DE10036068C2 - Process for the enzymatic production of rare monosaccharides, especially tagatose - Google Patents

Abstract

The invention relates to the purely enzymatic production of monosaccharides which are rare in nature and can only be chemically produced by complex and expensive processes, such as, for example, tagatose, fuculose and xylulose. It has been found that these monosaccharides can be produced enzymatically in good purity by using a polyol corresponding to the monosaccharide to be produced or a plant, vegetable or fruit extract which contains a polyol corresponding to the monosaccharide to be prepared, with polyol dehydrogenase from extremophilic red algae and NAD is incubated and the resulting monosaccharide is isolated. This enzymatic production is made possible by the use of polyol dehydrogenase from extremophilic red algae, especially from the thermo- and acidophilic red algae Galdieria sulphuraria.

Description

The invention relates to purely enzymatic production of those that are rare in nature and only chemically elaborate and cost-intensive processes that can be produced Monosaccharides such as Tagatose, Fuculose and Xylulose. It was found that these monosaccharides can be produced enzymatically in good purity, by a corresponding to the monosaccharide to be produced Polyol or a plant, vegetable or fruit extract which a polyol corresponding to the monosaccharide to be produced contains, with polyol dehydrogenase from extremophile red algae and NAD is incubated and the resulting mono saccharide is isolated. This becomes enzymatic Manufactured using polyol dehydrogenase from extremophilic red algae, especially from thermo- and acidophilic red algae Galdieria sulphuraria.

D-Tagatose, Fuculose and Xylulose are rare in nature occurring sugars that are used in the food industry Filler, as a sweetener without calories or in diet table foods are used. Even with higher ones Temperatures such as B. occur during baking these substances are stable and turn brown like sugar Coloring.  

In addition, D-Tagatose is known to have a taste amplify and grow health-promoting To support bacteria in the human intestinal tract (WO 99/43217, WO 99/34689, US 4,786,722.

So far, these rare monosaccharides have been produced by complex, costly chemical manufacturing processes. So large amounts of water and acid are necessary. No. 5,078,796 describes, for example, the preparation of the D-tagatose by the isomerization of a mixture of D-galactose, metal hydroxide, e.g. B. Ca (OH) ₂ and catalyst (inorganic salts such as CaCl ₂ ) at low temperatures to an intermediate metal hydroxide D-tagatose compound and subsequent neutralization with acid. The reaction mixture is deionized, alcohol is added and the tagatose is crystallized. Whey, deproteinized whey or lactose are possible starting materials. D-galactose is released from the lactose by acidic or enzymatic hydrolysis. The chemical production methods are not only complex and costly, but they are also unsatisfactory for production on an industrial scale from the point of view of environmental protection.

D-Tagatose is known to be microbial as well can be manufactured. Some bacteria are able to D-Tagatose form when they use dulcitol as external Find carbon source. This is how an arthro produces bacter globiformis culture under appropriate cultivation conditions D-Tagatose within approx. 15 days if the Medium dulcitol is added (JP 60/248196). The high Costs, the long duration and the susceptibility to contamination fermentation make such a process industrial production of D-Tagatose unfavorable.  

It is known from Phytochemistry (1997), Stein et al. That a dehydrogenase (EC 1.1.1.18) isolated from red algae can be. This enzyme catalyzes the reversible Oxidation of inositol to inososis.

In Plant Physiology (1997) Gross et al. Describe that the red alga Galdieria sulfuraria is able to metabolize different pentoses and on them to grow. The pentoses are in the red algae by a Reduced enzyme that has a molecular weight of approximately 80 kDa having.

Gross and Schnarrenberger disclose (Plant and Cell Physiology, 1995) that the red alga Galdieria sulfuraria various sugars and sugar alcohols as Can use carbon source. The individual tribes of the Red algae show - depending on the external conditions - a great variability. This versatility of The red alga G. sulphuraria becomes one in individual strains ideal system to select the metabolism of Studying sugars and sugar alcohols.

It is also known (European Journal of Phycology, 1999, Oesterhelt et al.) That the recording systems in Galdieria sulphuraria for the different sugars in her Partially overlap substrate specificity. The single ones Sugar transporters can be found in hexose, polyol and pentose Transporters can be divided. By co-induction of various transporters, the red algae are able to a wide range of organic carbons to record, especially in phases of limited Survive light supply.  

U.S. Patent 4,467,033 discloses a method of Oxidation of L-sorbitol to L-fructose is described, whereby mutant Pseudomonas strains as sugar-converting Microorganisms are used. The microorganism owns u. a. a D-iditol dehydrogenase that is used for oxidation is capable of L-sorbitol.

