DD200432A1 - PROCESS FOR PREPARING A PROTEOLYTIC ENZYME PREPARATION - Google Patents

Abstract

The invention relates to a process for the preparation of a proteolytic enzyme preparation. which is of potential use for the chemotherapy of human diseases, for the breakdown of protein-containing compounds and the digestion of complex substrates in the microbiological industry. In particular, the invention relates to a process for the preparation of a novel proteolytic enzyme preparation using a microorganism which has hitherto not been used for the production of enzymes of the type of proteinases. The microorganism, strain ZIMET 43647, is named Nocardiopsis dassonvillei for its taxonomic properties. The aim of the invention is to obtain a proteolytic enzyme preparation with fibrinolytic, biosludge-clarifying properties as well as the ability to exploit polypeptide-containing substrates of the fermentation industry to expand the range of such biocatalysts. The object is achieved in that the enzyme is formed by aerobic submerged fermentation of a microorganism in media with suitable C-N sources and mineral salts, concentrated by ultrafiltration technique in the culture filtrate and optionally iyophilized by conventional means.

Description

233125 3

Title of the invention

Process for the preparation of a proteolytic enzyme preparation

Field of application of the invention

Until the invention relates to a process for the preparation of a proteolytic Snzym preparation, aas due to its fibrinolytic properties for human medicine, due. its proteolytic effect is useful for the clarification of biosludge and the digestion of complex substrates in the sewage and microbiological industries _a In particular, the invention relates to the recovery of a proteinase using a microorganism hitherto unknown as a proteinase former *

Characteristic of the known technical solutions

Numerous representatives of the genera Bacillus , AsOergillus, and Streptomyces can synthesize proteinases under suitable fermentation conditions * which, for their proteolytic properties, are based on serine CB _- C * 3 "** 21), cysteine (S * C, 3. 4" 22) * Aspartic *. (S ₀ C ₉ 3o 4 · 23) and differentiate metalloproteinases (B * C » % 4, 24)«. Frequently, the known proteinase formers form mixtures of proteinases, z * B ₀ in this Palle alkaline and neutral proteinases are formed simultaneously «On the other hand limit the type of temperature and the pH optima of known proteolytic preparations their applications«

For certain applications, it is desirable that the proteolytic activity of an enzyme preparation is very low below 30 ^° C., but that it is fully unfolded at temperatures between 35 and 70 ^° C. "The use of mesophilic compounds

233125 3

treters of the genus ITocardiopsis as a proteinase- bildner is "not yet known, Ss is not known" that proteolytic enzyme preparations were obtained from Nocardiopsis with such a high temperature optimum, as otherwise only thermophilic microorganisms, z. " B ₀ Thermoactinomyces "," are known, fermentations with the latter are characterized by an unfavorable ratio of energy expenditure to Snzymertrag O

Object of the invention

The invention is intended for the preparation of a proteolytic Snzym preparation with a high temperature optimum with the help of a microorganism, which is preferable because of its mesophilic properties from the energetic point of view thermophilic microorganisms, Thus, the listed parts of previously known methods for preparing proteolytic enzyme Preparations can be avoided and the range of such preparations can be extended by a representative with a wide field of application.

Explanation of the essence of the invention

The invention has for its object _{} provide} a technologically simple method that allows to produce from easily accessible and inexpensive use materials in high yields, a proteolytic s enzyme preparation with high temperature optimum * without resorting to thermophilic microorganisms

According to the invention the object is achieved in that under aerobic and sterile culture conditions in a liquid nutrient medium with carbon and nitrogen sources and mineral salts of the microorganism described below or its variants are bred ·

The microorganism used in this process has been registered under the registration number ZIMST 4-3647 in the ZIMET Depository for Microorganisms, Central Institute for Microbiology and Experimental Therapy of the Academy of

