DE4341283A1 - Process for the production of amino acids - Google Patents

Abstract

Prodn. of aminoacids from proteins or peptides contg. proline (Pro) and/or hydroxyproline (Hypro) residues that are difficult to degrade is by using cells, pref. in an aq. medium, from Xanthomonas (X.) or its prod. which hydrolyses the protein or peptide. Pref., X. is X. maltophilia (malt.) or X. JCM 3857 (Ferm-BP-4475), X. malt NA-62 (FERM-BP-4479) or JCM 3807 (FERM-BP-4474). The proteins to be degraded are pref. collagen, casein or prolamine.Also claimed is method for prodn. of trans-4-hydroxy-L-proline by applying cells or a cell prod. of X. capable of hydrolysing the protein or peptide and isolating trans-4-hydroxy-L-proline.The ability to hydrolyse proteins or peptides that are difficult to degrade has the pref. meaning that all of benzyloxycarbonyl gp. (Z)-Pro-Pro, Z-Pro-hypro and Z-glycine-Pro can be hydrolysed.

Description

This invention relates to a method of manufacture of a (of) amino acid (s) that have a high utility value show (s), from a poorly degradable protein or Pep tid containing proline and / or hydroxyproline residues.

Known proteins containing numerous proline or Hy containing droxyproline residues are animal collagen, Ca etc., and vegetable prolamin etc. These proteins are used as starting compounds for the preparation of a Ge mixed dissolved amino acids by hydrolysis and / or Ab separating (a) certain amino acid (s) from the solution used.

The hydrolysis of these protein compounds was out finally chemically with an acid, an Alka li or a corresponding compound leads, since proline and / or Hydroxyprolinreste containing Peptides with conventional proteases inadequately hydrolyzed and often remain as poorly degradable peptides that do not know hydrolyzed (R.Walter, W.H. Sinirnons and T. Yoshimoto, Mol. Cell. Biochem. 30 (1980) 111). It was kept for virtually impossible to hy them completely with enzymes drolysieren.

It is also reported that chemical hydrolysis under severe reaction conditions to a lesser quality  the produced amino acids and that also during the implementation of carcinogenic substances arise (M.R. Williams and M.F. Dutton, Journal of Food Protection A5 (1988) 887). Therefore, it was desirable to ent ent a new process wrap, by which such protein compounds under milder Conditions can be hydrolyzed.

In recent years, several carboxypeptidases have been found which cause degradation of the C-terminal proline of a peptide chain. For example, (Kubota et al., J. Biochem 74 (1973) 757, Kubota et al., Protein * Nucleic Acid * Enzyme 28 (1983) 1407), carboxypeptidase C _N and carboxypeptidase C _U , available from Japan grown citrus fruits, such as Natsumikan (summer tangerine) and Unshumikan (Unshu tangerine), amino acids, including proline, which is particularly resistant to hydrolysis by proteases, release similar to that found by Zuber carboxypeptidase C (Nature 201 (1964) 613). However, the rates at which these carboxy peptidases release proline are very low. They are therefore unsuitable for industrial application. A carboxypeptidase derived from a microorganism belonging to the genus Pycnoporus can also release the C-terminal mineral proline of a peptide, but with low efficiency (Japanese Published Unexamined Patent Application No. 201987/1983).

Other known carboxypeptidases are, for example example, the carboxypeptidase P, from the culture broth of a obtained for the genus Penicillium microorganism (Yokoyama, Protein * Nucleic Acid * Enzyme 28 (1983) 1414), and the carboxypeptidase Y derived from a baker's yeast (Hayashi, Protein * Nucleic Acid * Enzyme 28 (1983) 1421). Both can be quite effective the C-terminal proline egg release the peptide, but have the disadvantage that their effect on a prolylproline binding of a peptide, in which several proline residues occur in succession, low is.

Therefore, all previously known carboxypeptidases  for the production on an industrial scale of amino acids, such as hydroxyproline and proline, from persistent Proteins or peptides, the proline and / or hydroxy contain proline residues, insufficient.

