FI59815C - FREQUENCY REFRIGERATION FOR ALPHA-AMYLES INHIBITORS WITH STRUCTURAL STRUCTURES - Google Patents

Description

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Patent · och registerstyraisen x 'Anattkan utlagd och utl. * Krtftan publkarad 30.06.81 (32) (33) (31) Privilege claimed— B «flrd prloritet (71) Hoechst Aktiengesellschaft, D-6230 Frankfurt / Main 80, Federal Republic of Germany t-avalt a-Förbun dsrepubliken Tyskland (DE) (72) Volker Oeding, Kelkheim (Taunus), Werner Pfaff, Hofheim am Taunus,

Laszlo Vertesy, Epps tein / Taunus, Hans-Ludwig Weidenmiiller, Hofheim am Taunus, Federal Republic of Germany Tyskland (DE) • (7U) Oy Kolster Ab (5U) E-amyl-inhibitor with peptide structure

The invention relates to a process for the preparation of a peptide-structured O-amylase inhibitor, characterized in that Streptomyces tendae strain U158 (ATCC No. 31210) is cultured in a culture medium containing at least one carbon source and one nitrogen source and inorganic salts, and that an inhibitor having a molecular weight of 5,000 to 10,000, an absorption maximum under UV light at 276 nm, an isoelectric point U, U and the following amino acid composition are isolated from the culture in a conventional manner: aspartic acid 5-6 isoleucine 1-2

threonine 5-6 leucine 3-U

serine 3-5 tyrosine U-5 glutamic acid 5-6 phenylalanine 0-2 proline 2-3 histidine 1-2 glycine 5-6 lysine 0-1 alanine 5-6 arginine 2-3 cysteine 3-U 'tryptophan 1-2 valine 5-6 2 59815

It is known to prepare glycoside hydrolase inhibitors from wheat (FI patent publication li-9,309). These inhibitors have a peptide structure.

DE-A-20 6U 092 discloses the preparation of amylase inhibitors from microorganisms, in particular from actinomycetes. In this DE publication, six groups of inhibitors are mentioned. The second group belongs to oligo- or polysaccharides of a chemical nature, and the second group is trypsin-degradable polypeptides with an inhibitory activity against amylase of 80 AIY / mg. DE-A-2 702 Ul7 discloses a glycopeptide which gives a positive color reaction of sugar with some reagents but does not give a positive reaction of peptides with the Folin-Ciocalteun peptide reagent; however, acid hydrolysis could detect lysine.

A peptide-type amylase inhibitor has been isolated from Streptomyces flavochromogenes strain No. 280 (U.S. Patent 3,86 k6l). It is a glycopeptide that contains four amino acids based on acid hydrolysis: glutamic acid, aspartic acid, serine, and alanine. It is stable in up to 1N hydrochloric acid for 15 min. time, while in 1 N sodium hydroxide solution at 100 ° C it quickly becomes ineffective. It is a potent enzyme inhibitor. In Chem. Abstr. butter. 83 (1975), 176 706 n describes an acidic polypeptide derived from Streptomyces fradiae strain FERM-P2303, which is also not an irreversible inhibitor. Said publication does not describe an amphoteric, irreversible? 2-amylase inhibitor having a peptide nature. Isolation of the 0β-amylase inhibitor from the Streptomyces tendae type strain ATCC 19812 has not previously been described in the literature.

A highly active inhibitor of pancreatic Ot amylase has now been found in the fermentation broths of Streptomyces tendae strain U158.

The inhibitor is characterized by a molecular weight of 5,000 to 10,000, an absorption maximum UV light at 276 nm, an isoelectric point and the amino acid composition shown below.

The molecular weight of the inhibitor according to the invention is quite high. It does not diffuse or diffuses only to a very small extent through commercial dialysis membranes such as Visking membranes. Determining the exact molecular weight is difficult and different assay methods give different results. If an analytical ultracentrifuge (Biochemisches Taschenbuch, 196U, Part 2, pages 7 ^ 6-767 »is used to determine the molecular weight, values of about 10,000 are obtained. or even a lower value.According to currently available research results, λ is considered to have a molecular weight of 5,000-10,000.