233125 3

Sciences of the DDS, 69 Jena, Beutenbergstraße 11, deposited ₃ The tribe. ZIMBT 4364-9 assigns to the species ufocardiopsis the son- Tillei (Brocq-Eousseu) Meyer (InW J * Syst "Bacteriol _e 26 (4): 487 bi3 493 (1976)). * Sr was isolated from moldy straw." The strain is aerobic, Grampositi and catalase positivity »Sr has the cell wall chemotype III (meso-diaminopimelic acid» arabinose and galactose are absent) «lycolic acids and madurese, absent, * The substrate enzyme develops well at 23 ⁰ G on the organic medium 79 (Prauser and Palta * 1968» Z * Allgs Mikrobiol * 8 (i): 39 Ms 46) and on the ISM media 2 _f 3 ₃ ^ and (Gottlieb and "Shirlinge 1966 _O Inta J" Syst "Bacteriol * j1§ (3) j 313 bis 3 ^) * 23s is nearly colorless to pale yellowish-brownish (Medium 79) "Superficial substrate mycelial hyphae usually grow in a very succulent manner colonial margin after aiSen" they are more or less right-angled branching "Soluble pigments are missing in the agar * Luftaycel becomes best on your medium 79 ", 3s is floury, white with black oh yellowish-greyish tint "On the other" media, aerial mycelium is only developed on the colony edges or the luxirnsycelph tp- phens are only recognizable with the microscope * The aerial mycelhyphene fragment into elongated elliptical spores with a smooth surface * ZIMBT 43647 does not form melanoid pigments <, It reduces Hitrate _a peptonized milk, * liquefies gelatin, hydrolyzes starch and breaks down xanthine and Aesculin _a The strain grows on D-glucose * D-mannose, maltose ^. D-mannitol _t D-fructose, sucrose and glycerine · Sr does not grow on L-arabinose, D-XyIose, L-Bhamnose., Lactose, Haffinose, Adonitj, dulcitol and meso-inositol The contrary to the description of Meyer (loccit ") lack of growth on arabinose, Zylose * Lactose and Ehamnose is not taken as occasion for the taxonomic treatment of the genus Nocardio? sis to describe a new species of this genus au «

Cultivation of the ZIMET 43,647 strain and its mutants and variants is carried out under aerobic conditions. Mycelium fragments lyophilised in glucose gelatin are inoculated into suitable agar media and subsequently into liquid, previously sterilized nutrient media and the resulting mycelium

233125 3

is cultured in a sieve known manner at a temperature between 25 and 37 ⁰ C (preferably 28 ⁰ O) over a period of 2 to 10 days (preferably 6 days) at an acidity at the beginning of the fermentation process between pH 6.5 and pH 7> 2 "The nutrient medium consists of carbon and nitrogen sources, as well as inorganic salts." Carbon sources can be starch, glucose, glycerine, mannitol, dextrin, sucrose, soybean oil and soybean meal. " other than the above-mentioned nitrogen-containing substrates, dry yeast, meat peptone and casein in question _Λ

Good results can be achieved with the addition of mineral salts. The latter favor the course of the fermentation in. Depending on the used curative medium "In complex media * containing various flours, additions of calcium carbonate _i; Sodium nitrate or "Potassium phosphate advantageous" · Fermentation of the former can be carried out in steep breast bottles and round-bottomed flasks of various contents, in the glass fermenter and in "2A" tanks

The determination of the proteolytic activity of the crude caustic broths of strain ZIMET 43 64? the type lüocardiopsis dassonvillei and obtained therefrom enzyme Präparates- ZIMBT E-43647 was done with a method described by M _a Kunitz 1946 UY spectroscopic method (J "· Gen Physiol * ,. 29» 14-9) ^ ² ^ s Isolation of the proteolytic enzyme preparation is carried out by separating the culture solution into jfycelium and potassium fluorate in the usual manner, concentrating the culture filtrate by means of the ultrafiltration technique and subsequently lyophilizing _e

The lyophilized new Snzym preparation is a brownish, amorphous substance that dissolves well in water | ^; The proteolytic enzyme preparation was tested for its degradative activity on various substrates. The preparation can be characterized by the following substrate profile:

233125 3

Substrates Proteolytic action

from. B-ZIMBT 43 64?

