to the invention, can be such as starch, either amylose and amylopectin, glycogen, and their products partial degradation, in particular dextrins, hydrogenated dextrins, maltotriose, etc.

Thus one can use raw materials much more easily accessible than those of the prior art mentioned above.

 Although it is possible to carry out the process of the invention by direct culture of the appropriate strain e.champignon on a glucan solution, the preferred embodiment consists in first extracting the desired enzyme from a mushroom culture, purify it, and then use it to produce AF from the glucan solution, under optimal, determined conditions.

 The actual preparation of AF, according to the invention, is therefore generally preceded by an operation of culturing the selected strain, activated subsequently by a plasmolytic treatment which unmasks the enzymes present in the cells of the fungus. Such a treatment can consist in raising the temperature of the culture to the optimum growth temperature, in the action of vapors of organic solvents, such as for example chloroform, ether, acetone, ethyl acetate, etc. or by aerating the aqueous medium with an aromatic hydrocarbon, in particular toluene; other means may be freezing followed by thawing or further grinding. It may moreover be good to apply several of these treatments at the same time, for example freezing and then melting of the aqueous medium, followed by grinding.

 The action of the enzyme on the glucan solution, with a view to obtaining the AF, according to the invention, is generally carried out in an aqueous medium with a pH of about 4 to 8 and preferably 5 to 7, at a temperature of 250 to 400C or better between 300 and 370C. The preferred margins, if indicated, are particularly suitable, for example, when an enzyme extract of Morchella vulgaris is used with starch.

forme pyrannique forme pontez carbonylée en 2 en 2-6
Suivant un trait de la présente invention, c'est cet AF purifié qui est appliqué à la production de l'antibiotique, la microthécine.Pour cela, à une solution aqueuse odrganique d'AF on ajoute une enzyme produite par une des souches de groupe (a) indiquées plus haut, ou bien l'enzyme de Microthécium ou deMélanospora selon la publication de brevet japonais 79-122796, pour faire transformer l'AF en l'antibiotique. Celui-ci est alors obtenu avec un rendement bien meilleur que par le procédé du brevet japonais ; il est également plus pur et il est plus aisé de pousser davantage sa purification finale. Les avantages de cette application suivant l'invention résultent, entre autres, de ce que celle-ci rend possible d'utiliser un milieu beaucoup plus pur, grâce à la purification de l'AF employé, et plus concentré en ce dernier.When the AF prepared by the method described above is purified, an anhydro1,5D-fructose is obtained which has never been prepared in this form or used in the past. It is a white, amorphous solid, very soluble in water, heat stable, non-toxic to mice, orally, at a dose of 5gJkg. Taste slightly sweet in aqueous solution. Not metabolized by baker's yeast. In aqueous solution, this fructose gives rise to a balance between at least two forms

pyrannic form bridge form carbonylated in 2 in 2-6
According to a feature of the present invention, it is this purified AF which is applied to the production of the antibiotic, microthecin. To this, to an aqueous odrganic solution of AF, an enzyme produced by one of the group strains is added. (a) indicated above, or else the enzyme of Microthecium or of Melanospora according to Japanese patent publication 79-122796, to transform AF into the antibiotic. This is then obtained with a much better yield than by the method of the Japanese patent; it is also purer and it is easier to further push its final purification. The advantages of this application according to the invention result, inter alia, from the fact that it makes it possible to use a much purer medium, thanks to the purification of the AF used, and more concentrated in the latter.

 The invention is illustrated without limitation by the examples which follow, and the main characteristics of which, but not exclusive, are given below.

Nature of the strain: any of the species or strains (a) and (b), mentioned above, constitutes a potential source of enzymatic activity.

Culture method: on agar medium or in liquid medium.

Preparation of the active enzyme extract: the activating plasmolysis of the strain, necessary, is carried out here by a combination of freeze-thaw, grinding and stirring in an aqueous medium, supplemented with toluene.

The degree of purification of the extract can be variable depending on the objective; with the crude extract, experience shows that even a coarse preparation may be suitable.