The compound of the invention is a colorless substance. However, it absorbs ultraviolet light at a maximum point of 276 nm with a shoulder at 281 nm.

3,59815 1 ί E, = 16. The absorption spectrum is shown in Figure 1.

1 cm

Due to its chemical nature, the inhibitor is a peptide. It can be separated into amino acids by hydrolysis. To date, however, no components other than amino acids have been identified. In determining the amino acid composition according to the method described in St. Moore and W.H. Stein (Methods in Enzymology, Part VI, pages 819_831, Colovick and Kaplan, Academic Press, New York, London 1963) »the following composition is obtained: aspartic acid 5-6 trainine 5-6 serine 3-5 glutamic acid 5 ~ 6 proline 2- 3 alanine 5-6 glycine 5-6 cysteine 3- ^ valine 5-6 isoleucine 1-2 tyrosine U-5 phenylalanine 0-2 histidine 1-2 lysine 0-1 arginine 2-3 tryptophan 1-2

The value of tryptophan was evaluated by the absorption of ultraviolet light. It is natural that the results of an amino acid study do not always imply a single, integer ratio, and that measurements like this usually involve certain errors. Thus, the limits of variation given in the list do not mean the non-uniformity of the inhibitor, but the measurement inaccuracy necessarily associated with the method of analysis. The inhibitor according to the invention is characterized in that the amino acids aspartic acid, glutamic acid, threonine, glycine, alanine and valine form a higher than average molecular weight of the peptide and that methionine appears to be completely absent from the pure substance. Since methionine is a very common amino acid, its complete absence is a good feature of the inhibitor of the invention and this can also be used especially in the enrichment method to determine the purity of the product.

The inhibitor of the invention reacts positively with peptide reagents but negatively with phenolic sulfuric acid.

The oL-amylase inhibitor of the invention does not contain a sugar component. Based on this, as well as its amino acid composition, molecular weight, and isoelectric point, it differs from all known α 2 -amylase ε inhibitors. The activity of the pure inhibitor preparation is 2-3 x 10 AIY / g.

and 59815

As a peptide-structured substance, the thermal stability of the amylase inhibitor according to the invention is remarkably good. Even cooking in a neutral or weakly acidic medium (pH about U-8) for one minute does not significantly affect its glucose hydrolase inhibitory properties.

Pepsin »trypsin or chymotrypsin causes the inhibitor to convert proteolytically inactive only very slowly compared to other proteins. Thus, no significant decrease in gastrointestinal activity is expected during the therapeutic duration.

The specificity of action of the inhibitor is high. In contrast, pancreatic amylase is highly inhibited, whereas bacterial ° C amylases, for example those obtained from Bacillus subtilis, are not measurably inhibited. Similarly, no effect against β-amylases has been observed.

Even small doses of the amylase inhibitor according to the invention cause a complete inhibition of the enzymatic activity of the pancreatic amylase. This finding cannot be explained by classical inhibition mechanisms, where the inhibition of enzymatic catalysis depends on the quantitative relationship between the inhibitor and the medium (starch). Rather, the irreversible inactivation of pancreatic amylase by the inhibitor of the invention must be assumed.

According to the invention, Streptomyces tendae strain U158 is used to prepare the amylase inhibitor. This strain differs from the original S. tendae strain ATCC 1912. (Ettlinger et al., Arch. Microbiol. 31, 351 (1958)) with respect to their spore morphology. The spores are spherical and have a diameter of 1 micro-1 meter. The characteristics of the stock are as follows:

Substrate mycelium color: yellowish brown, no color change due to pH shift

Spore-containing air felt-gray-brown (slightly greyish reddish-brown; ton color: ISCC methods of designating colors)

Spore chain morphology: Retinaculum apertum (RA)

Spore morphology: spherical spores with an average diameter of 1 μa. Smooth or slightly warty surface

Melanin formation in pepton medium: positive

Nitrate reduction: positive

Substrate use spectrum: glucose ++ arabinose + sucrose + xylose + inositol + mannitol ++ fructose + rhamnose ++ raffinose + - cellulose 5 5981 5

Strain Streptomyces tendae U15O is deposited in the American Type Culture Collection (ATCC) under accession number 31210, except for the above differences, the description of Streptomyces tendae ATCC 31210 corresponds to the description of the original S. tendae strain (Ettlinger et al., Arch. Microbioi). 3J_, pp. 351-352, 1958.

