DK143414B - METHOD OF PREPARING A GLYCOSIDE HYDROLASE INHIBITOR - Google Patents

Description

| J | (12) PUBLICATION <ιυ 143414 B

(is) DENMARK

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 159 ^ / 78 (51) lnt.CI.3 C 12 P 21/00 (22) Filing Day Π. April 1978 C 07 0 7/00 (24) Race day 11 Apr. 1978 (41) Aim. available 12 · Oct. 1979 (44) Posted 17 Aug. 1981 (86) International application no. - (86) International filing day - (85) Continuation day - (62) Master application no. - (30) Priority - (71) Applicant HOECHST AKTIENGESELLSCHAFT, 6230 Frankfurt / Main 80, DE.

(72) Inventor of Volker Oeding, DE: Werner Pfaff, DE: Laszlo Verte = she, DE: Hans-Ludwig Weidenmueller, DE.

(74) Associate Engineering Company Budde, Schou & Co.

(54) Process for Preparing a Glycoside Hydrolase Inhibitor ·

The present invention relates to a process for the preparation of a novel inhibitor of glycoside hydrolases in the digestive tract, particularly of pancreatic α-amylase.

Inhibitors of glycoside hydrolases from microorganisms, especially 3D Actinomycetes, are known. The substances so far investigated are chemically part of the oligo or polysaccharide class. Similar inhibitors with probable peptide character are described 2 as heat-labile, more or less easily inactivated with trypsin '1 - and, first of all, relatively less active.

It has now been found in culture mixtures of Streptomyces tendae, 3 strain 4158, a highly active pancreatic α-amylase inhibitor, which chemically belongs to the peptides.

2 1434 14

Thus, the present invention relates to a process for producing a peptidic glycoside hydrolase inhibitor having a molecular weight of 5000-10,000, an absorption maximum in UV light at 276 nm, an isoelectric point of 4.4, and an amino acid composition as The method below is characterized by cultivating Streptomyces tendae 4158 (ATCC No. 31210) in a culture medium containing at least one carbon source, at least one nitrogen source and inorganic salts, and from the culture the inhibitor is recovered.

As mentioned, the present inhibitor has a relatively high molecular weight. It does not or only diffuses very little through conventional dialysis membranes, such as "Wiping" hoses. Determination of the exact molecular weight is consequently difficult and various methods of determination are obtained for divergent results. If the analytical ultracentrifuge (cf. Biochemisches Taschenbuch, part 2, pages 746-767) is used to determine the molecular weight, values of approx. 10,000. By contrast, if molecular sieves such as "Sephadex" G-50 superfine are used, a molecular weight of 5000 or even less is found. Accordingly, on the basis of the present study results, it is assumed that the molecular weight is between 5000 and 10,000.

The compound of this invention is a colorless substance.

However, as mentioned, it absorbs ultraviolet light with a maximum of 276 nm at 1%, exhibiting a shoulder at 281 nm. E7 ° = 16.

33 '1 cm -

The drawing shows an absorption spectrum.

According to its chemical structure, the inhibitor is a peptide.

It can be cleaved into amino acids by hydrolysis. So far, it has not been possible to detect other components besides the amino acids. Is the amino acid composition determined by that of Moore and W. H. Stein (cf. Methods in Enzymology, Vol. VI, pages 819-331, published by Colo-vick and Kaplan in Academic Press, New York, London 1963), the following composition is noted

Aspartic acid 5-6

Threonine 5-6

Serine 3-5

Glutamic Acid 5-6

Proline 2-3

Alanine 5-6 3 143414

Glycine 5-6

Cysteine 3-4

Selected 5-6

Isoleucine 1-2

Leucine 3-4

Tyrosine 4-5

Phenylalanine 0-2

Histidine 1-2

Lysine 0-1

Arginine 2-3

Tryptophan 1-2 The value of tryptophan is evaluated based on the absorption of ultraviolet light. It is natural that the results of the amino acid study do not always indicate a specific integer ratio and that such measurements are usually subject to certain errors. Accordingly, the range of variation shown in the set does not depend on the disequilibrium of the inhibitor but on the inevitable measurement inaccuracy of the assay method. However, it is characteristic of the present inhibitor that the amino acids aspartic acid, glutamic acid, threonine, glycine, alanine and valine constitute an above average crude alkyl part of the peptide and that methionine appears to be completely lacking in the pure substance. Since methionine is a widespread amino acid, its extensive absence is a good characteristic of the present inhibitor and, especially in enrichment methods, may also serve to determine the purity of the product.