On the other hand, hydroxyproline and proline are the reason substances for the production of various medicines especially useful. For example, trans-4-hydroxy-L is Proline is a hydroxyproline found in hard-to-digest proteins or peptides containing proline and / or hydroxy contain proline residues, and as a raw material for the manufacture Use of carbapenem antibiotics useful (Japanese Publ unpublished patent application No. 236980/1993).

An application of a carboxypeptidase containing the "Hydrolysis of prolylproline binding of peptides in which multiple proline residues occur consecutively, catalyze " can, and a method of making them is at japa Patent Office (on 17 September 1993 Published, Japanese published, unaudited Patent Application No. 236960/1993).

An object of the present invention is the Provision of a method for producing an amino acid by hydrolysis of a protein or peptide, the difficult to break down and proline and / or hydroxy contains proline residues, for example collagen, casein or Prolamin under mild conditions without use of a Acid or an alkali and of sharp reaction conditions.

A particular object of the present invention is the provision of a very efficient method Preparation of hydroxyproline, proline or an amino acid mixed by hydrolysis of such a hardly degradable Protein or peptide according to an enzyme method, d. H., by working a microorganism or a treated one Products of cells.

A particular object of the present invention is the provision of a very efficient and efficient process selective production of trans-4-hydroxy-L-proline, referred to as  a raw material for the production of carbapenem antibiotics is particularly useful by hydrolysis of such a heavy degradable protein or peptide according to an enzyme method.

These objects of the present invention are solved by a process for producing an amino acid, looking at a protein or peptide that's difficult to get rid of and contains proline and / or hydroxyproline residues, Cells of a microorganism of the genus Xanthomonas, the has the ability to hydrolyze the protein or peptide or a treated product of the cells with the ge called abilities and the generated amino acid gains.

The "poorly degradable protein or peptide, the pro contains lin- and / or Hydroxyprolinreste, "is in the before a protein or peptide (the one May include amino acid residue whose amino group with a benzyloxycarbonyl group is protected), the proline and / or hydroxyproline, with conventional proteases is not hydrolyzed and as a natural protein be especially an animal collage, casein or similar Protein or a plant prolamine or a corre sp enclosing connection. The peptide is for example a polypeptide or oligopeptide formed by partial hydro lysis of the proteins described above is obtained, or a commercially available, synthetic peptide Z-Pro. Hyp, Z-Pro-Pro, Z-Gly-Pro or similar compounds (Z: Benzyloxycarbonyl group, hyp: hydroxyproline).

"An ability to hydrolyze a difficult to break down To possess protein or peptide "should also mean that besides the ability to hydrolyze common peptide bonds at least one, preferably two or all of the amino acids of Z-Pro-Pro, Z-Pro-Hyp and Z-Gly-Pro can be hydrolyzed NEN.

Everyone belonging to the genus Xanthomonas and those before standing described possess possessing microorganism can be used in the present invention. From Xanthomonas maltophilia -derived microorganisms with the  The ability described above are preferred, in particular Xanthomonas maltophilia NA-62 (FERM-BP-4479) and Xanthomonas maltophilia JCM No. 3807 (FERM-BP-4474). The Xanthomonas species JCM No. 3857 (FERM-BP-4475) is also prefers.