The culture can be performed at 25-35 ° C, preferably 28-30 ° C, either as a submerged shake culture or in culture vessels of different sizes with stirring and aeration.

The culture medium is a particularly suitable combination of carbon and nitrogen sources. Such a culture medium contains, in addition to the inorganic salts commonly used in the cultivation of microorganisms, at least one carbon source such as starch, glucose, cane sugar, fructose, lactose, glycerin or molasses and at least one nitrogen source such as soybean meal, cornmeal powder, corn liquor , milk powder, nitrates or ammonium salts.

Utilization of carbon sources is described in Bergey's Manual of Determinative Bacteriology, 8th Edit., Pp. 772, 197 ^ ·

The optimal composition has been shown to be: (by weight # solution) 3-5% soluble starch, 0.2-0.6% corn solubilizer, 0.5-1.5% glucose, 0.5-1% (NH 2 ^, 0.3-0.6% soy flour, 0.5-1.5% casein peptone Also when using other starchy nutrient media, for example a composition containing 2-6% peanut flour, 1-3% potato starch, 3-5% oatmeal , 3-5% milk powder, 1-2% whey syrup, 1-2% milk sugar, the amylase inhibitor is obtained in good yields, so that the choice of nitrogen source and buffer component is less important if the physiological range is maintained. , the yield of inhibitor is greatly reduced.

If, on the other hand, the starch content is raised substantially above 7 #, the oxygen uptake of the microorganisms is not optimal due to the high viscosity. As a result, the yield of inhibitor decreases.

In the fermentation broth, the formation of inhibitor begins regularly between 10 and 30 hours after the start of culture. Over the next 20 hours, it will substantially advance. Longer culture times do not have a detrimental effect on inhibitor yield, however, it no longer increases significantly. As a result, cultivation must be carried out for about 30 to 70 hours.

To isolate the inhibitor, the cell mass is removed from the culture medium by centrifugation, filtration or suction filtration, as most of the active ingredient is regularly present in the clear culture medium.

If, due to the special culture conditions, part of the inhibitor remains in the cell material, it is not difficult to remove the active ingredient from it by suitable methods, for example by mixing with a water-miscible organic solvent, whereby further dissolution of the cell mass is advantageous. The active ingredient, 59815 6 dissolved in the extractant, can be separated by centrifugation or filtration. la insoluble cellular material and treat it as a clear culture medium.

The inhibitor can be isolated from the culture medium in ways known per se in protein and peptide chemistry. Thus, doping with water-miscible organic solvents such as acetone, isopropanol or other lower alcohols or salts such as ammonium sulfate can be used.

It is further possible to absorb the inhibitor in a suitable carrier, for example activated carbon, and remove it by filtration or centrifugation from aqueous solution. This can take place over a wide pH range of 2 to 10, however, the pH is preferably h-6. Ion exchangers can also be used to separate the oi-amylase inhibitor of the invention. Since the desired substance has both acidic and basic properties, it is thus amphoteric and can react with both the cation and anion exchanger and can be removed from the culture medium by them. In this case, methods can be used, the principles of which are described in the chapter on ion exchangers in Biochemisches Taschenbuch, published by H.M. Rauen, Springer-Verlag '\ 96k) 2. Teil, pages 808-82U.

When treating culture batches containing a substance of the invention in a culture medium, it is often appropriate to concentrate it. In this case, known methods such as distillation, ultrafiltration, spray or freeze-drying or other known methods can be used. From the concentrate thus obtained, the inhibitor can then be separated as described above. From the concentrates corresponding to the concentrated culture filtrates, the inhibitor can also be obtained by removing essential impurities. Extraction with organic solvents is suitable for the removal of fatty substances. By dialysis or, if desired, ultrafiltration (C.J.O.R. and P. Morris, "Separation methods in Biochemistry", Ditman Publishing, London 1976, pages 9-950), the solution can be purified from small molecules. High molecular weight substances such as nucleic acids, polysaccharides or several proteins can be separated by partitioning by salt formation or by the addition of a water-miscible solvent such as acetone or a lower alcohol. The addition of precipitant is dosed in these cases in such a way that easily precipitating substances with molecular weights greater than 100,000 precipitate, while o <-amylase remains in solution. The enrichment steps shown can be combined and varied in an optional order. In this way, solutions are obtained in which the inhibitor is highly enriched.