The present inhibitor reacts positively to peptide reagents but negatively to phenolic sulfuric acid.

The α-amylase inhibitor disclosed herein contains no sugar content. Thereby, as well as by its amino acid composition, its molecular weight and its isoelectric point, it differs from all known α-amylase inhibitors. Pure preparations of the inhibitor exhibit an activity of 2-3 x 10 5 AIE / g.

Of being a substance of peptide nature, the subject amylase inhibitor exhibits remarkable thermal stability. Even boiling in a neutral or slightly acidic medium (pH about 4-8) for one minute has no appreciable effect on its gly coside hydrolase inhibitory properties.

4

U3AU

The inhibitor is only proteolytically inactivated with pepsin, trypsin, or chrymotrypsin very slowly compared to other proteins. Therefore, during the therapeutic period, a significant reduction in activity in the digestive tract cannot be expected.

The inhibitor exhibits a high specificity of action. The α-amylase from the pancreas is greatly inhibited, whereas bacterial α-amylases, e.g. those of Bacillus subtilis, are not measurably inhibited. Similarly, no effect against β - amy laser has been observed.

Even low doses of the amylasein inhibitor in question lead to a complete inhibition of the enzymatic activity of pancreatic amylase. This study result cannot be explained by the classical inhibitory mechanisms by which the inhibition of enzymatic catalysis depends on the relative amount of inhibitor to substrate (starch) ratio. Rather, it is to be assumed that the present inhibitor causes an irreversible inactivation of pancreatic amylase.

In order to prepare the amylase inhibitor, according to the invention, the strain Streptomyces tendae 4158. is used. This strain differs from the original strain S. tendae (cf. Ettlinger et al., Arch. Microbiol. 31 The spores are spherical and have a diameter of 1 ^ i. The properties of the strain are as follows: 143414 5

Substrate mycelium color: j yellow-brown, no color change j at pH shift

The trace-encrusted light grayish

mycelium color: reddish brown according to ISCC

methods of designating colors) Morphology of the spore chains: Retinaculum apertum (RA)

Spore morphology: spherical spores with an average diameter of 1 p.

Smooth to slightly warped surface.,

Melanin formation on peptone positive J

medium:! , ..... .......... ..... ...... -------—_____ '. ... 'j

Nitrate Reduction: Positive: —-—-----: - 1:

Substrate Utilization Glucose ++ I

spectrum: arabinose + i

Sucrose + I

Xylose + I

Inositol ++! 1

Mannitol ++]

Fructose +

Rhamnose ++

Raffinose + -

cellulose

The strain Streptomyces tendae 4158 is deposited with the American Type Culture Collection (ATTC) under registration number 31210.

The fermentation can be carried out at 25-35 ° C, preferably at 28-30 ° C, either submersed in shaking culture or in fermentation vessels of various sizes under stirring and aeration. In addition to the inorganic salts commonly used in the cultivation of microorganisms, the culture medium contains at least Sn carbon source such as starch, glucose, cane sugar, fructose, lactose, glycerol or molasses and at least Sn nitrogen source such as soy flour, corn mold, yeast extract, cotton flour, peanut flour, milk powders, nitrates or ammonium salts.

6

U34U

The optimum composition has been found to be (% by weight in solution) 3-5% soluble starch, 0.2-0.6% maize cast water, 0.5-1.5% glucose, 0.5-1% (NE ^), 0.3-0.6% soybean meal and 0.5-1.5% casein peptone. Also on other starchy nutrient media, e.g. consisting of 2-6% peanut flour, 1-3% potato starch, 3-5% oatmeal, 3-5% whey powder, 1-2% whey syrup and 1-2% milk sugar, the amylase inhibitor is obtained in good yield, with the choice of nitrogen source and buffer proportion , insofar as one is in physiological areas, is not so crucial. However, if you reduce or completely iron out the starch content, the inhibitor yield drastically declines. On the other hand, if the starch concentration is increased significantly above 7%, the oxygen supply of the microorganisms becomes suboptimal due to the high viscosity. Accordingly, the inhibitor yield decreases.

In the fermentation mixture, inhibitor formation usually begins between the 10th and 30th hour after the start of the fermentation.

Over the next 20 hours, it will be largely completed. Longer fermentation times have no adverse effect on the inhibitor yield, but this is also not significantly increased. From this it appears that the fermentation mixtures should be kept in operation for approx.