In the above description, NA-62 is a pri vate marking, JCM an acronym for Japan Collection of Microorganisms and FERM-BP an international backer at the National Institute of Bioscience and Human Tech nology, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, Japan, according to the provisions of the Budapest Treaty on the laying of microorganisms. The microbial properties of the genus Xanthomonas and Xanthomonas maltophilia also in Krieg, N.R. et al., "Bergey's Manual of Syst matic Bacteriology ", Vol. 1, Williams & Wilkins, Balti more / London, pp. 140-219 (1984). Besides that is Xanthomonas maltophilia NA-62 the same strain as the one in the aforementioned Japanese published, Unexamined Patent Application No. 236960/1993 Xanthomonas NA-62, wherein the patent application also a Description of its microbial properties. It It was found that the strain NA-62 to Xanthomonas maltophilia, where it is characterized in that branched fat in the fatty acid composition of the cell acids, for example, large amounts of isopentadecanoic acid and Isoheptadecanoic acid and branched hydroxy fatty acids, at For example, 3-hydroxyisoundecanoic acid and 3-hydroxyisotride canic acid, are included. In this context one holds such a fatty acid composition for a feature of Xanthomonas maltophilia (see, for example, Swings, J. et al., Int. J. Syst. Bac. 33 (1983), 409, C. Wayne Moss et al., J. Bac. 114 (1973), 1018). It was further confirmed that the fatty acid composition of the cell to a microorganism which was known to be Xanthomonas belongs to maltophilia.

An amino acid can be produced according to the invention  by (hereinafter referred to as the first method be records) a microorganism of the genus Xanthomonas, the hydrolyze a poorly degradable protein or peptide can be bred in a medium that is a heavy degradation contains bar protein or peptide, the proline and / or Having hydroxyproline (hereinafter "becomes a heavy degradable protein or peptide, the proline and / or Hydroxyproline "contains, sometimes just as" a poorly degradable protein or peptide ").

An amino acid can also be produced according to the invention be by microbial cells, by breeding a Microorganism of the genus Xanthomonas, which is a heavy off buildable protein or peptide can hydrolyze in one usual medium and then collecting the cells be obtained with the hardly degradable protein or Be brought into contact peptide in an aqueous medium (hereinafter referred to as the second method).

First, the first method will be described. On such microorganism is grown in a medium the carbon sources, nitrogen sources used as main Part of a hard degradable protein or peptide umfas sen, inorganic substances and micronutrients, under aerobic conditions at a set temperature, a set pH, etc.

As carbon sources are usually verschie used carbohydrates, for example glucose, sac Charose, Mannose, Starch, Starch Hydrolysates and Restme but it can also vary depending on assimilability various organic acids, such as Brenztrau Benzenic acid, fumaric acid, lactic acid and acetic acid, alcohols, for example, ethanol, glycerin and polyalcohol, and Hydrocarbons, for example n-paraffin, used become. As nitrogen sources, one or more can be difficult degradable proteins or peptides are used, others However, nitrogen sources can also be used. When other nitrogen sources may be used: ammonia, various inorganic and organic ammonium salts,  for example, ammonium chloride, ammonium sulfate, ammonium car carbonate and ammonium acetate, urea and other nitrogen containing compounds and various nitrogen ent holding natural products, for example peptone, Meat extract, yeast extract, corn steep liquor, caseinhydro lysates, fishmeal or its digestion product, defatted Soybean cake or its digestion product and chrysalis hydrolysates. There is no certain common upper limit the amount of hardly degradable protein or peptide for everyone Nitrogen sources, but the amount is preferably 10 to 100 wt .-%, in particular 50 to 100 wt .-%, based on the Dry matter.

As inorganic substances can be primary Kaliumphos phat, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium carbonate, etc. be used. Although it is necessary to use the medium Nete, for the growth of the microorganism he used required amounts of micronutrients (vitamins, Nuclein acids, etc.), these substances are given if already with the natural, as nitrogen source ver added products.

Although it is common for the entire amount of poorly degradable protein or peptide at the beginning of the Breeding is completely present in the medium, the Total also gradually or to a specific Time during breeding (eg during the logarith mixing phase) are added. Although it is not specific Upper limit for the concentration of the hardly degradable Protein or peptides in the medium are commonly 0.1 to 20 g / l used.