To obtain a semi-crude extract, the interesting enzymatic activity is precipitated by polyethylene glycol (P.E.C.) or by ammonium sulphate from a suspension of cellular ground material in a buffer.

Nature of the substrate of the enzymatic reaction: starches of different botanical origins, in particular wheat, corn, rice, amylopectin, amylose, soluble starch, maltodextrins, glycogen, etc.

Carrying out the enzymatic reaction and purifying the AF: the substrate previously dissolved is incubated with the enzymatic extract, it can be operated in the presence of an antibiotic, for example 0.1% chloramphenicol, to inhibit microbial proliferation. To remove glucose, which accompanies AF, we can make baker's yeast work, which does not metabolize it. Several possibilities are then offered, among which: to practice a dialysis which separates the products of the reaction, that is to say AF and dextrins of low molecular weight, of the macromolecules of the reaction medium, either untransformed substrate, the enzyme and figurative elements, yeasts, particles possibly coming from the substrate, etc.

centrifuge to remove the figured elements, then evaporate to dryness and take up the residue of methanol, which dissolves the AF but also other compounds.

Further purification of the AF can be carried out by column chromatography either on a gel-filtration support or on an ion exchange support, with elution with water, which gives a pure product at approximately 958; in order to obtain practically 100% pure AF, the process is completed by preparative layer chromatography on silica gel.

AF detection and assay can be performed by C.C.M., C.L.H.P.

TLC can be performed on silica plate
Macherey-Nagel Sil G / UV254, with elution by chloroform-methanol mixture 70:30 and development by heating for 10 min, at 1000C after spraying with the reagent with anisaldehyde (anisaldehyde, 0.5 ml; sulfuric acid, 0, 5 ml; ethanol, 9 mol); the Rf is 0.5.

 The detection and the assay can be carried out by the C.L.H.P. on a Partisil Carbohydrate 10 column of 25 cm x 4.6 mm (Whatnan), with the acetonitrile-water mixture 80:20 at a flow rate of 1.5 ml / min, with refractometric detection.

The retention time is 4.4 min.

EXAMPLE. 1
Obtaining a crude enzyme extract from a microthecin-negative strain of Morchella vulgaris, cultivated on agar medium.

 The mycelia of the fungus are cultivated at 24-250C in Petri dishes 10 cm in diameter, containing 20 ml of glucose-agar medium agar with yeast extract; glucose, 20 g; Mérieux yeast extract, 5 g agar, 20 g; distilled water, q.s.p. I 1, covered with a sterile cellophane film which facilitates the subsequent recovery of the mycelium. The inoculation is done by depositing in the center of the box a mycelial washer 0.5 cm in diameter, taken with a sterile cookie cutter from a growing culture. After ten days, when the mycelia occupy the entire surface of the Petri dishes, they are harvested and frozen.

At the time of use, they are thawed by maceration, at room temperature and for 1 hour, in distilled water (5 ml per round of mycelium) the maceration water, which causes various impurities, is rejected. The mycelia are then drained and ground in 0.05 M sodium phosphate buffer, pH 6 (5 ml per mycelium), in the presence of 10 Al of toluene per mycelium. The ground material is centrifuged for 30 minutes at 20,000 g, and the supernatant constitutes the enzymatic reagent. One can carry out, before this centrifugation, a freezing-thawing of the ground material; this operation, as well as the presence of toluene, is not essential, but it improves the yield of AF by completing the unmasking of the enzymes caused by the freezing-thawing of the mycelia and by the grinding itself.

EXAMPLE 2 Obtaining a semi-crude syntactic extract from the same material as in Example 1.

 The centrifugation supernatant obtained according to Example 1 is labeled with polyethylene glycol 6000 in an amount of 3 g / 10 ml. The precipitate which forms is collected by a new centrifugation for 30 minutes, at 20,000 g, and taken up in 0.05 M sodium phosphate buffer of pH 61 made 0.2 M in NaCl, ie 1/10 of the volume of the initial supernatant, that is to say approximately 0.5 ml per round of mycelium. After a new centrifugation under the same conditions, a second precipitation is carried out by the same P.E.G. at the concentration of zig / 10 ml. The precipitate is collected by centrifugation as above and taken up in 0.05 M sodium phosphate buffer of pH 6 (1/20 of the volume of the starting supernatant, ie approximately 0.25 ml per mycelium); this solution constitutes the enzymatic reagent.