Furthermore, from the prior art (Derwent Abstr. 1999 - 61 37 85/53) announced that microorganisms of the taxonomic groups: Achromobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azotobacter, Brevibacterium, Corynebacterium, Enterobacter, Erwinia, Flavobacterium, Micrococcus, morganella, nocardia, planococcus, proteus, Propionibacterium, Pseudomonas, Rhodococcus, Sporosarcina, Staphylococcus and Vibrio are able to use D-Arabitol Convert D-xylulose. The resulting D-xylulose can be used as Starting material for the production of dietary sweeteners be used.

It is also known (Derwent Abstr. 1994 - 00 21 77/01 and Derwent Abstr. 1993 - 13 95 76/17), sugar, like for example L-Tagatose or L-xylulose with the help of Alcaligenes denitrificans and Klebsiella spec. u. a. out to produce cheap sugar alcohols. The microorganisms are cultivated for this in a medium which Potassium hydrogen phosphate, magnesium sulfate, yeast extract and Sorbose or xylitol comprises.

The object of the invention was to provide a manufacturing method for rare monosaccharides, especially for D-Tagatose, Fuculose and xylulose, to develop that easy feasible and suitable for industrial production is guaranteed high product purity and none Recycling of environmentally harmful substances is required.

The object of the invention is achieved by an enzymatic Solved manufacturing process, in which one adjusting monosaccharide corresponding polyol or Plant, vegetable or fruit extract, the one for that containing corresponding monosaccharide polyol, with Polyol dehydrogenase from extremophilic red algae and NAD is incubated and the resulting monosaccharide is isolated becomes. Depending on the desired monosaccharide Production of Fuculose Fucitol as a starting material set, for the production of xylulose arabitol or xylitol or fruit or vegetable extracts containing them, and for the production of Tagatose dulcitol or a dulcitol plant extract.

Dulcitol, fucitol, arabitol or xylitol itself can in turn be produced from extremophilic red algae and NADPH by reduction from galactose, fucose, arabinose or xylose using an aldose reductase. Since it is practically not found in nature in free form, galactose must be obtained from galactosides (galactose-containing oligosaccharides). The galactose contained in the galactosides can be released through the use of galactosidases (see Fig. 1).

What is new about the method according to the invention compared with previously known procedures is that the manufacture of the rare monosaccharides not by chemical means as before Process takes place, but solely on implementation reactions based on enzymes. This is made possible by the use  novel enzymes from extremophilic algae, especially from Galdieria sulphuraria from the Rhodophyceen group.

For the production of the rare monosaccharides, in particular Tagatose can also be used according to the invention galactosides or a galactoside-containing plant extract with galactosidase, Aldose reductase and polyol dehydrogenase from extremophiles Red algae, NADPH and NAD are incubated and the desired one Monosaccharide, e.g. B. the Tagatose, isolated and crystalline be lized. Galactosides are wide in nature spread and come z. B. in legumes, preferably in Soy, cabbage, sugar beet, ulmaceae or tuber. Stachyose, a galactoside from 2 galactose, one glucose and a fructose unit, and raffinose (D-galactose, D- Glucose and D-fructose) come e.g. B. in Ulmaceen as most important form of transport besides sucrose. Soy or Generally legume seeds also contain Sucrose and verbascose especially a lot of stachyose and Raffinose. So ripe soybeans contain little starch, however, 4.1% stachyose and an additional 1.4% raffinose. Cabbage also has a high content of oligosaccharides such as Stachyose and raffinose, as well as sugar beet. The at the sugar production from sugar beet has molasses a raffinose content of approx. 3%. Is the leader Stachys Sieboldii Miq. (Nodule, Lamiaceae), the 14- Contains 18% stachyosis. As a starting material for galactose can also floridiside or isofloridoside from red algae be set.

According to the invention, the process for producing the rare monosaccharides, especially Tagatose, as Starting materials are either the plant ex containing galactoside tract, which is obtained by usual methods, or the Galactoside itself (e.g. raffinose, stachyose or Melibiosis) can be used. To the galactoside too  extract, the plants with water after konven tional methods.

The galactose contained in the galactosides is by Galactosidase released. According to the invention can be commercial available galactosidase can be used. It is also possible, galactosidase from different organisms purify. Melon (Citrullus battich and Cucumis melo) Aspergillus niger, Bifidobacterium adolescentis, Mortierel la vinacea, Phanerochaete chrysosporium, thermotoga neapolitana, Pyrococcus furiosus, Penicillium spec., Coffee beans, Phaseolus vulgaris, Aspergillus nidulans, Bacillus stearothermus, Klebsiella spec., Aspergillus ficuum, Aspergillus tamarii, Saccharum officinarum possible sources for galactosidase. According to the invention galactosidase from thermo- and acidophilic unicellular red algae are used.