τ 59815

The final purification can be carried out by various suitable methods, such as using gel or ion exchange chromatography, or similar methods whose separation principle is not based solely on ion exchange, such as, for example, separation with hydroxylapatite. The solvent can still be used! salt precipitations, preparative electrophoresis or other methods known per se. Particularly suitable are separations with ion exchangers containing diethylaminoethyl (DEAE) groups and fractional precipitations with ammonium sulphate or ethanol, whereby even a crystalline material is obtained.

The properties of the inhibitor of the invention are of interest in its use as a therapeutic agent in the treatment of diabetes and precursor diabetes and obesity and as a dietary support.

Starchy nutrients and nutrients cause an increase in blood sugar in animals and humans and thus also an increase in the secretion of pancreatic insulin. Hyperglycemia occurs when starch is broken down in the gastrointestinal tract by amylase and maltase into glucose.

In diabetics, this hyperglycemia is particularly severe and prolonged.

In obese people, increased insulin secretion contributes to lipogenesis and lowers lipolysis.

Alimentary hyperglycemia as well as hyperinsulinemia after ingestion of starch can be reduced by the amylase inhibitor of the invention. This effect depends on the dose. The amylase inhibitor of the invention can thus be used as a therapeutic agent in diabetes, in the pre-diabetes and in cases of obesity, and in support of diet.

In this case, it is especially appropriate to enjoy it at meal times. The dosage to be adjusted according to the weight of the patient and the individual need is about 10,000 to 300,000 AIY, however, in limited individual cases it may be either higher or lower.

The amylase inhibitor according to the invention is particularly suitable for oral administration. It can be used as a pure substance or also as a pharmaceutical preparation, in addition to the customary auxiliaries and carriers.

Also, combined use with other drugs, such as blood glucose lowering or lipid lowering agents, may be preferred. Since the high molecular weight peptides as such are not or not significantly absorbed from the gastrointestinal tract, no toxicologically questionable side effects are to be expected with the substance according to the invention. Due to the conventional amino acid composition of 8,59815, any proteolytic degradation products are also harmless. Thus, when the amylase inhibitor was administered orally in large amounts to experimental animals, no special symptoms were observed. Also, when the inhibitor of the invention was administered intravenously to mice (1 g / kg), no toxicological effect was observed during the 2-hour observation period.

To investigate the pharmacological effect of the amylase inhibitor, fasted male Wistar rats weighing between 200 and 250 g were administered the inhibitor of the invention simultaneously with 2 g of starch per kg of body weight orally, followed immediately by blood sampling for baseline blood glucose. Blood samples were taken further at 15 and 30 minutes and at 1, 2, 3, and 5 hours after the tail vein. Blood glucose assay was performed according to Hoffman's method on an autoanalyzer (J. Biol. Chem. 120, 51 (1937)).

NZO mice have impaired glucose tolerance. They are therefore particularly well suited to studies that affect blood sugar levels. The experimental setup was the same as in rats. Blood sampling was performed from the orbital vascular network. Blood glucose levels were monitored for more than three hours.

Similarly, the drug was studied in NMRI mice. Blood sampling was also performed from the orbital vascular network and the course of blood glucose levels was monitored for more than three hours.

In these experiments, animals treated with the inhibitor of the invention showed a smaller, delayed increase in blood glucose compared to untreated animals.

amylase

An amylase inhibitor unit (AIY) is defined as the amount of inhibitor that is capable of inhibiting two amylase units (AY) by 50- # under experimental conditions. According to an international agreement, an amylase unit is the amount of enzyme that breaks down 1 μ-equivalent glucosidic bonds in starch in one minute. The u-val value of diffused glucosidic bonds is determined photometrically with dinitrosalicylic acid as the reducing sugar. Results are calculated in μmoles of maltose, determined from the maltose calibration curve.