Gelatin © +

Casein +

Hinder se rumalbumin +

Gamma globulin -.

Collagen *

Fibrin * ·

The optimum temperature of the proteolytic enzyme preparation S- ZDflBT -43647 is between 35 and yO ⁰ G (preferably 60 ⁰ O) s if at. pH '7 * 6 was used as substrate casein or * diazo-casein (Abbe .. 1) »The pH = -Qptimum is smschen pH 6.0 and pH 10.0 (preferably pH 9 * 0), when as' temperature 60 ⁰ G were selected (Fig. 2) *

The thermostability of the proteolytic Snzym preparation B-ZIMBiD 43 64? was examined at 35 and 60 ⁰ C for a period of 6 hours at pH 9j.O "It is found that in a 2« hour preincubation of the preparation at 35 ° C no activity sabf all _s. after preincubation for 6 hours, on the other hand, the proteolytic activity drops to 90 %. "

In contrast, a decrease in the already proteolytisc-hen activity to 35% and after 6 hours of preincubation observed at about 8% after 2 hrs preincubation of the preparation at 60 ⁰ C (Fig. 3) ''

Both fresh lotion solutions of the ZIMEI 43 647 and the enzyme preparation B-ZIMST 43 647 obtained from them have special properties for the solubilization or "partial hydrolysis of animal, vegetable, but also human proteins." This results in various fields of application in agriculture and industry and Human Medicine »

The proteolytic activity of the preparation E-ZIMST 43 647 on gelatin was determined by the method described in Taufel et al * (J, Chromate, 93 , 48 Ms 490, 1974). The effect of Snzym preparation on casein was determined by the method

233125 3

M Q M

according to Kunitz (J * Gen * Physiol, 22, 149, 1946). In addition, the proteolytic activity of the enzyme preparation has also been studied on the diazo gasein by a conventional method (J * Biolo Chem., 171 t 501, 1947). In the case of the substrates bovine rumalbumin and gamma-globulin, too, the presence or absence of proteolytic activity of E-ZIMBT 43 has been measured by the tyrosine release determined by DY spectroscopy according to the Kunitz method. The proteolytic action of the enzyme preparation Collagen was tested using the Hide-and-the-Azur technique. In Hide Powder Azure, each free hydroxyl group of the Koilagens is associated with a dye residue, so that a large number of dye molecules are formed in the enzymatic cleavage of a single molecule (Remazolbrilliantblau) go into solution "Thus Hide-Powder-Azur acts as an" amplifier substrate "(Rinderknecht et al., ₃ Olin, Ohim *, Acta, 21, 197 to 2O3 _t, 1968).

The fibrinolytic activity was determined by means of a Pibrinagar plate technique according to Astrup and Müllertz (In: Coagulation Laboratory in Clinic and Practice (Edit, Ε · Perlick and Α _Λ Bergmann, Verlag Georg Thieme-Leipzig _? 1971 j S, 374)) »

embodiments

1 * Inoculum for the inoculation of a pre-breeding culture lyophilised mycelial fragments of Uocardiopsis dassonvillei , strain ZIMET 43 647, are used in glucose gelatin. Ü As pre-breeding medium, a liquid nutrient medium of the following composition is suitable:

Glucose 1.5% soybean meal 1.5%

CaCO- 0.1 % FaGl

Tap water, sterilization: 35 min, at 115 G * acidity between pH 6.5 and. pH 7.0

The inoculum amount for 80 ml pre-breeding medium in a 500 ml steep-breast bottle consists of 1 ml resuspended mycelial fragment lyophilisate "After 2-day pre-breeding

233125 3 " ⁷ "