30-70 hours.

For isolation of the inhibitor, by centrifugation, filtration or separation through the suction filter, the cell mass is removed from the fermentation solution, since most of the active substance is usually in the clear culture liquid.

In the event that part of the inhibitor remains in the cell material due to special fermentation conditions, it is of no difficulty using suitable methods to extract the active substance therefrom, e.g. by stirring with a water-miscible organic solvent, such as methanol, whereby further maceration of the cellular material is advantageous. The active substance dissolved in the extractant can be liberated by centrifugation or filtration from the undissolved cell components and further processed as the clear culture liquid.

The inhibitor can be isolated from the culture fluid in a manner known per se in the protein and peptide chemistry. In addition, precipitates with water-miscible organic solvents such as 7 are suitable

14341A

acetone, isopropanol or other lower alcohols, or also with salts such as ammonium sulfate.

Furthermore, it is possible to adsorb the inhibitor on corresponding carriers, e.g. on activated carbon, and separate this from the aqueous solution by filtration or centrifugation. This can occur within a wide pH range between 2 and 10, but preferably the range between 4 and 6. To separate the α-amylase inhibitor in question, ion exchangers can also be used.

Since the substance of the present invention has both acidic and basic properties, that is, amphoteric, it can react both with cation and anion exchangers and by means of these is removed from the fermentation solution. In this way, the techniques described in principle in the chapter on ion exchangers in Biochemisches Taschenbuch, published by Η, can be used. M. Rauen in Springer-Verlag 1964, Part 2, pages 808-824.

When working up fermentation mixtures containing the subject matter in the culture liquid, it is often appropriate to concentrate these. For this purpose, the known methods can be used for distillation, ultrafiltration, spray and freeze drying or other known methods. From the concentrate thus prepared, the inhibitor can again be separated by the methods described above. Of concentrates, such as the evaporated culture filtrates, the inhibitor can also be enriched by removing the major impurities. Thus: extraction with organic solvents to separate fatty substances is advantageous. By dialysis or possibly ultrafiltration (cf. C. J. O. R. and P. Morris, "Separation Methods in Biochemistry", Ditman Publishing, London 1976, pages 944-950), the solution can be freed of low molecular weight substances. High molecular weight substances such as nucleic acids, polysaccharides or many egg whites can be removed by fractional precipitation by salting out or by the addition of a water miscible solvent such as acetone or a lower alcohol. In these cases, the addition of the precipitant is dosed such that the easily separable substances with molecular weights above 100,000 are precipitated while the α-amylase inhibitor remains in solution. The enrichment steps described can be combined and varied in any order. In this way, solutions are strongly enriched with inhibitor.

The final purification can be carried out by various non-ionic methods, such as gel and ion exchange chromatography, or by related processes whose separation principle does not depend solely on ion exchange, such as separation by hydroxylapatite.

143414 8

Furthermore, solvent or salt precipitates, preparative electrophoresis or other methods known per se can be used. Particularly suitable are separation by diethylarainoethyl (DEAE) group-bearing ion exchangers as well as fractional precipitates with ammonium sulfate or ethanol, thereby obtaining crystalline material.

The properties of this inhibitor are interesting for its use as a therapeutic agent for diabetes and pre-diabetes, as well as obesity and dietary support.

Starchy foods and pleasures lead to an increase in blood sugar in animals and humans and thereby also to increased insulin secretion in the pancreas. Ilyperglycemia results from cleavage of the starch in the digestive area to glucose under the action of amylase and maltase.

Kos diabetics, this hyperglycemia is particularly pronounced and long-lasting.

In adipose, the increased insulin secretion acts on the lipogenesis and decreases the lipolysis.

The alimentary hyperglycemia as well as hyperinsulinemia after starch uptake can be reduced by the amylase inhibitor in question. This effect is dependent on the dosage. Therefore, the present amylase inhibitor can be used as a therapeutic agent in diabetes, pre-diabetes and obesity as well as in support of diet. For this purpose, an administration is recommended, especially for meals. The dosage, which depends on the patient's weight and individual needs, amounts to approx. 10,000-300,000 AIE, but in justified individual cases it may also be above or below.

The amylase inhibitor in question is particularly suitable for oral administration. It can be used as a pure substance but also in the form of a pharmaceutical composition using conventional adjuvants and carriers. Also, a combined use with other drugs, such as blood sugar lowering or lipid lowering drugs, may be advantageous.

