DE3038130A1 - Alpha amylase inactivators for Streptomyces tendae - useful for lowering blood sugar levels - Google Patents

Abstract

The alpha-amylase inactivators HOE 467-A and HOE 467-B, and their mixts. are new. The amino acid compsn. of HOE 467-A is Asp 6; Thr 8; Ser 5; Glu 7; Pro 3; Gly 7; Ala 7; Val 8; Cys 4; Ile 2; Leu 4; Tyr 6; His 2; Lys 1; Arg 3; Tp 1, and its isoelectric point is 4.38-4.68. The inactivators are prepd. by cultivating Streptomyces tendae ATCC 31210 or HAG 1266 (the second claimed as a new organism) and recovery by adsorption on resins or by reverse-phase chromatography. The inactivators are used to regulate the level of sugars in the blood, esp. for treating diabetes, pre-diabetic states or adiposity. They are resistant to enzymatic degradation; produced no toxic signs in mice when given at 1 g per kg (intraveneously) and are pref. administered at doses of 10000-300000 amylose-inhibiting units.

Description

d -Amylal% naktivatoren and process for their production In the DE-OS 27 01 890 describes an NC amylase inhibitor which is produced by fermentation obtained from Streptomyces tendae 4158 (ATCC 31210) and its variants and mutants will. In terms of its chemical structure, it is a peptide and possesses the amylase ability to inactivate irreversibly. Because of these properties, it can be used for regulation blood sugar levels can be used.

It has now been found that by using an improved method for the extraction and purification of the i -amylase inactivator, hereinafter also referred to as HOE 467, the inactivator produced in a simpler way in greater purity can be, and that the high-purity substance made up of 2 components, hereinafter referred to as HOE 467-A and HOE 467-B.

The invention therefore relates to an α-amylase inactivator consisting from the components HOE 467-A and HOE 467-B, the two components HOE 467-A and HOE 467-B, as well as a method for its manufacture and separation into the two Components.

For the - amylase inactivator according to the invention as well as for the two Individual components meet the substance description given in DE-OS 27 01 890 except for the following major differences: It possesses an increased enzymatic effectiveness and consists of the 2 components HOE 467-A and HOE 467-B, which are characterized by the following data: HOE 467-A has the following amino acid composition labeled: Asp 6 Glu 7 Ala 7 Tyr 6 Lys 1 Thr 8 Pro 3 Val 7 Ile 2 Arg 3 Ser 5 Gly 7 Cys 4 Leu 4 His 2 Try 1 HOE 467-B has the following amino acid composition: Asp 5 Glu 7 Ala 7 Tyr 6 Lys 1 Thr 6 Pro 3 Val 6 Ile 2 Arg 3 Ser 5 Gly 7 Cys 4 Leu 4 His 2 Try 1 The isometric points of the two components are different slightly, they depend on the ionic strength and the methods of determination (Biochemical paperback, edited by H.M. Rauen, Srpinger Verlag, 1964). The isoelectric focusing (R.C. Allen, H.R. Maurer: Eletrophoresis and Isoelectric Focusing in Polyacrylamide Gel, W. de Gruyter, Berlin, 1974) Values are: HOE 467-A: 4.35 + 0.15 + HOE 467-B: 4.53 - 0.15 for the two Components HOE 467-A and HOE 467-B is the specific inhibitory effect against amylase from pig pancreas, each 1.7 107 107 AIE / g.

The inhibitory effect was determined in the amylase test described below determined: amylase test An amylase inhibitor unit (AIE) is defined as the Amount of inhibitor which, under the test conditions, contains two amyase units (AU) to 50% able to inhibit. According to international convention, an amylase unit is the Amount of enzyme which in one minute produces 1 A equivalent of glucosidic bonds in starch splits. The ßVal of cleaved glucosidic bonds become reducing as Val Sugar determined photometrically with dinitrosalicylic acid.

The information is calculated as ßmole of maltose, which is based on a maltose calibration standard be determined.