Breeding by an aerobic culture method is optimal, for example by a Schüttelkulturverfahren or an aerating stirring culture method, but it can also a liquid state culture method combined suitable become. Although the culture conditions vary according to the nature of the Differentiate microorganism is usually a breed temperature of 15 to 45 ° C, especially from 25 to 35 ° C,  and a pH of the medium of 5 to 8 suitable. It will under such conditions usually 10 hours to 10 Days, preferably 24 hours to 6 days, bred. In the The method described above, the main part of Amino acids are enriched extracellularly.

The second method will now be described. First, a micro used in this invention organism in the manner corresponding to the first method bred. Although under these conditions usually no difficultly degradable protein or peptide as nitrogen it can be used.

After a sufficient increase of the microorga nism, the breeding is stopped and the cells pass through Centrifugation, filtration or appropriate procedures isolated, if necessary, washed and the obtained Cells or a treated product, which by their treatment is obtained with a poorly degradable protein or Peptide in an aqueous medium brought into contact.

Treated products are destroyed by destruction of Cells (products which are broken up, dried, etc. of the cells are obtained). It can be enzymes, for example, crude enzymes, purified enzymes and immobili sierte enzymes, which from the by breaking up the cells he were obtained and the hardly degradable Hydrolyze protein or peptide. The breaking up of the Cells can be performed according to a conventional method the, for example, using a cell mill under Shaking, a high pressure, zeros cells interfering device (Hughes press, French press or similar Ches), one with (Ultra) -Schall working, cells destroyed machine or equivalent equipment. It can also Under certain circumstances, a drying process or a Cell wall degrading method can be used. After Removal of cellular debris from cell destruction holding products or their aqueous suspension can Crude enzyme or a purified enzyme by a Required combination of the following steps he  salting out using ammonium sulfate or corresponding compounds, solvent precipitation under Use of ethanol, acetone, methanol, isopropanol or corresponding products, precipitation on isoelectric Point, fractional adsorption using a Adsorbent, for example, acid clay, bentonite, Kaolin, activated carbon or aluminum C-γ gel, different Chromatography, electrophoresis, etc, as well as dialysis se, ultrafiltration, concentration, removal of nuclei acid, etc. There may be different chromatography be suitable for use, for example a Gel chromatography, in which the fractionation by means of mol particle size using the molecular sieve effect (Gelfil tration), ion exchange chromatography using cellulose or dextran to which an ion-exchanging group is bonded, or a Ion exchange resin, adsorption chromatography under Ver use of hydroxyapatite, affinity chromatography under Use of a specific binding reaction between a carrier (cellulose or dextran gel or an ent talking product) containing a substrate attached thereto, a specific inhibitor, a coenzyme or a derivative and the target enzyme and hydrophobic chroma tography using a hydrophobic binding reaction tion between the hydrophobic part of the column filling and the Enzyme. As electrophoresis, zone electrophoresis can be used in a carrier gel or electrofocusing by electro phoresis can be used in a pH gradient.

In the methods described above, the Movement of the target enzyme using the specific Activity can be tracked as a marker.

The destruction of the cells and the enzyme purification can for example, by a "Jikken Nogei Kagaku" (Experi mental Agricultural Chemistry), last volume, 3rd edition, edited by Agricultural Chemistry Classroom, Depart ment of Agriculture, Tokyo University, published by Asakura-shoten (March 1992), pages 42-95  Procedures are performed.

A specific example in which a purified Enzyme was obtained by working up this invention, is below with regard to the use of Xantho monas maltophilia NA-62. The cleaning of a Enzyme from another used in this invention Microorganism can also be done in about the same way be performed.

First, a culture broth of Xanthomonas malto philia NA-62 centrifuged and the precipitated cells who recovered, suspended in 50 mM Tris-HCl buffer (pH 7.5) and broken up by ultrasound. After centrifugation This fractionated product is the cell extract gewon subsequent work steps are carried out at 4 ° C leads.

First, this cell extract is mixed with ammonium sulfate fractionated and the fractions with 35 to 50% saturation won. Subsequently, the following chromatography carried out, whereby proteins by absorption at 280 nm be detected.