The in the inventive method for producing the Tagatose in a first step by galactosidase released galactose is from the invention Aldose reductases from extremophilic red algae to dulcitol implemented by the polyol dehydro according to the invention recovered from extremophilic red algae to Tagatose becomes.

Other monosaccharides such as fuculose or xylulose will be produced by fucitol or arabitol or xylitol or Plant, vegetable or fruit extracts that ent hold with polyol dehydrogenase from extremophilic red algae and NAD incubated and the sugars subsequently cleaned become. Arabitol and xylitol, of course, come in many Fruits and vegetables, so Xylitol z. B. in strawberries, Plums or pears, Arabitol z. B. in wine or avocado  in front. Fucitol can be extracted from the Fucose of the alga Fucus vesiculosis can be produced.

According to the invention, 1 to 40 g of lyophilized plants extract dissolved in 50 ml buffer. Ever galactosidase, aldosere ductase and polyol dehydrogenase are 1 to 1,000 U at Presence of 0.1 to 10 mM NADPH and NAD and a pH Incubate value from 5 to 9. Incubation can occur at Temperatures of 10 to 50 ° C take place over 1 to 24 h.

It has been shown that the processes according to the invention with these enzymes, namely galactosidase, aldose Reductase and the polyol dehydrogenase from the thermo- and acidophilic red algae Galdieria sulphuraria especially run advantageously and pure monosaccharides are formed. All the enzymes from the MSh strain of Galdieria sulphuraria, deposited on July 12, 2000 under the deposit number 1372/1 with the CCAP in Great Britain. The deposit with this The depository meets the requirements of the Budapest Contract.

Isolation, cleaning and activity determination of the Enzymes can and are made using conventional methods described in more detail in the examples. Especially before According to the invention, L-iditol or L-fucitol dehydro is added genase isolated and used as a polyol DH.

In the process according to the invention for the production of tagatose from galactosides or galactoside-containing plant extract the three enzymes are incubated at 10-50 ° C, preferably at about 20 ° C for 1 to 24 hours, preferably for 5-13 hours, particularly preferably for 9 hours.

A good yield is achieved according to the invention if, for. B. 1 part by weight of lyophilized galactoside-containing plant extract  each with 1-100 U, preferably with 5-20 U, particularly preferably with about 10 U galactosidase, aldose reductase and Polyol dehydrogenase is added.

It is also possible according to the invention to start with galactose or dulcitol (cf. Fig. 1), the latter specifically from red algae or the celstraceae, there again preferably from Euonymus europaea. Dulcitol as a direct starting material for synthesis occurs only to a significant extent in a few plants, such as the Pfaffenhütchen and some red algae. Dulcitol can be extracted using conventional methods.

The manufacturing method for rare Monosaccharides, especially for Tagatose, have opposite the chemical processes of the prior art Advantage of having enzymes as biocatalysts from them promoted reactions emerge unchanged and neither disposed of nor laboriously recycled. In addition, their specificity ensures that high product purity, which is <98% for Tagatose.

The Tagatose obtained according to the invention is suitable in especially as a glaze for bakery and luxury items such as Chocolates, petit four or cake.

The invention is illustrated below using exemplary embodiments explained in more detail:  

embodiments example 1 Production of Tagatose

To extract dulcitol or galactoside, the Planted with water using conventional methods be closed (e.g. ball mill or press). Samples are deproteinized, the water-soluble galactosides optionally by HPLC on Rezex RSO oligosaccharide enriched, lyophilized and in reaction buffer for the enzymatic conversion added.

Cultivation of Galdieria sulfuraria

Strain MSh from Galdieria sulfuraria was grown autotrophically in culture tubes aerated with 5% CO ₂ enriched air at 20 ° C in a mineral medium in continuous light (80 µEm ^-2 s ^-1 ; L40 W / 30-1 Osram).

enzymes

After growing the organisms according to conventional methods the respective enzyme is isolated from the culture (see below). The enzyme in question can be made using conventional methods the cells are extracted. So cell homogenates by 2 or more techniques such as ammonium sulfate precipitation, hydrophobic interaction chromatography, ion exchange Chromatography and gel filtration to be purified To obtain enzyme preparations that only have an electro have phoretically detectable bands or the enzyme in  contain the necessary degree of purity. Preparations of the Enzymes can be prepared using conventional methods such as z. B. adsorptive, ionic, covalent bond or matrix be immobilized. These products can then repeated in batch process or continuously Filling into columns or membrane reactors can be used.

enzyme evidence

The activity of the polyol dehydrogenase is determined as follows: 800 µl 50 mM Tris / HCl, 50 µl 4 mM NAD, 50 µl 1 M Polyol and 100 µl of an appropriate enzyme extract solution are mixed and the formation of NADH at 340 nm with followed by a photometer.