The tests are carried out as follows: Ct amylase from porcine pancreas and the solution to be tested are mixed together in 1.0 ml of 20 mM phosphate buffer, pH 6.9 + 10 mM NaCl preincubated for 10-20 min at 37 ° C.

The enzymatic reaction is made by adding 1.0 ml of soluble starch according to Zulkowski (0.25% in the specified buffer). After exactly 10 minutes. the reaction is carried out with 2.0 ml dinitrosalicylic acid color reagent (according to Boehringer Mannheim: Biochemica-Information II) stopped and for 5 min. In the boiling temperature for color development Heated water bath. After cooling, the absorbance at 546 nm is against the reagent blank measured. The 50% inhibition is compared to the uninhibited enzyme reaction Use of different amounts of inhibitor graphically using the probability plot certainly.

In the preparation of the inventive beni-amylase inactivator as well as the two components are used according to the invention from the strain Streptomyces tendae 4158 (ATCC 31210) or the derived strain Streptomyces tendae HAG 1266. The inactivator is obtained from the culture solution. The application of the Streptomyces strain tendae HAG 1266 is preferred.

The invention also relates to the Streptomyces tendae HAG strain 1266.

The new strain differs from the strain Streptomyces tendae ATCC 31210 due to important properties.

The different morphological characteristics are striking of both strains and also new ones for fermentative production and isolation the amylase inactivator according to the invention has important properties, such as the faster Growth and the approximately 40% increased & amylase inactivator production capacity Melanin formation, which in Streptomyces tendae ATTC 31210 is represented by a brown-black Color of the culture solution and the culture filtrate manifested and the work-up before poses certain cleaning problems, was with the new strain HAG 1266 reduced to about 1/10 with the help of genetic interventions and also qualitatively changed by a color shift. The melanin pigments are now showing yellow-reddish color spectrum. The elimination of this type of melanin during processing is unproblematic.

The two A-amylase inactivator-producing strains are compared in the following table: Table properties of the strains ATCC 31 210 and HAG 1266 ATCC 31210 HAG 1266 The color of the substrate mycelium is yellow-brown, reddish Color of the shipped gray-brown white-yellow Luftmycels Morphology of the retinaculum flexibilis (F) Spore chains apertum (RA) Spore morphology spherical, slightly spherical, sharp warty to smooth-edged warts N size QI 1 tim ~ size # 1.3 µm Nelanin formation to positive negative Peptone medium Nitrate reduction positive positive Substrate- glucose ++ recovery fructose # ++ spectrum sucrose + Maltose + + Lactose # - Galactose + Rhamnose # - Sorbose - Xylose + Arabinose + ++ Inositol ++ Mannitol + + Raffinose # - Cellulose - The strain Streptomyces tendae HAG 1266 has been deposited with the German Collection of Microorganisms (DSM) under registration no. DSM 1912.

The fermentation is expediently carried out in a manner analogous to that in DE-OS 27 01 890 described, carried out.

The finished fermentation solutions of Streptomyces tendae ATCC 31210 and HAG 1266 contain'Enzyme that the active ingredient concentration during the work-up can reduce considerably. In addition, substances such as

Melanin may be present, its complete separation with the known Procedure is difficult to achieve.

It has now been found that the required separations of the Inactivator by using so-called adsorption resins (DE-PS 12 74 128, OS-PS 35 31 963) can be carried out. Commercially available resins are suitable such as polystyrene resins. For use, the resin is brought into contact with the culture filtrate brought. The pH of the culture liquids is 2 to 8, preferably 4 to 6, discontinued. The solid resin selectively adsorbs the inactivator HOE 467, whereas the unwanted enzymes as well as most of the impurities from filtration can be removed as they remain unbound in solution. The recovery The HOE 467 can be removed from the resin either by washing with suitable aqueous buffer solutions such as phosphate buffers or with aqueous solutions of organic solvents such as lower aliphatic alcohols, acetone, acetonitrile or others. The further purification of the now enriched inactivator HOE 467 can after known procedures.

