DD141323A1 - METHOD FOR PRODUCING A PROLYLPEPTIDE COLLECTING ENZYME - Google Patents

Abstract

The invention relates to the production of a prolyl peptide-cleaving enzyme by culturing a microorganism the bacterial species Escherichia coli B and isolation of the formed Cell contents. The new enzyme is used to break down protein and Peptides down to the individual amino acids. amino acid mixtures of certain amino acid composition are e.g. for the manufacture of Diet and special nutrition used. The aim of the invention is therein, a more favorable prolyl peptide-cleaving enzyme source tap. It is therefore the task of a microorganism and to show its breeding and culture conditions, the one effective enzyme yield with the desired degradation properties of Enzyme ensured. According to the invention the strain Escherichia coli B 181, deposited in the central collection of the GDR in the ZIMET / Jena, used. The nutrient medium consists of yeast extract, Meat extract and peptone and there is a special mechanical Digestion of the cell mass to release the cell contents.

Description

Field of application of the invention

The invention relates to a process for the preparation of a prolyl peptide-cleaving enzyme by cultivating a microorganism of the bacterial species Escherichia coli B and isolating the cell constituents formed. The novel enzyme is particularly suitable for use in an enzyme reactor which is supported by means of a carrier. Enzymes Proteins and high-molecular-weight peptides broken down to the individual amino acids, suitable "It is possible to obtain amino acid mixtures whose composition corresponds to the amino acid spectrum of the natural proteins. These amino acid mixtures are said to be used for the preparation of diet and special foods as well as infusion solutions with certain amino acid composition for parenteral nutrition, especially after severe operations, in diseases of the esophagus, the gastrointestinal tract or biliary tract.

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It is known that a mixture of endopeptides and exopeptidases, when in immobilized form and arranged in an appropriate combination in an enzyme reactor, contain various proteins, e.g. Insulin ~ B-chain S-sulfonate, S-aminoethyl-Ijysozyme, bovine serum albumin, to hydrolyze to the amino acids.

So becomes ζ. Example, using the carrier-fixed endopeptidases pronase, trypsin and a protease from Thermoactinomyces spec, the protein molecule is broken down into short-chain peptides. Due to the specific, likewise carrier-fixed exopeptidases leucine aminopeptidase, carboxypeptidase A and prolidase, they are split up to the individual amino acids.

Virtually all the amino acids of the protein are recovered in the hydrolyzates prepared in this way.

The prolidase has a special function in protein degradation: it is the only peptidase that cleaves the imido linkage at the N-terminal end of the proline, which can not be broken down by nonspecific peptidases. Prolidase hydrolyzed according to the literature, only dipeptides, and those with the amino acid proline at the C-terminal end.

It is also known that prolidase from animal tissues, eg. B. from pig kidney, liver, intestine and intestinal mucous membranes can be isolated.

Furthermore, the preparation of an enzyme similar to that of prolidase by culturing a microorganism of the bacterial species Escherichia coli B, which the authors refer to as "aminopeptidase P", is already described in the literature. Aminopeptidase P cleaves the same peptide binding (x-Pro) as animal prolidase, but with higher molecular weight peptides being much better decomposed than low molecular weight peptides. The peptide glycyl-proline is hydrolyzed at a cleavage rate of only 25 % compared to poly-L-proline as a substrate. Other animal, microbial and plant prolyl peptide-specific enzymes are described in the literature, but only cleave peptides with proline at the N-terminal end ("proline iminopeptidase", "prolineiminodipeptidase").

The prolidase is only very low in animal tissues.

gene quantities so that large output volumes for

an extraction is necessary. The "known microbial aminopeptidase P" has relative to prolin-containing dipeptides a relatively successful specific activity (16 m units per mg protein), so that the enzyme preparations thus prepared are very expensive.

Aim of the invention

The object of the invention is to develop a more favorable enzyme source in contrast to the relatively expensive production of prolidase from animal tissue. "

Explanation of the essence of the invention

The invention is based on the object of selecting a prolyl peptide-cleaving microorganism and of demonstrating conditions of its cultivation and culture which can also be achieved on an industrial scale and lead to an effective enzyme yield "The enzyme should correspond to the animal prolidase in all characteristics essential for protein degradation"

The object is achieved in that according to the invention for the production of a prolyl peptide-cleaving enzyme of the microorganism Escherichia coli B 181 - (deposited in the central strain collection de-r DDR in ZIMET / Jena) - is used, the nutrient medium of yeast extract, meat extract and peptone and the cell constituents are released by mechanical treatment of the cell mass formed, brought into solution and separated from the cell debris. "

After a special procedure, the cell mass formed is separated by centrifugation of the nutrient solution and disintegrated by means of glass ball Vibrationshomogenisatcr or ultrasound and extracted the prolyl peptide-cleaving enzyme.