The aldose reductase activity is determined as follows: In a reaction batch designed for a volume of 1 ml, consisting of 50 mM potassium phosphate, pH 7.0, 0.3 mM NADPH, 50 mM galactose and a suitable amount of enzyme extract, the formation of NADP tracked.

The galactosidase activity is determined as follows: In 1 ml Test solution consisting of 40 mM Hepes-KOH, pH 6.5, a suitable amount of substrate p-nitrophenol α-d-galacto sid and a suitable amount of enzyme extract is at 30 ° C. the formation of p-nitrophenol using a photometer 360 nm tracked.

Cleaning of aldose reductase

All cleaning steps were carried out at 0-4 ° C. In a beadbeater (Biospec Products, Bartlesville, OK) were 10-20 g of algae with 50 ml of digestion buffer from 20 mM potassium phosphate, pH 8.0 and 2 mM DTT and 0.5 mM  EDTA open minded. The extract was at 37,000 x g for 60 min centrifuged, the supernatant on a DEAE Fractogel column (22 × 2.5 cm), equilibrated with 20 mM Tris-HCl, pH 8.3 and Given 0.5 mM DTT. The aldose reductase does not bind the matrix and can be eluted with 50 ml buffer. The unbound proteins were placed on a hydroxyapatite column (16 × 1.5 cm), equilibrated with 10 mM Tris-HCl, pH 8.3, loaded. After rinsing with 30 ml column buffer, the Proteins with a linear gradient of 200 ml from 0.1- 0.8 M potassium phosphate, pH 7.5 eluted. The two activity Peaks were separated separately against 2 l of 20 mM Hepes-KOH, pH 6.5 and 0.5 mM DTT dialyzed. The samples were each on a cation exchanger column (Econo-Pak High S, Biorad), equilibrated with 20 mM Hepes-KOH, pH 6.5, loaded. The column was washed with 10 ml column buffer and Proteins with a linear gradient of 0-1.5 M NaCl eluted in column buffer.

Aldose reductase fractions were pooled and 1 ml Aliquots on a gel filtration column (Superdex 200 HiLoad, 1.6 x 60 cm, Pharmacia), equilibrated at 50 mM Potassium phosphate, pH 7.0 and 0.25 M NaCl loaded. proteins were eluted isocratically with column buffer, fractions with Aldose reductase combined. The gel electrophoretic Analysis of these samples showed only one protein band.

Purification of the L-Idit dehydrogenase

All cleaning steps were carried out at 0-4 ° C. In a beadbeater (Biospec Products, Bartlesville, OK), 10-20 g of algae were digested with 50 ml of digestion buffer consisting of 20 mM Tris-HCl, pH 8.0 and 1.4 mM β-mercaptoethanol and 1 g of polyvinylpolypyrrolidone / 100 ml. The extract was centrifuged at 4000 x g for 15 min. The supernatant was centrifuged for a further 60 min at 40,000 × g. The crude extract was adjusted to 10% ammonium sulfate saturation and the mixture was centrifuged at 32,000 × g for 15 min. Proteins from the supernatant were precipitated by increasing the ammonium sulfate saturation to 40% and pelleted by centrifugation again. The resulting precipitate was taken up in 20 mM Tris-HCl pH 8.5 and desalted against this buffer by dialysis for 2 h. The dialysate was loaded onto a DEAE Fractogel column (2.5 × 20 cm) equilibrated with 20 mM Tris-HCl, pH 8.5. The column was washed with 3 times the column bed volume of column buffer and proteins were eluted with a linear gradient of 0-150 mM KCl in column buffer. Fractions with polyol dehydrogenase were combined and dialyzed against 10 mM potassium phosphate, pH 6.8 for 2 h. The dialysate was loaded onto a hydroxyapatite column (1.5 × 16 cm) equilibrated with 10 mM potassium phosphate, pH 6.8. The column was washed with 3 times the column bed volume with the same buffer and proteins were eluted with an ascending gradient of 10-200 mM potassium phosphate, pH 6.8. Fractions with polyol dehydrogenase activity were combined and dialyzed against 1 l of 20 mM Tris-HCl, pH 7.5 for 2 h. The dialysate was brought to 40% saturation with ammonium sulfate and placed on a decyl agarose column (1.5 × 16 cm) equilibrated with 20 mM Tris-HCl, pH 7.5 and 40% ammonium sulfate. The column was washed with three times the column bed volume of buffer (see above) before proteins were eluted with a descending gradient of ammonium sulfate (40-0% saturation) in 20 mM Tris-HCl, pH 7.5. Fractions with polyol dehydrogenase activity were combined and 1 ml aliquots were loaded onto a gel filtration column (Superdex 200 HiLoad, 1.6 × 60 cm, Pharmacia) equilibrated with 20 mM Tris HCl, pH 7.5. Proteins were eluted isocratically with the same buffer and fractions with polyol dehydrogenase activity were pooled.