The described separation with adsorption resins is based on the principle the distribution of different polar connec-.

fertilize. More can be used for the required rapid separation Use methods of distribution. Furthermore, distributions are in the sense of the reverse phase Chromab tography possible (G Schwendt, Chromatographic Separation Methods, Georg Thieme Verlag, Stuttgart, 1979), using either commercially available carriers or self-made e.g.

silanized carriers can be used. Finally liquid -be also referred to the liquid separation process e.g. with aqueous polyethylene glycol, aqueous phosphate buffer systems as in principle, for example in DE-OS 26 39 129 are described.

It has been described in DE-OS 27 01 890 that ion exchangers, such as DEAE cellulose for cleaning the c -ATnylase inactivator HOE 467. As usual in ion exchange chromatography, pEI values are selected, which ensure the required degree of ionization. It was now surprising found that when one cleans - contrary to the rule - near the isoelectric Point, i.e. with a low degree of ionization, HOE 467 in two components is separated. The pH range from 4.4 to 6 is suitable for separation, preferably from 4.8 to 5.3. One component of the active ingredient is removed from the ion exchanger and can only be removed from this by changing the pH value or by increasing it the salt concentration are dissolved.

This substance is called HOE 467-A. The other component remains more or less unbound by the ion exchanger under the conditions mentioned and can thus be easily washed by the wearer. This component becomes HOE 467-B called.

The & amylase inactivator according to the invention and its two components HOE 467-A and HOE 467-B influence the blood sugar level and can therefore be used as Medicines used to regulate blood sugar levels.

Both the individual components and the Mixture are used.

Example 1 The strain Streptomyces tendae HAG 1266 is placed on slant tubes inoculated with a nutrient medium of the following composition: oat flakes 50 g ad. 1000 ml H20 pH 7.2 The inoculated tube is incubated for 10 days at 280C and then at Stored at + 4 ° C. The spores, which are easily detached from the yellowish aerial mycelium, become in 10 ml of sterilized agua dest. suspended with 0.2 ml Tween 80. 108 - 109 spores, i.e. 1 ml of the suspension is used to inoculate a 300 ml Erlenmeyer flask, the with 35 ml of sterilized nutrient solution and a pH of 7.2 and the following composition loaded is: 1% glucose 1% soy flour 0.25% NaCl The sterilization time is 45 min. At 1210C and 1 bar.

The flask is shaken at 250 rpm for 40 hours at + 300C shaken at an amplitude of 3.5 cm.

5 ml each of this preculture are transferred into several Erlenmeyer flasks, which are charged with 50 ml of sterilized nutrient solution and have a pH of 7.4 to have. The composition of this main culture is as follows: Soluble starch 4 96 Soy flour 0.4% Cornsteep liquid 0.4% Skimmed milk powder 0.7% Glucose 1.0% (NH4) 2 1.2% The sterilization time is 45 minutes at 121 ° C and 1 bar.

The main cultures were grown at 250C at 250 rpm. on a shaker shaken with an amplitude of 3.5 cm for 96 - 120 hours. On the third, fourth and fifth day of culture, the content of o-amylase inactivator according to the test procedure certainly. HAC The strain Streptomyces tendae1266 provides under the described experimental and culture conditions at a final pH of 6.4 1900 AIE / ml.

Example 2 Approach as in Example 1, however, the main fermentation is carried out in a fermenter of 15 l total volume with filling 10 l. It will the following nutrient solution composition is used: starch 4% soy flour 0.4% cornsteep liquid 0.4% skimmed milk powder 0.7% glucose 1.0% (NH4) 2HPO4 1.2% The pH value should be 6.8 after sterilization and is also sterilized as required Acid (2n H3P04) or lye <2 n NaOH) set to this value. Is inoculated the main stage with 1 1, corresponding to 10%, one as described in Example 1 Preculture.

It is fermented for 50 to 70 hours at 300C. The ventilation is 300 l / h with a stirring of 250 rpm and an overpressure of 0.3 bar.

By taking the sample, the fermentation process with regard to the Inhibitor activity, carbohydrate degradation, biomass development and physical-technical Behavior of the culture solution (surface tension, viscosity, density, osmotic Pressure) monitored and tracked.