The product obtained by the ammonium sulfate fractionation Precipitate is dissolved in 20 mM Tris-HCl buffer (pH 7.5), dialyzed and equilled to one with the same buffer bred Q-Sepharose column (2.6 × 10 cm, Pharmacia Co.) carry. Anion exchange chromatography using eluted from a concentration gradient of 0 to 0.3 M NaCl is carried out to obtain an active fraction becomes.

Subsequently, ammonium sulfate is added to a seed 30% dissolved in this active fraction, the Solution on a phenyl supersol column (0.5 × 5 cm, Pharmacia Co.), containing 50 mM to 30% with ammonium sulfate saturated bis-Tris-HCl buffer (pH 6.5) is equilibrated, and a hydrophobic chromatography performed. The elu tion is decreasing with a linear gradient Ammonium sulfate concentration and the active Won fractions.  

Subsequently, this active fraction is dialyzed against 10 mM potassium phosphate buffer containing 0.4 mM CaCl ₂ (pH 6.8) and applied to a hydroxylapatite column (0.8 × 10 cm, Tonen Corporation) equilibrated with the same buffer. After washing the column with the buffer used for equilibration, elution is performed with a linear gradient between 10 mM potassium phosphate buffer containing 0.014 mM CaCl ₂ (pH 6.8) and 0.4 M potassium phosphate buffer containing 0.014 mM CaCl ₂ contains, (pH 6.8) performed. The active fractions are recovered and subjected to the gel chromatography described below.

The active fractions are placed on a Superdex 200 Column (1.6 × 60 cm, Pharmacia Co.), which was filled with 50 mM bis-Tris-HCl buffer containing 200 mM NaCl (pH 6.5) is equilibrated, eluted at a flow rate of 0.5 ml / min and the eluate fractionated in 1 ml portions. The active one Substance becomes as a single in fractions 69-71 Gained protein peak. The molecular weight is considered 160,000 certainly. The obtained enzyme further showed both in the isoelectric focusing as well as SDS electrophoresis a single gang and is thus as a unified To look at protein.

As a method for the immobilization of enzymes can a carrier binding method may be mentioned in which a Enzyme through a covalent bond, ionic bonding, adsorption or is bound to a carrier in a corresponding manner, a cross-linking process in which enzyme molecules through kova lente binding are mutually linked, a real estate Inclusion method by inclusion of an enzyme in the Network structure of a macromolecule, etc. included is. Each of these methods can be used in the production of immobilized enzymes in the refinement of this inventions be used. The use of an immobilized Enzyme is beneficial because it is after the enzymatic Implementation of substrate and product separated and again and can be used repeatedly.

Although the use of a larger amount mikrobiel  ler cells or the treated product of the cells for the Implementation would be advantageous, is too large amount wirt economically disadvantageous. As well as the microorganisms and the treated products of the cells to varying degrees difficultly degradable proteins or peptides can hydrolyse, the quantity to be used is not fixed. In the However, use of microbial cells is common an amount of 1 to 100 mg of persistent protein or peptide per mg cells (dried cells) and at the Use of the treated product of the cells a lot from 0.01 to 5 g of degradable protein or peptide per Unit enzyme activity (1E) suitable for use. In In this context, E denotes an enzyme activity which is 1 μMol of a substrate Z-Pro-Hyp in 1 minute at 37 ° C hydroly Siert. Further, the concentration suitable for use of the hardly degradable protein or peptide However, it is suitably 0.01 to 20 g / dl.

As the aqueous medium, for example, water, a Buffer or the like may be mentioned. Further, when a through Disruption of cells as an enzyme It already contains an aqueous solution Medium, and it is not always necessary, in addition to that to use an aqueous medium. As a buffer Phos phosphate buffer, succinate buffer, citrate buffer, Tris-HCl buffer, Borate buffer, acetate buffer, glycylglycine buffer, etc. ver be used.