Process implementation

50 ml starting material containing galactoside (5 g lyophili based plant extract (see above) dissolved in 50 mM potassium phosphate, pH 7.5) were obtained commercially with 10 U each Licher galactosidase from Apergillus niger or greens Coffee beans, Aldose Reduc purified from G. sulphuraria tase (see above) and polyol purified from G. sulphuraria Dehydrogenase, 4 mM NADPH and 4 mM NAD added and 9 h at Incubated at 20 ° C. The reaction approach was deproteinized and sugar using HPLC on a REZEX RCM monosaccharide Column that is isocratic at 80 ° C with distilled water was eluted separately. Based on comparisons with sugar The Tagatose peak was identified by standards. The Fractions were concentrated and the Tagatose after conventional methods crystallized.

Purification of the Tagatose

The reaction mixtures contain in addition to the desired one Product also undesirable substances like others Oligosaccharides, monosaccharides, sugar alcohols, etc. Im In general, these can be combined less Purification techniques are removed, e.g. B. Proteins through filter techniques, insoluble components through Centrifugation, salts with ion exchangers in the H or OH-form. As a final step, sugar can be left be separated by means of HPLC or selectively with alcohol or other solvents. chromatography with silica gels also produces high purity saccharides. The cleaned Tagatose can then be used with conventional Methods are crystallized.

Claims (15)

1. A process for the enzymatic production of rare monosaccharides, characterized in that a polyol corresponding to the monosaccharide to be produced or a plant, vegetable or fruit extract which contains a polyol corresponding to the monosaccharide to be produced is incubated with polyol dehydrogenase from extremophilic red algae and NAD is and the resulting monosaccharide is isolated. 2. The method according to claim 1, characterized in that Polyol dehydrogenase from thermophilic and acidophilic Red algae Galdieria sulphuraria is used. 3. The method according to claim 2, characterized in that Polyol dehydrogenase from the MSh strain, Hinterle CCAP 3172/1 is used. 4. The method according to any one of claims 1 to 3, characterized in that L-idite or L-fucite dehydrogenase is used. 5. The method according to any one of claims 1 to 4, characterized in that for the production of fuculose fucitol or a fucitol plant extract is used.   6. The method according to any one of claims 1 to 4, characterized in that for the production of xylulose arabitol or xylitol or a fruit or vegetable extract that contains them, is used. 7. The method according to any one of claims 1 to 4, characterized in that for the production of Tagatose Dulcitol or a Plant extract containing dulcitol is used. 8. process for the enzymatic production of tagatose, characterized in that Galactoside or a plant containing galactoside extract with galactosidase, aldose reductase from extre mophilic red algae, polyol dehydrogenase from extreme mophilic red algae, NADPH and NAD are incubated and the monosaccharide is isolated. 9. The method according to claim 8, characterized in that Polyol dehydrogenase from thermophilic and acidophilic Red algae Galdieria sulphuraria is used. 10. The method according to claim 9, characterized in that Polyol dehydrogenase from the MSh strain, Hinterle CCAP 1372/1 is used.   11. The method according to any one of claims 8 to 10, characterized in that L-idite or L-fucite dehydrogenase is used. 12. The method according to claim 8, characterized in that as galactoside floridoside, isofloridoside, raffinose, Stachyose or melibiosis can be used. 13. The method according to claim 8, characterized in that a plant extract from Legumi containing galactoside noses, cabbage, sugar beet, ulmaceae or tuber, is used. 14. The method according to claim 13, characterized in that as a galactoside-containing plant extract from legumes a soy extract is used. 15. Process for the enzymatic production of tagatose, characterized in that Galactose or a plant extract containing galactose with aldose reductase from extremophilic red algae, Extremophilic red algae polyol dehydrogenase, NADPH and NAD is incubated and the resulting tagatose is isolated.