The solution and extraction of Zeilinhaltsstoffe is advantageously carried out by means of a buffer solution, in particular with TrJB-HCl buffer.

When practicing the method according to the invention, it was found that by cultivating the special strain in the complex nutrient medium an increase of the cell mass by 100% per volume unit is reproducibly guaranteed. «

The nature of the cultivation and the method of cell disruption (eg ₄ shaking in a vibratory homogenizer) increase the specific activity of the crude enzyme extract up to 2-fold compared with the known aminopeptidase P recovery (300 m units per mg protein). ,

The enzyme isolated from Escherichia coli B 181 best cleaves from the tested substrates the dipeptide glycyl-proline, other substrates tested. u. a. Alanyl-prolyl glycine, glycyl-prolyl-glycine, glycyl-prolyl-alanine, poly-L-proline, with increasing chain length of the peptides, continuously decreases the cleavage rate. The cleavage-specificity corresponds to that of the animal protease, although not only dipeptides but also relatively high-molecular-weight peptides can be decomposed.

The strain Escherichia coli B 181 is deposited in the central collection of the GDR in the Central Institute for Microbiology and Experimental Therapy in Jena. In the same way, mutants of this strain can be used. The stock is maintained in slant tube cultures on the Lab-Lemco medium containing 0.5% peptone and 0.5% meat extract. , ·

The cultivation of the enzyme-forming agent is carried out as follows?

Incubated at 30 ⁰ C Schrägager culture (Lab-Lemco) is first in a shaking medium consisting of peptone, meat extract, z. B. Walfleischextrakt, beef extract and yeast extract, z. B. from feed yeast or bakers yeast, inoculated.

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This culture is shaken for about 15 hours and then transferred to a nutrient medium of the same composition (working medium). In the logarithmic growth phase of cultivation, the prolyl-peptide-specific enzyme forms in the cells, which can be detected after the cells have been destroyed «

Exercise session

By the following examples, the invention will be explained;

example 1

A slant tube (medium: Lab-Lemco) with a 72 h culture of Escherichia coli B 181 is washed off with 5 ml of sterile physiological saline, transferred to 100 ml shaking medium (preculture) (500 ml round-bottomed flask) and 15 h at 30 ⁰ C on a 20 ml of this culture solution in 500 ml of the Fährmediums of the same composition as the shaking medium (0.7% peptone, 0.7% yeast extract, 0.7% meat extract) are then inoculated. Culturing is carried out at 37 ^° C. with shaking (300 rpm) in an incubation shaker for 4-5 hours, and the cell mass formed is separated from the culture solution by centrifugation. By shaking the cell mass with glass balls in a vibratory homogenizer, the cells are disrupted and the cell contents are subsequently dissolved in Tris.HCl buffer (pH 8.6). After separation of the cells

Glass beads and cell debris, the aqueous solution contains the prolyl peptide-specific enzyme with a specific activity of 300 m units per mg protein.

Example 2

Cultivation and preculture are carried out as in Example 1 "From the preculture is inoculated into 30 1-fermentors containing 20 1 Nährflüs sigke.it (medium s, above); the

Inoculum is 2%. The cultivation is carried out at 37 ⁰ C, the number of revolutions of the stirrer is 350 U / min, the air is fed in a ratio of 1 part to 2 parts of nutrient solution per minute. After 4 to 5 h, the contents of the suspension vessel are freed from the cell mass by means of a separator. The digestion of the cells is carried out in the same manner as described in Example 1. The specific activity is also 300 m units per mg protein.

Claims (1)

invention claims 1, Process for the preparation of a prolyl peptide-cleaving enzyme by culturing a strain of the bacterial species Escherichia coli B in a liquid nutrient medium, characterized in that the microorganism Escherichia coli B 181 - deposited in the central strain collection of the GDR in ZIIvIBT / Je na. - Is used . , The nutrient medium consists of yeast extract, meat extract and peptone and the cell contents are released by mechanical treatment of the cell mass formed, brought into solution and separated from the cell debris 2 _e method according to item 1, characterized in that the cell mass formed by centrifugation from the nutrient solution separated and disintegrated by means of glass-sphere vibration homo-gen or ultrasound and extracts the prolyl peptide-cleaving enzyme is, 3- process according to item 1 and 2, characterized in that the extraction of the enzyme takes place by means of Tris-HCl buffer, 4, "method according to item 1 to 3 _f, characterized in that a mutant of the strain Escherichia coli B 181 is used ·