As high molecular weight peptides as such are not or not appreciably resorbed from the digestive tract, toxicological adverse effects are not expected from the subject matter here. On the basis of the not unusual amino acid composition, any proteolytic cleavage products must also be considered physiologically acceptable. Correspondingly, striking symptoms have not been observed with oral administration of even high doses of the amylase inhibitor to experimental animals.

Also, by intravenous administration to mice (1 g / kg), the inhibitor disclosed herein is tolerated at a 24-hour observation time with no appreciable toxic effect.

To study the pharmacological action of the amylase inhibitor, the present curing agent is orally administered to male Wistar fasting rats weighing between 200 and 250 g at the same time as 2 g of starch per day. kg of body weight, after taking a blood test immediately before to determine the starting blood sugar value. Additional blood samples are taken from the tail vein after 15 and 30 minutes and after 1, 2, 3 and 5 hours. The blood glucose assays are performed in an Autoanalyzer according to the method described by Hoffman (cf. J. Biol. Chem. 120, 51 (1937)).

NZO mice exhibit a disturbed glucose tolerance. Therefore, they are particularly well suited for studies in which blood glucose levels are affected. The test order is similar to that described for the rats. The blood samples are taken from the orbital vein plexus. Blood glucose monitoring is followed for 3 hours.

Analogously, the detection of active substance in NMRI mice is provided. The blood samples are also taken from the orbital vein plexus and the blood sugar course is monitored for 3 hours.

In these test series, the animals treated with the inhibitor in question relative to untreated animals exhibit an inferior, protracted rise in blood sugar.

Amylaseforsøg

An amylase inhibitor unit (AIE) is defined as the amount of inhibitor which, under the experimental conditions, is able to inhibit two amylase units (AE) to 50%. An amylase unit is, by international agreement, the amount of enzyme that in 1 minute cleaves 1 ju equivalent of glucosidic bonds in the starch. The pollen cleaved glucosidic bonds are determined with dinitrosalicylic acid photometrically as unvaluating reducing sugar. The indications are calculated as µmol of maltose detected by means of a maltose measuring line.

The experiments are carried out as follows: α-amylase from pig pancreas and the solution to be tested are preincubated together in 1.0 ml of 20 mM phosphate buffer, pH 6.9 + 10 mM NaCl for 10-20 minutes at 37 ° C. The enzymatic reaction is started by adding 1.0 ml of soluble starch according to Zulkowski (0.25% in the indicated buffer). After exactly 10 minutes 10

1434U

The reaction is quenched with 2.0 ml of dinitrosalicylic acid dye (according to Boehringer Mannheim: Biochemica-Information II) and heated for 5 minutes in a boiling water bath for color development. After cooling, the extinction is measured at 546 nm relative to the reagent bee value. The 50% inhibition is determined graphically by probability paper relative to the uninhibited enzyme reaction using different inhibitor amounts.

Example 1

The Streptomyces tendae 4158 strain is seeded on inclined tubes with a nutrient substrate of the following composition: 50 g oat flakes ad. 1000 ml H2 O pHA 7.2

The seeded tube is incubated for 7 days at 30 ° C and then kept at 4 ° C. The spores are flushed away from the inclined tube with 10 ml of sterilized distilled water or a physiological NaCl solution. 1.0 ml of the suspension is used to inoculate a 300 ml Erlenmeyer flask containing 35 ml of sterilized nutrient solution having a pH of 7.7 and the following composition: 1% glucose 1% soybean 0.25% NaCl pH 7.7.

The flask is shaken at 220 rpm and 30 ° C for 48 hours on a shaker at an amplitude of 4 cm. Next, 5 ml portions of this preculture are transferred into several Erlenmeyer flasks containing 35 ml of sterilized nutrient solution having a pH of 8.3. The composition of this main culture is as follows: 4% starch 0.4% corn cast water 1.0% glucose 0.8% (NH4) 2HPO4 0.4% soybean 1.0% peptone pH ^ 8.3.

The main cultures are also shaken at 30 ° C at 220 rpm on a shaker with an amplitude of 4 cm for 2-3 days. At first,

U3AU

On the second and third day of cultivation, the content of α-amylase inhibitor is determined according to the experimental regulations.

The Streptomyces tendae 4158 strain yields an average of 100 AEI / ml, under the described experimental and culture conditions, at a final pH of 5.2.