Culture "at 28 ⁰ G on a shaking platform at an oscillation frequency of 180 / min ·. 80 mL of a production culture in 500 ml of reagent bottles with 8 ml of the culture Vorzucht" inoculated *

As a production medium, a liquid nutrient medium of the following composition is suitable;

Sucrose Q, .25 %

O ₃ 2%

0.1 %

₄ pO 0 _f P5 %

KCl "~ 0 * 05 %

Peso ₂ ^ 0.001%

Aqua the more sterilization; 35 min, at 115 ⁰ O acidity between pH 7 * 2 and pH 7 * 8

The duration of the fermentation is S up to 10 days at 28 ⁰ G on a shaking table with a vibration frequency of 180 / min · _s before the maximum proteolytic activity is reached,

2e ^: 'The procedure is as in Example 1 except that the production medium has the following composition

Soya flour 2 * 0 %

Glucose 2.0 %

CaGOo · 0.3 %

HaCI. 0.5 %

In tap water «sterilization 35 Miru

at 115 ⁰ G * Acidity at pH S _t 5 «

The method differs from that of Example 1 in that the inoculum used to inoculate the breeding culture is grown on a solid agar medium of the following composition for a period of 10 days

Glucose 1 * 0 %

Peptone 1.0 %

Yeast extract 0.2 %

Gasamino acids 0.1%

NaGl 0.6 %

233125 3

Agar-agar 1 _t 5%

Tap water. Sterilization: 35 min

115 ^° C. acidity pH 7.0

The method differs from that of Example 1 in that a 1 _O pre-culture as described in Example 1, is applied, but subsequently a 2 »Vorkuitur by inoculating 400 ml of the pre-growing medium in 21-glass bottles with 10 ml suspension bred from the 1st preculture for a period of 24 hours.

400 ml of the 2 × preculture obtained in this way are used to inoculate HO 1 of the production medium described in Example 1, which is contained in a 321-Giasfermenter * During the fermentation ^ at 28 ⁰ O with a stirring speed of 400 Ü / With an air supply of 15 l / M in ρ, foaming can be controlled by the addition of small quantities of sunflower oil or silicone antifoaming agent. * The activity of the proteolytic enzyme obtained after approximately 120 hours of fermentation corresponds to the activity; 0.9 to 1 _t 4 / umol liberated tyrosine / ml in the supernatant of the culture solution of the wild strain

5 * After separating the üycels from the obtained according to Example 4 Fennentationslösung by means of separator 20 l KuI-turfiltrat be concentrated by ultrafiltration to 3 * 5 1 ♦ For ultrafiltration a Sigenbau apparatus is used which, with cellulose acetate membranes type UI is equipped. The concentration factor is 5 * 7 "The proteoiytische type formed per 1 ml native culture solution causes the release of 0.9 / umol tyrosine in a Oaseinlösung after 20 Mi utes at +60 ⁰ O · The prote - 01ytische activity per 1 ml of concentrate is on the other hand, 4.0 / umol liberated tyrosine under the same conditions »The concentration factor with respect to the proteinase activity is 4.4 * The enzyme yield after concentration is 77» 7 % of the starting solution «The filtrate

233125 3

current density reaches a value of 16 1 per hour and m and the selectivity rf> »(1-

After ultrafiltration, the proteolytic enzyme preparation is iyophil dried and stored at -20 ⁰ G "

Claims (1)

Process for the preparation of a proteinaceous enzyme preparation by microbiological means, characterized in that the microorganism Noougiopsis dassonvillei «. Strain ZIMBI 43 547 j under aerobic conditions in liquid media containing a carbon and nitrogen source and mineral salts, cultivated at a temperature of between 25 and 37 ⁰ O for a period of 5 to 10 days, and this concentrated proteolytic enzyme Ε-Ζ1ΜΞ2 4-3 647 concentrated in the culture filtrate using the ultrafiltration technique and optionally subsequently lyophilized * Hierzo ^ 2_Seit8n drawings