The experiments were performed as follows: γ-amylase obtained from porcine pancreas and the test solution 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 initiated by the addition of 1.0 ml of soluble starch according to Zulkowski (0.25% of the buffer used). After exactly 10 minutes, stop the reaction with 2.0 ml of dinitrosalicylic acid dye reagent (according to Boehringer Mannheim: Biochemica-information II) and heat for five minutes in a boiling water bath to develop the color. After cooling, the extinction is measured at 51 + 6 nm against the void value of the reaction components. The 50% inhibition is determined by comparing the uninhibited enzyme reaction by adding different amounts of inhibitor graphically using the probability method.

Example 1

Streptomyces tendae strain 1 + 158 is inoculated in oblique tubes into a medium having the following composition: 50 g of oat flakes ad. 1,000 ml H 2 O pHA = 7.2 • .... O .....

The inoculated tubes are incubated for seven days at 30 ° C and then kept at + 1 + ° C. The spores are expelled from the oblique tubes with 10 ml of sterilized distilled water or physiological NaCl solution. 1.0 ml of suspension is used to inoculate a 300 ml Erlenmeyer flask containing 35 ml of sterilized nutrient solution with a pH of 7.7 and a composition of: 1% glucose 1% soy flour 0.25% NaCl pH 7.7

The flask is shaken on a shaker at 220 rpm at + 30 ° C with an amplitude of k cm for 1 + 8 hours. 5 ml of this preculture are then transferred to several Erlenmeyer flasks containing 35 ml of sterilized nutrient solution at pH 8 each. 3. The composition of this main culture is as follows: 1+% starch 0, k% corn extraction solution 1.0% glucose 0.8% (NH 3 ^ HPO ^ 0.1+% soy flour 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 1+ cm for 2-3 days On the first, second and third days of culture, the concentration of β-amylase inhibitor is determined according to the experimental protocol.

Strain Streptomyces tendae 1 + 158 gives an average of 100 AIY / ml at the end of the pH under the experimental and culture conditions shown.

l0 5981 5

Example 2

The batch is as in example t, but a fermenter with a total volume of 300 liters is added, in which 200 liters of sterilized nutrient solution having the following composition have been placed: 1 +% starch 0, k% corn extract solution 1.0% glucose 0.8% (NH 4 KPO 1% Desmophen ', (polyether, manufactured by Bayer AG, Leverkusen) PHa 8.3-8.5

The sterilization time for the batch is 1 + 5 minutes at 121 ° C and 1 bar, during which the glucose is sterilized separately and, after cooling to the operating temperature of the fermentation broth, it is added sterile.

After sterilization, the pH must be 6.8 and adjusted to this value, if necessary, with sterilized acid (2 n H2O) or base (2N NaOH). The main stage is inoculated with 20 liters, which corresponds to 10% of the preculture shown in Example 1 and prepared in a small culture apparatus with a total volume of 30 liters.

The culture is performed for 50-70 hours at 30 ° C. Aeration is 6 m 2 / h with stirring at 250 rpm and an overpressure of 0.3 bar.

Sampling is used to monitor and control the course of fermentation in terms of inhibitor activity, substrate degradation, biomass formation and physico-technical behavior of the culture medium (surface tension, viscosity, density, osmotic pressure).

Maximum inhibitor activity is reached after 60 hours of culture and averages 105 AIY / ml. The contents of the fermentation vessel are then transferred to further processing.

Example 3

The charge is the same as in Example 1 and Example 2, but the main step is a bioreactor with a total volume of! + 000 liters, in which 2,500 liters of the following nutrient solution have been charged: 6% starch 1.0% glucose 0.1+% corn extraction solution 0.8% (NHU) 2HP0u 1.0 % soy peptone 0.1% Desmophen pHA 7.0-7.3 5981 5 11

The sterilization time for this batch is 60 minutes at 121 ° C and 1 bar, but the glucose is sterile filtered separately and added to the fermentation broth after cooling to operating temperature under sterile conditions.