Example 2

The same procedure as in Example 1 is used, however, for the main culture, a fermentation vessel having a total volume of 300 liters is selected containing 200 liters of a sterilized nutrient solution of the following composition: 4% starch 0.4% corn cast water 1.0% glucose 0.8% (NH4) 2HPO4 1.0% casein peptone 0.1% "Desmophen ® 3600" pKA 8.3-8.5

The sterilization time of the mixture is 45 minutes at 121 ° C and 1 bar, whereby the glucose is sterilized separately and after cooling the fermentation vessel to operating temperature is added sterile.

The pH should, after sterilization, be 6.8 and, if necessary, be adjusted to this value with sterilized acid (2 n H ^ PC ^) or base (2 n NaOH). The main culture is seeded with 20 liters corresponding to 10% of a pre-culture prepared as described in Example 1 and in a small fermentation vessel with a total volume of 30 liters.

Ferment for 50-70 hours at 30 ° C. The aeration 3 is 6 m / h with a stirring of 250 rpm and an overpressure of 0.3 bar.

When sampling, the fermentation process is monitored and monitored for the culture solution's inhibitory activity, substrate degradation, biomass evolution and physico-technical conditions (surface tension, viscosity, density and osmotic pressure).

The maximum inhibitory activity is obtained from the 60th culture hour at an average of 105 AIE / ml. The contents of the fermentation vessel are then processed for reprocessing.

1434U

Example 3 12

A method is used as in Examples 1 and 2, however, as a main step, a bioreactor with a total volume of 4000 liters / in which 2500 liters of nutrient solution of the following composition is filled is used: 6 S starch 1.0% glucose 0.4% corn cast water 0 , 8% (NH4) 2HPO4 1.0 S soy peptone 0.1% "Desmophen R 3600" pH & 7.0-7.3.

The sterilization time of this mixture is 60 minutes at 121 ° C and 1 bar, whereby the glucose is sterile filtered separately and after cooling the fermentation vessel to operating temperature is pumped to it under sterile conditions.

The pH is adjusted - if necessary - with sterile acid (H 2 PO 4) or base (NaOH) to an initial value of 7.0-7.3.

200 liters of a preculture described and prepared in Example 2 are seeded.

The fermentation time is 70 hours, thereby fermenting at a temperature of 30 ° C, an aeration of 60 Nm / h, an overpressure of 0.5 bar and at 180 rpm.

When sampling - as described in Example 2 - all important process, organism and activity data are followed throughout the fermentation time.

After 70 hours of fermentation, an average activity concentration of 55 AIE / ml is achieved. The fermentation is then discontinued and the culture solution brought to work up.

Example 4 10 liters of a filtered culture liquid prepared according to Examples 1-3 with an activity of 100 AIE / ml are dried in a spray-drying apparatus and 400 g of light brown powder are obtained. For degreasing, this is thoroughly stirred with a mixture of 1 liter of methanol and 1 liter of chloroform and then separated on suction filter. The still solvent-free, undissolved product containing the active substance is dissolved in 3 liters of water and adjusted to a pH of 6.5, and 4.5 liters of methanol are added. This creates a light, fluffy precipitate which is removed by centrifugation and discarded as it contains only negligible amounts of the α-amylase inhibitor. The dark liquid above

U3AU

The methanol precipitate containing the desired inhibitor is then freed from methanol in vacuo and the now concentrated to 2.5 liters of solution is dialyzed salt-free against distilled water. The retenate contains 9 g of dry matter (specific effect 96 AIE / mg). The retenate is then brought to a saturation of 50% with ammonium sulphate at a pH of 5.5, to which 79 g of salt is needed. At the salting out, a precipitate is formed containing approx.

90% of the active substance. After centrifugation, the above liquid is discarded. The precipitate is redissolved in distilled water and at a pH of 8 ammonium sulphate is again added, but this time only to a saturation of 15% and after removal of the precipitate further to a saturation of 35%. The precipitate from the salting between 15 and 35% contains the major amount of the amylase inhibitor. This fractional precipitation is repeated.

After dialysis and drying, 360 mg of product with a specific activity of 992 AIE / mg is obtained. 350 mg of this crude product is then divided into "Sephadex G-50 fine" in a glass column 100 cm long and holds 1.2 liters and enriched. As the swelling and eluent is used an aqueous 5 millimolar phosphate buffer solution having a pH of 8 to which is added 0.02% sodium azide. The crude product is dissolved in 15 ml of the same buffer and applied to the column. The elution takes place over two days, taking 15 ml fractions.