The maximum inhibitory activity is on average from the 60th culture hour 1800 AIE / ml reached. The contents of the fermenter are then sent for processing.

Example 3 6.5 1 culture filtrate, obtained by suction filtering the after Example 2 obtained Streptomyces culture, with glacial acetic acid with stirring to pH 4.9 and applied to a prepared glass column. The pillar included 230 g of adsorption resin (e.g. Diaion (R) HP 20) loosened in water. The column dimensions measured 22 x 5 cm corresponding to a volume of 430 ml. The liquid flow is regulated so that 6.5 liters have passed through after 2 hours. Then there was the adsorption process finished and the resin could first be washed with pure water and then eluted with water to which increasing amounts of isopropanol have been added. The flowing liquid was caught in fractions of 1 l each and up enzyme-inhibiting effect tested. The active fractions were collected and in vacuo concentrated to 100 ml distilled. The concentrate contained 110,000 AIE / ml.

The resulting, now salt-proof solution was applied directly to a 1/30 phosphate buffer, pH 7.5, applied to prepared DEAE ion exchange column and cleaned according to DAS 27.01 890, Example 5. 4 g of substance resulted at 2500 AIE / mg.

Example 4 A glass column 2 cm in diameter was used for further purification and 22 cm high, corresponding to 70 ml content, filled with DEAE cellulose, which was previously equilibrated with 1/10 M sodium acetate buffer, pH 4.9 and 0.02% sodium azide is. Now the Su}) obtained according to Example 3 was punched in 10 ml of the same buffer dissolved and applied to the column. First, you washed the column content with 100 ml acetate buffer, then common salt was gradually added to this eluent in such a way that that a continuous gradient was guaranteed. With fractional collection of the column flow, HOE 467-B was in the still saline-free catch fractions, while the HOE 467-A component was detectable in the fractions in which the *) Polystyrene ~ NaCl concentration was 0.25 to 0.3 molar. Thief- and the fractions containing A components were collected separately, against dialyzed distilled water and freeze-dried. The colorless substances each had an effectiveness of 1.7 10 AIE / mg. The amino acid analysis of the products after 20 hours of hydrochloric acid hydrolysis with the aid of a Beckman multichro analyzer the following percentage compositions: HOE 467-A HOE 467 B aspartic acid 8.51 7.90 Threonine 10.07 8.17 serine 5.41 5.98 glutamic acid 11.30 12.39 proline 3.28 3.39 glycine 5.61 6.19 alanine 6.85 7.41 cysteine 4.68 3.83 valine. 8.69 8.54 methionine isolencine 2.93 3.28 Leucine 5.91 6.61 Tyrosine 11.28 12.69 Phenylalanine Histidine 3.49 3.72 Lysine 1.78 2.05 arginine 6.12 6.52-

Claims (4)

Claims: 1. α-Amylase inactivator, consisting of HOE 467-A with the amino acid composition: Asp 6 Glu 7 Ala 7 Tyr 6 Lys 1 Thr 8 Pro 3 Val 7 Ile 2 Arg 3 Ser 5 Gly 7 Cys 4 Leu 4 His 2 Try 1 - and the isoelectric Point 4.35 f 0.15 or consisting of HOE 467-B with the amino acid composition Asp 5 Glu 7 Ala 7 Tyr 6 Lys 1 Thr 6 Pro 3 Val 6 Ile 2 Arg 3 Ser 5 Gly 7 Cys 4 Leu 4 His 2 Try 1 and the isoelectric point 4.53-0.15 or consisting of the two Components HOE 467-A and HOE 467-B. 2. Process for the preparation of the s-ainylase inactivator according to claim 1, characterized in Streptomyces tendae ATCC 31210 or HAG because one 1,266 cult fourth and separates the inactivator from the culture with the aid of adsorption resins or reverse phase chromatography and then purifies. 3. The method according to claim 2, characterized in that the separated inactivator with the help of ion exchangers in the pH range between 4,4 and 6 cleans and here in the two components HOE 467-A. and HOE 467-B separates. 4. Streptomyces tendae HAG 1266