EXAMPLE 3
Obtaining a semi-crude enzyme extract from a microthecin-negative strain of Morchella vulgaris cultivated in a liquid medium.

Preculture
The strain is cultivated at 24-250C in the form of mycelia in tubes containing 15 ml of inclined agar of the same formula as in Example 1. Scraping in sterile distilled water of a tube provides mycelial debris which are used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of the medium of the same formula as the previous one, except for agar. These flasks are incubated on a rotary shaker-incubator at a temperature of 260C, at the speed of 50 rpm, for 4 days.

Under these conditions, the fungus grows by producing, in each flask, a single mycelial ball or a small number of balls. The grinding of this preculture serves as an inoculum for the culture itself.

Culture proper
It is carried out in 25 vials of 250 ml containing 100 ml of the medium of the following formula: soluble amindon, 20 g; corn-steep liqueur, 10 ml; K2HPO4, 1 g; MgSO4, 7 H2O, 0.5 g; distilled water, 1 liter.

Each flask is inoculated with 2 ml of the above ground material, then the flasks are incubated on a rotary shaker-incubator at a temperature of 26 "C, at a speed of 140 rpm, for 8 days. Under these conditions, the fungus develops in the form of small balls of approximately 4 mm in diameter which are collected by decantation and frozen until use.

Preparation of the enzyme extract
The preceding mycelial biomass is thawed, then ground in 100 ml of 0.05 M sodium phosphate buffer, pH 6. The following operations, centrifugations and precipitation with polyethylene glycol are carried out under the same conditions as in Example 2.

EXAMPLE 4
Preparation of AF from Merck 1253 soluble starch and an enzymatic extract obtained according to the method of Example 1, with the intervention of dialysis, chromatography on an ion and exchange column final purification on a chromatoplate.

 1 g of soluble starch is dissolved in 40 ml of water containing 40 mg of chloramphenicol; incubated for 24 hours at 35 "C in the presence of 2 ml of the enzyme extract, then about 400 mg of baker's yeast is added; after 24 hours of contact at the same temperature, three successive dialyses are carried out against 1 distilled water; all of the dialysates (approximately 1 l) are evaporated to dryness, the residue is taken up in 2 ml of distilled water and this solution is introduced at the top of a column of 16 mm x 1 m containing 180g of Dowex 50 WX 4 resin (AFNOR particle size 17 to 20); the elution is carried out with distilled water at a flow rate of 0.4 ml / min and 4 ml fractions are collected. fractions shows that the AF is contained in fractions 30 to 40; they are combined and lyophilized, which gives 500 mg of white product.

This residue is taken up in 2 ml of methanol and the solution is distributed in strips on preparative silica gel plates (Kieselgel Merck 60 PF254) at a rate of 0.5 ml per plate. Chromatographic development is carried out with a 70:30 chloroform-methanol mixture; the strips containing AF, visually identified by their matt white appearance or by reduction, on a control strip, of triphenyltetrazolium (triphenyltetrazolium chloride, 2 g; 0.5 N NaOH, 100 ml), are scraped off and the powder obtained is exhausted by the same solvent. After evaporation to dryness, the residue is taken up in distilled water; this solution is filtered through a membrane with a porosity of 0.22 Am, then lyophilized, which provides approximately 300 mg of practically 100% pure AF.

EXAMPLE 5
Preparation of AF from a raw wheat starch and a crude enzymatic extract obtained according to the method of Example 1, with the intervention of dry evaporation, recovery with methanol and a chromatography on a gel-filtration column.