If necessary, adjust the pH to an initial value of 7> 0-7 »3 with sterile acid (H 2 PO 4) or base (NaOH).

Inoculation is performed with 200 liters of the preculture shown and prepared in Example 2,

The culture time is 70 hours at 30 ° C with aeration of 60 Nm 3 / h, an overpressure of 0.5 bar and a speed of 10 ° rpm.

Sampling - as in Example 1 - monitors all important process, organism and activity values throughout the culture.

After 70 hours of culture, an average activity of '95 AIY / ml has been reached. The culture is then stopped and the culture solution is transferred to further processing.

Example U

10 liters of filtered culture medium with an activity of 1Q0 AIY / ml are dried in a spray dryer to give 100 g of a light brown powder. This is then thoroughly degreased with a mixture of 1 liter of methanol and 1 liter of chloroform and then filtered off with suction. The still solvent-insoluble product containing the active ingredient is dissolved in 3 liters of water, its pH is adjusted to 6.5 and 1.5 liters of methanol are added. This produces a pale, flaky precipitate which is separated by centrifugation and discarded because it contains only an insignificant amount of od-amylase inhibitor. The dark overhead portion obtained in methanol precipitation containing the desired inhibitor is freed from methanol in vacuo and then dialyzed against 2.5 liters of concentrated solution against desalinated water. The retentate contains 9 g of dry matter (specific activity 96 AIY / mg). The retentate is then brought to 50% saturation with ammonium sulphate at pH 5.5, requiring 79 g of salt for 250 ml. Upon addition of the salt, a precipitate is formed which contains about 90% of the active substance. After centrifugation, the upper part is discarded. The precipitate is redissolved in distilled water and at pH 8 ammonium sulphate is added again, but this time first to a $ 15 saturation and after removal of the precipitate to a further $ 35 saturation. The precipitate formed during the addition of salt between 15 and 35% contains the major part of the amylase inhibitor. This fractional precipitation is repeated.

12 5981 5

The product obtained after dialysis and drying is 360 mg and has a specific activity of 992 AIY / mg. 350 mg of this crude material is separated and then enriched using Sephadex v; G-50 fine in a 100 cm long and 1.2 liter glass column. An aqueous 5 millimolar phosphate-buffer solution, pH 8, to which 0.02% sodium azide has been added is used as starting and eluent. The raw material is dissolved in 15 ml of the same buffer and applied to the column. Elution takes place over two days, taking 15 ml fractions. The four most active fractions are combined, dialyzed against salt and freeze-dried. 60 mg of a white powder with an activity of 2520 AlC oC-amylase inhibitor / mg are obtained.

Example 5 2,200 liters of culture broth are cooled to 6 ° C and 2% silica is added and filtered using a chamber press. The filter cake (about 1 + 50 kg when wet) is discarded, the clear filtrate (1750 liters, activity 95 AIY / ml) is concentrated after adding 500 g of sodium azide in a drop-flow evaporator to a volume of 120 liters. The concentrate obtained is cooled to 1 [deg.] C. and 120 liters of precooled acetone are slowly added with stirring. To complete the precipitation, stir for a further 1 / k hour and, after adding 1 kg of diatomaceous earth, clarify in a plate press. The solid with the filter aid is discarded. The acetone-containing filtrate containing the active ingredient is stirred under cooling with 380 liters of acetone to give an acetone suspension of about 80- $. The precipitate formed (2 ..) contains the desired amylase inhibitor. It is recovered by allowing the precipitating solution to stand without stirring. In this case, the precipitate settles to the bottom and the liquid above can be removed. Small amounts of precipitate can be separated from the liquid above this second acetone precipitate by centrifugation. This solid is dissolved at pH 7 and added to the precipitate solution (see below). The main precipitate remaining in the precipitation vessel is dissolved at pH (r) in 7,120 liters of water and the resulting solution is clarified in a flow-through centrifuge (Cepal, manufactured by Karl Padberg, Zentrifugenbau GmbH, Lahr / Black Forest, 1700 rpm).