The four most active fractions are collected, dialyzed, salt-free and freeze-dried. They provide 60 mg of white powder with an activity of 2520 AIE α-amylase inhibitor / mg.

Example 5 2200 liters of culture liquid prepared according to Example 3 is cooled to 6 ° C and filtered through a chamber filter press after the addition of 2% silica gel. The filter cake (about 450 kg, moist) is discarded, the clear filtrate (1750 liters with an activity of 95 AIE / ml) is evaporated via a drop stream evaporator to 120 liters after the addition of 500 g of sodium azide.

The resulting concentrate is cooled to 1 ° C and 120 liters of pre-cooled acetone are added slowly and with stirring. For complete precipitation, stir for a further 1/4 hour and after addition of 1 kg of silica gel is clarified via a filter press. Discard the solid with the filter aid. The acetone filtrate containing the active substance is mixed with stirring and cooling with 380 liters of acetone to give an approx. 80% acetone suspension. The resulting (2nd) precipitate contains

1434U

14 the desired amylase inhibitor.

It is extracted by allowing the trap bag to stir well. Thereby the precipitate sinks down and the above liquid can be foamed off. From the above liquid from this second acetone precipitate, small amounts of precipitate can be separated by centrifugation. This solid is dissolved at a pH of 7 and added to the precipitate solution (see below). The scalp residue remaining in the precipitation vessel is dissolved in 120 liters of water at a pH of 7 and the resulting solution is clarified with a through-flow centrifuge (Cepa, 1700 opra). This removes 90 g of inactive suspended particles. The clear aqueous phase is now concentrated up to 15 liters with a DDS ultra filtration system (membrane # 800) and dialyzed. To complete the desalination, the retenate is diluted with distilled water and concentrated again. After repeating two to three times, the retenate containing the amylase inhibitor is practically salt-free. Upon addition of 19.5 kg of ammonium sulphate, the inhibitor is then precipitated at a pH of 5.5 from 50 liters of retenate solution and centrifuged and the supernatant is discarded. The precipitate is redissolved in 40 liters of water and this time precipitates at a pH of 7.5 with 12.5 kg of ammonium sulphate. After separation of the clear phase in a tube centrifuge, the precipitate is fractionally precipitated as in Example 4:

From 40 liters of the redissolved substance, at a pH of 5.5, with the addition of 3.2 kg of ammonium sulphate, an inactive precipitate is first precipitated and separated and discarded. Next, the major part of the active substance is separated from the liquid by further concentration of the liquid phase with 7.4 kg of ammonium sulfate. This precipitate is collected, dialyzed against distilled water and lyophilized. 114 g of brown powder are obtained with an activity of 1100 AIE / mg.

For further division, fill a glass column with a diameter of 5 cm and a height of 50 cm (holds about 1 liter) with DEAE "Sephadex" A-25, previously equilibrated with 1 molar phosphorus buffer to a 30 pH value of 7.5 and 0.02% sodium azide. Now 10 g of substance is dissolved in 100 ml of the same buffer and applied to the column. Next, the column is washed with 1 liter of the same buffer, after which, to this eluant, boiling salt is gradually added in such an amount as to provide a continuous gradient. If the column eluate is fractionally trapped, the amylase inhibitor is in the fractions in which the NaCl concentration is approx.

0.5 molar.

15

U34U

These fractions are collected and dialyzed against distilled water. Freeze drying gives 5.6 g of light brown substance with an activity of 2200 AIE / mg.

In addition, a gel chromatographic purification is also performed, as described in Example 4. Of the material of 5.6 g, 4.1 g of colorless powder is obtained, the activity of which is 2800 AIE / rag. Upon amino acid analysis of the product, after a 20-hour hydrochloric acid hydrolysis, the following composition is obtained by a Beckman multichrome analyzer:

Aspartic acid 7.72%

Threonline 7.66%

Serine 5.35%

Glutamic Acid 10.05%

Proline 3.43 l

Glycine 4.93%

Alanine 5.84% 1/2 cysteine 4.26%

Selected 7.72%

Methionine 0.41%

Isoleucine 2.30%

Leucine 5.18%

Tyrosine 10.24%

Phenylalanine 3.35%

Histidine 3.01% bysin 1.45%

Arginine 5.16%