 Isg suspended starch in 40 ml of water containing 40 mg of chloramphenicol and brought to boiling for 2 min. After cooling, this mixture is added with 2 ml of the enzymatic extract prepared according to Example 1 and the incubation is carried out with shaking for 24 hours at 35 "C. Then approximately 400 mg of baker's yeast is added; after 24 contact hours at the same temperature, centrifuged for 20 minutes at 15,000 rpm.

The supernatant is evaporated to dryness and the residue is extracted by stirring with 2 x 20 ml of methanol; after evaporation of this solvent, the new residue is dissolved in 2 ml of distilled water.

The solution is then introduced at the top of a 26 mm x 1 m column packed with a suspension of Fractogel TSK
HW-40 (F) Merck (ref. 14982). Elution is carried out with distilled water at a flow rate of 0.5 ml / min and fractions of 5 ml are collected. Their control shows that the AF is contained in fract ons 42 to 48, which are combined and evaporated to dryness. About 400 mg of pure AF at around 90% is thus obtained.

Claims (10)

Claims  1. Process for the preparation of anhydrous -1.5D-fructose by the enzymatic route from a carbohydrate, characterized in that an aqueous solution of an alpha 1-4 glucan is incubated, or d a degradation product of such a glucan, with an enzyme produced by a Macro-Ascomycete or Discomycete fungus, the anhydro-1,5D-fructose formed is then separated from the solution and purified.  2. Method according to claim 1, characterized in that the enzyme is produced by one of the strains Disciotis venosa, Gyromitra esculenta, Helvella crispa, Helvella lacunosa, Leptopodia elastica, certain strains of Morchella, Verpa digitaliformis.  3. Method according to claim 1, characterized in that the enzyme is produced by one of the strains Discina perlata, -Discina parma, Gyromitra gigas, Gyromitra infula, Mitrophora hybrida, Morchella conica, Morchella costata, Morchella elata, Morchella hortensis, Morchella rotunda, Morchella vulgaris, Peziza aurantia, Peziza badia, Sarcosphaera eximia, but its activity to convert anhydro-1,5D-fructose to microthecin is previously inhibited, in particular with the help of E.D.T.A.  4. Method according to one of the preceding claims, characterized in that the alpha 1-4 glucan is starch, glycogen, dextrin or maltotriose.  5. Method according to one of the preceding claims, characterized in that the incubation takes place in an aqueous medium of pH 4 to 8, preferably 5 to 7, at a temperature of 250 to 400C, preferably between 300 and 370C.  6. Method according to one of the preceding claims, characterized in that a culture of the fungus capable of producing the enzyme to be used is first carried out, the fungus is subjected to a plasmolytic treatment, then the enzyme is extracted from the culture and it is introduced into the solution of alpha 1-4 glucan or of a degradation product of such a glucan.  7. Method according to claim 6, characterized in that the plasmolytic treatment comprises freezing and then thawing of the culture medium, raising the temperature above the normal temperature for growth of the fungus, the action of vapors d 'an organic solvent, stirring the aqueous medium with an aromatic hydrocarbon, or / and a thorough grinding.  8. Anhydro-1,5D-fructose, characterized in that it consists of a white, amorphous solid, very soluble in water, heat stable, not metabolized by baker's yeast, giving a pyrannic form, because tonylated in 2, and a bridged form in 2-8, when it is in solution in water.  9. Application of anhydro-1,5D-fructose according to claim 8 for the production of the antibiotic, micro thecine, characterized in that a solution of this fructose is subjected to the action of an enzyme extracted from the cultivation of a fungus from one of the Discina perlata strains, Discina parma, Gyromitra gigas, Gyromitra infula, Mitrophora hybrida, Morchella conica, Morchella costata, Morchella elata, Morchella hortensis, Morchella rotunda, Morchella vulgaris, Peziza aurantia, Peziza badia, Sarcosphaera eximia.  10. Application of anhydro-1,5D-fructose according to claim 8 for the production of the antibiotic, microthecin, characterized in that a solution of this fructose is subjected to the action of an enzyme extracted from the culture of a fungus from one of the Microthecium or Melanospora strains.