In this case, 90 g of inactive suspended particles are removed. The clear aqueous phase is then concentrated using a DDS ultrafiltration apparatus, manufactured by De Danske Sukkerfabrikker Aktieselskapet, Nakskov, Danmark (membrane no. 800), to 15 liters and dialyzed. To complete desalting, the retentate is diluted with distilled water and concentrated again. After two or three repetitions, the retentate containing the amylase inhibitor is substantially salt-free. The inhibitor is then precipitated from a 50 liter solution of retentate at pH 5> 5 by adding 19.5 kg of ammonium sulphate, the precipitate is removed by centrifugation and the upper liquid is discarded. The precipitate is redissolved in 1 + 0 liter of water and this time precipitated at pH 7.5 to 12.5 kg of ammonium sulphate. After separation of the clear phase in a tubular spinner, precipitation is fractionated as in Example 1+; From 1 + 0 liters of reconstituted substance first at pH 5.5 by adding 3.2 kg of ammonium sulphate an inactive precipitate which is separated and discarded. By reconcentrating the liquid phase, 7.1+ kg of ammonium sulfate are separated from the liquid for most of the active substance. This precipitate is collected, dialyzed against distilled water and freeze-dried. 11 U g of a brown powder with an activity of 1,100 Al / mg are obtained.

For further separation, a DEAE-Sephadex A-25 7 (dextran-based ion exchanger, manufactured by Pharmacia Fine Chemicals AB, Uppsala, Sweden) is filled from a glass column with a diameter of 5 cm and a height of 50 cm (volume approx. 1 L) with a pre-filled adjusted to pH 7.5 with 1/30 molar phosphorus buffer and equilibrated with $ 0.02 sodium azide. 10 g of the substance are then dissolved in 100 ml of the same buffer and applied to a column. First wash the column with 1 liter of the same buffer, then gradually add common salt to this eluent to ensure a continuous gradient. When fractional recovery is recovered, there is an amylase inhibitor in those fractions with a NaCl content of about 0.5 molar.

These fractions are collected and dialyzed against distilled water. Freeze-drying gives 5.6 g of a light brown powder with an activity of 2,200 Al / mg.

Further gel chromatographic purification is then performed as shown in Example U. 5.6 g of material give 1 +, 1 g of a colorless powder with an activity of 2,800 AIY / mg. Amino acid analysis of the product after 20 hours of hydrochloric acid hydrolysis using a Beckman multichrome analyzer gives the following composition: aspartic acid 7.72% methionine 0, U1% threonine 7.66% isoleucine 2.30% serine 5.35% leucine 5.1 δ% glutamic acid 10 .05% tyrosine 10,2U% proline 3.1 + 3% phenylalanine 3.35% glycine 1+, 93% histidine 3.01% alanine 5.81+% lysine 1.1 + 5% 1/2 cysteine 1+ .26% arginine 5.16% valine 7.72%

Claims (3)

5981 5 lU A process for preparing a peptide-structured β1-amylase inhibitor, characterized in that Streptomyces tendae strain 1 * 158 (ATCC No. 31210) is cultured in a culture medium containing at least one carbon source and one nitrogen source and inorganic salts, and that the culture is isolated by conventional means. inhibitor with a molecular weight of 5,000 to 10,000, an absorption maximum under UV light at 276 nm, an isoelectric point and the following amino acid composition: aspartic acid 5-6 isoleucine 1-2 threonine 5-6 leucine 3 - * + serine 3-5 tyrosine U- 5 glutamic acid 5-6 phenylalanine 0-2 proline 2-3 histidine 1-2 glycine 5-6 lysine 0-1 alanine 5-6 arginine 2-3 cysteine 3- ^ tryptophan 1-2 intermediate 5 -6 Process according to Claim 1, characterized in that the nutrient contains 3-5% of soluble starch; 0.2-0.6% corn extraction solution; 0.5-1.5% glucose; 0.5-1% NagHPO 4; 0.3-0.6% soy flour and 0.5-1.5% casein-peptone. Process according to Claim 1, characterized in that the nutrient contains 2 to 6% of peanut flour; 1-3% potato starch; 3-5% oatmeal; 3-5% whey flour; 1-2% whey syrup and 1-2% milk sugar.