DE3317064A1 - Process for the preparation of cyclooctaamylose - Google Patents

Abstract

The invention relates to a process for the preparation of cyclooctaamylose by enzymatic cleavage of starch and subsequent fractionation of the cleavage products, in which cyclodextrin glycosyltransferase is added to an aqueous preparation of starch, and bromobenzene is added to the starch preparation at least after the amount of initially formed cyclodextrin no longer increases.

Description

Process for the preparation of cyclooctaamylose

The invention relates to a process for the preparation of cyclooctaamylose, hereinafter also referred to as y-cyclodextrin, by enzymatic cleavage of Starch and subsequent separation of the fission products.

y-cyclodextrin is relatively soluble in water and is hydrophobic Torus about 10 Angstrom in diameter, in which guest molecules are enclosed can. Because of these properties, γ-cyclodextrin is in itself a sought-after starting material e.g. in the pharmaceutical sector, in the fields of plant protection, cosmetics or in the food industry.

According to EP-OS 45 464, cyclodextrins from starch hydrolysates are state of the art separated by precipitation with e.g. bromobenzene. From the mentioned publication it is also known to convert γ-cyclodextrin from starch by enzymatic cleavage win and from the reaction product using chromatographic methods isolate.

In general, it should be noted that the known production methods for γ-cyclodextrin are so complex and thus so costly that γ-cyclodextrin so far not the broad industrial scale based on its properties Could find use.

The object of the invention was to provide a process for the preparation of cyclooctaamylose to develop that provides improved yields.

The invention relates to a process for the preparation of cyclooctaamylose by enzymatic cleavage of starch and subsequent separation of the cleavage products, which is characterized in that a) an aqueous preparation of starch cyclodextrin glycosyl transferase is added and b) at least after the amount of primarily formed cyclodextrin no longer increases, bromobenzene is added to the preparation according to a).

In principle, according to the invention, any type of starch, including native starch or starch partial hydrolysates can be used. examples are Potato starch, corn starch, naniok starch, etc.

All aqueous preparations can be used as aqueous preparations of starch are used, which have already been used for the enzymatic-n cleavage of starch could be used.

These are in particular 5 to 30% strength by weight aqueous solutions of gelled starch. In the simplest case, they become more appropriate by cooking Amounts of starch obtained in water.

The preparations mentioned mostly contain enzyme stabilization smaller amounts of calcium chloride, especially approx.

5 mmol calcium chloride / l.

According to the invention, they preferably contain an enzymatic cleavage reaction Starch preparations to be subjected also to alkali salts of short-chain alkanoic acids. The amounts are expediently 5 to 300 mmol / l starch preparation. Examples for the alkali salts mentioned are sodium formate, potassium formate, sodium acetate, Potassium acetate, sodium propionate, potassium propionate and others, especially sodium acetate. This measure promotes the formation of cyclooctaamylose.

The enzyme known per se is now used in the starch preparations mentioned Cyclodextrin glycosyl transferase added. The source of this enzyme are microorganisms, such as Bacillus macerans, Bacillus megaterium, Bacillus circulans, Bacillus stearo-thermophylus, Micrococcus spp., Alkalophilic Bacillus (e.g. Bacillus No. 38-2 or 17-1), Klebsiella pneumoniae, especially Klebsiella pneumoniae.

The enzyme is preferably added in such amounts that the weight ratio of enzyme: starch is 1: 2000 to 1: 10,000, in particular 1: 4000 to 1: 5000.

The cleavage reaction is carried out at temperatures of 30 to 55 "C, in particular 40 to 45 "C carried out with stirring. The pH of the starch preparation is 4.5 to 7.9.

Preferably, part of the amount of enzyme, about 10 to 40% by weight, in particular 25 to 35% by weight, of the total amount of the enzyme used, even at one temperature of the reaction mixture from 60 to 70 "C. The reaction mixture is then added cooled with stirring to a temperature of 30 to 55 "C and finally the remaining amount of enzyme metered in.

The progress of the enzymatic cleavage reaction can, for example monitored by sampling and chromatographic analysis. As a benchmark this is based on the amount of cyclodextrins that are primarily formed. Depends on the Most of this is derived from cyclodextrin glycosyltransferase a- or B-cyclodextrin. For example, forms cyclodextrin glycosyl transferase from Bacillus macerans and from Klebsiella pneumoniae predominantly a-cyclodextrin, while the cyclodextrin glycosyl transferase of the alkalophilic Bacillus is primarily predominant S-cyclodextrin forms.

The enzymatic cleavage reaction eventually leads to a state in which the amount of these primarily formed cyclodextrins in the reaction mixture is not more increases. At least after this stage is reached, according to the invention Bromobenzene added. Typical incubation times to reach this stage are 5 to 9 hours.

Appropriately, the addition of bromobenzene only takes place if more than 50% by weight, in particular more than 70% by weight, of primarily forming cyclodextrin is present in the reaction mixture.

Adding bromobenzene causes ß-cyclodextrin and z-cyclodextrin to fall as poorly soluble clathrates. The used. Amount of bromobenzene is based on the amount of cyclodextrins present and developing in the reaction mixture at least equimolar. However, based on the amount mentioned, more is preferably obtained Cyclodextrins a 1.5 to 10-fold, in particular a 2 to 3-fold molar excess added to bromobenzene.

Thereafter, the reaction mixture is at 30 to 50 ° C, especially at 40 to 45 "C, stirred until no increase in target product (cyclooctaamylose) more is noted. The reaction can be monitored, for example, by taking samples and chromatographic analysis. Typical total incubation times up to The end point of the reaction is 24 to 48 hours.

To work up the reaction mixture, the solids are first used separated by, for example, decanting, filtering, centrifuging and the like. That The target product is a solid (bromobenzene clathrate) mixed with bromobenzene clathrate of ß-cyclodextrin and long and short-chain, also formed as by-products, non-cyclic cleavage products.

The cyclodextrins are now used to separate the mixture by destroying them the corresponding bromobenzene clathrate brought back into solution. A suitable one The method for this is the treatment of the solid mixture mentioned with hot or boiling water or steam, being in the manner of steam distillation Bromobenzene is removed from the mixture. From the finally bromobenzene-free, aqueous solution can remove the long-chain, non-cyclic cleavage products by precipitation be separated with alcohols such as methanol.

The main amount of the ß-cyclodextrin is now through from the target product Separated crystallization. For this purpose, the aqueous solution is expediently concentrated and most of the β-cyclodextrin formed at a temperature of 2 to 10 "C, in particular approx. 4" C, crystallized out. There remains a supernatant aqueous solution of z-cyclodextrin, the target product, with smaller amounts of ß-cyclodextrin and short-chain, non-cyclic cleavage products. The removal of the short-chain, non-cyclic cleavage products is often not required as these products mostly only available in small amounts. If separation is desired, will the bromobenzene clathrates in the same procedure as already described of ß- and γ-cyclodextrins formed and isolated. The short-chain, non-cyclic ones Cleavage products remain in solution. Then by expelling the bromobenzene brought the cyclodextrins back into solution from the mixture.

When evaporating the resulting solution to dryness or by spray drying and the like, as a target product, is crude cyclooctaamylose with a purity content received by> 90%.

If a purer product is desired, the crude cyclooctaamylose will be used treated with pyridine at a temperature of 20 to 60 "C, in particular approx. 50" C. Cyclooctaamylose is precipitated by adding alcohol, e.g. methanol, to the pyridinic suspension as an easily sedimentable precipitate. As a further cleaning step offers the reprecipitation of cyclooctaamylose from aqueous solution with e.g. ethanol.

The method according to the invention makes it possible, starting from starch, To produce cyclooctaamylose in greatly improved yields and in high purity. The process product is used, among other things, as a component of plant protection products, Medicines, cosmetics or food.

The invention will now be explained in more detail by means of examples: Example 1 150 g of potato starch were in 1 1 of water, which is still 200 mmol of sodium acetate and 5 mmol calcium chloride contained, suspended and heated to 120 "C inside gelled by 30 minutes. The pH was 6.9. After the mixture has cooled down At 70 ° C., 10 mg of cyclodextrin glycosyltransferase were obtained with stirring and further cooling from Klebsiella pneumoniae, dissolved in 20 ml of a 20 millimolar triethanolamine hydrochloride buffer (pH 7.2; 5 mmol calcium chloride) was added. With further stirring, the temperature was increased to 40.degree cooled and then another 20 mg of cyclodextrin glycosyltransferase from Klebsiella pneumoniae in the same preparation as described above was added.

After an incubation time of 7 hours at 40 "C, chromatographic Analysis no further increase in the α-cyclodextrin formed was found.

26 g of bromobenzene were then added to the reaction mixture.

After a further incubation period of 15 hours at 40 "C below With stirring, 61.4% by weight of the starch had degraded to cyclodextrins. The ratio of cr- / ß / cyclodextrin was 1: 4.1: 2.23.

The sparingly soluble constituents of the reaction mixture were now separated by centrifugation, suspended in 1 l of water and heated to boiling, bromobenzene distilling off as an aceotrope with water.

The slightly cloudy solution obtained was cooled to room temperature and treated with 1.2 l of methanol. After separating the precipitate formed was the methanol is distilled off and the aqueous solution is concentrated to such an extent that the concentration of cyclodextrin was 20% by weight. The concentrate obtained was then 12 Stored at 4 "C for hours.

48 g of colorless crystals of β-cyclodextrin were obtained which separated from the solution and washed once with 200 ml of ice-cold water.

7.2 g of bromobenzene were added to the combined supernatants and Stirred for 6 hours at room temperature. The resulting colorless precipitate was then centrifuged off, taken up in 150 ml of water and boiled under aceotropic distillation of the bromobenzene brought into solution.

The aqueous solution obtained was finally lyophilized.

There were 30 g of a mixture of 2.4 g of β-cyclodextrin and 27.6 g Obtained z-cyclodextrin.

The y-cyclodextrin yield was accordingly 98.4%, based on the z-cyclodextrin content of the conversion mixture.

The mixture was used for further purification after drying for 2 hours suspended at 105 "C in 100 ml of pyridine and stirred at 50" C for 2 hours. Afterward 200 ml of methanol were added. The colorless precipitate obtained was in 150 ml of water suspended and brought into solution by heating. The -Cyclodextrin was precipitated with 450 ml of ethanol and dried in vacuo after reprecipitation with ethanol.

There were 27.2 g of γ-cyclodextrin with a purity of 98.2 % obtain. The yield based on the z-cyclodextrin content of the conversion mixture was 95.2%.

A repetition of the cleaning process described resulted in 25.6 g of r-cyclodextrin with a purity of 99.8%.

The yield of pure product, based on the starch used, was 17%.

Example 2 200 g of cassava starch were added to 1 1 of water with an additional Suspended content of 200 mmol sodium acetate and 5 mmol calcium chloride and through 30 minutes of heating to 120 "C gelled. The pH of the reaction mixture was 6.9.

After cooling to 70 ° C., the mixture was stirred and cooled further 13 mg of cyclodextrin glycosyltransferase from Klebsiella pneumoniae in the same Preparation as described in Example 1 added. With continued stirring was cooled to 43 "C and then another 27 mg of cyclodextrin glycosyltransferase from Klebsiella pneumoniae was added in the same preparation as described above.

After an incubation time of 8 hours at 43 "C, it was determined by chromatographic Analysis determined, no further increase in initially formed a-cyclodextrin determined.

Then 32.6 g of bromobenzene were added to the reaction mixture. To a further incubation time of 40 hours at 43 "C was 59 wt .-% of the starch degraded to cyclodextrins.

The ratio of a / B / cyclodextrin was 1: 10.6 3.92.

The insoluble constituents of the conversion mixture were through Separated centrifugation and taken up in 1 l of water.

The suspension was heated to the boil, with bromobenzene as an aceotrope distilled off with water.

The slightly cloudy solution obtained was then concentrated to such an extent that the concentration of γ-cyclodextrin was 20% by weight. The concentrate obtained was Stored at 4 ° C for 12 hours.

74 g of crude β-cyclodextrin were obtained, which were separated from the solution and washed once with ice cold water.

The combined supernatants were described as in Example 1 worked up.

The following were obtained: 29.2 g of crude γ-cyclodextrin, corresponding to one Yield of 98.6% based on the z-cyclodextrin content of the conversion mixture 27 g of y-cyclodextrin with a purity of 99.7%, corresponding to a yield, based on to used strength of 13.5%.

Example 3 The procedure of Example 2 was repeated, with the modification that instead of in the presence of 200 mmol of sodium acetate in the presence was gelified by 200 mmol of sodium formate.

After a total incubation time of 48 hours, 57% by weight was the Starch broken down into cyclodextrins. The proportion of γ-cyclodextrin was 14% by weight.

Example 4 The procedure according to Example 2 was repeated with the modification that instead of in the presence of 200 mmol of sodium acetate in the presence was gelified by 200 mmoles of potassium propionate.

After a total incubation time of 48 hours, 56.7% by weight were the starch is broken down into cyclodextrins. The proportion of cyclodextrin was 13.8%.

Claims (3)

Claims. "1. Process for the preparation of cyclooctaamylose by enzymatic Cleavage of starch and subsequent separation of the cleavage products, d a d u r c h g e -k e n n n z e i c h n e t that a) an aqueous preparation of starch Cyclodextrin glycosyl transferase is added and b) at least after the amount in primarily formed cyclodextrin no longer increases, the preparation according to a) with Bromobenzene is added. 2. The method according to claim 1, d a d u r c h g e -k e n n z e i c h n e t that the aqueous preparation of starch prior to the addition of cyclodextrin glycosyltransferase is mixed with alkali salts of short-chain alkanoic acids. 3. The method according to claims 1 and 2, d a d u.r c h g e k e n It should be noted that in the separation of the cleavage products from cyclooctaamylose pyridinic suspension is obtained by adding alcohol.