The contact of microbial cells or behan defective product of the cells with the hardly degradable protein or peptide in an aqueous medium, depending on the nature of Microorganisms performed differently, however, will usually at a pH of 5 to 8 and at a Temperature of 20 to 80 ° C, preferably 30 to 70 ° C, carried out. The contact time is usually 0.5 to 48 Hours, preferably 0.5 to 24 hours.

In each embodiment of the first and second Process becomes the reaction after completion of the reaction  terminated by a suitable method, for example by Removal of cells or inactivation of the enzyme by Heating the reaction mixture or lowering the pH Value (addition of an acid, such as chlorine water Stoffstoff), and a membrane treatment using an ultrafiltration membrane or filtration membrane for reverse osmosis or equivalent, adsorption treatment using an adsorbent, at For example, activated carbon, and other known Ver Driving are used in a suitable combination to obtain a solution of an amino acid mixture. Further can according to the invention a desired specific amino acid (For example, hydroxyproline, proline or peptides) from such Amino acid mixture using, for example, a Ion exchange chromatography process can be isolated. The According to the invention obtained hydroxyproline is trans-4-hydroxy L-proline, which is used as a raw material for drugs, such as For example, carbapenem antibiotics, is useful as before and trans-4-hydroxy-L-proline can be described in FIG of the present invention particularly efficient and selective are prepared (see Test Examples 4 and 5).

The examples illustrate the invention.

Reference Example 1

Each of the three shown in Table 1, to Gat Xanthomonas strains were dissolved in 100 ml of an in Table 2 shown liquid medium in a conical, with a chicane-equipped 500 ml bottle inoculated and Bred for 72 hours at 30 ° C with shaking. After that was the culture broth is centrifuged (15,000 rpm, 20 minutes), the precipitated cells are collected and made using a Ultrasonic Homogenizer (US-150, Nippon Seiki Seisaku-sho Co.). The cell debris was purified by centri fugation (15,000 rpm, 15 minutes) from the surviving suspen sion, the supernatant obtained to 80% saturation adjusted with ammonium sulfate and the precipitate as  Crude product by centrifugation (15,000 rpm, 20 minutes) collected. The precipitate was dissolved in 1 ml of 50 mM bis-tris- HCl buffer (pH 6.5, containing 200 mM sodium chloride) sus to give a crude enzyme solution.

Test Example 1 Test for hydrolysis activity of the synthetic peptide

One by dissolving a commercial synthetic one Peptides Z-Pro-Hyp, Z-Pro-Pro or Z-Gly-Pro in 50 mM bis- Tris-HCl buffer (pH 6.5) at 1 mM concentration solution was used as a substrate solution. 25 μl of in the reference example 1 obtained crude enzyme solution and 25 ul 50 mM bis-Tris-HCl buffer (pH 6.5, 200 mM sodium chloride ent holding) was added to 50 μl of this substrate solution and the mixture after sufficient stirring for 60 minutes at 37 ° C. implemented. 4 μl of 1N hydrochloric acid were added, to finish the implementation.

The concentration of a free amino acid (proline or hydroxyproline) in the reaction solution was added Using an Automatic Amino Acid Analyzer (Hita chi L-8500) and a hydrolysis rate by Verglei of the value obtained with the concentration (theoreti shear value) at the time of complete hydrolysis per calculated (free) amino acid calculated.

As a comparative example, 25 .mu.l of a liquid by suspension of a commercially available Protease preparation "actinase" (from Streptomyces griseus originating, Kaken Pharmaceutical Co.) instead the crude enzyme solution described above (25,000,000 tyrosine units thereof were added per mmol of the synthetic peptide added). This enzyme preparation has an extremely strong one commonly used in proteolysis proteolytic force.

The results are shown in Table 3.

As can be seen from Table 3, all showed  Crude enzyme solutions used by the three in this test th trunks of the genus Xanthomonas wa hydrolytic activity in Z-Pro-Hyp and Z-Pro Pro, and it was found that these strains contain the enzyme can produce. Further, the hydrolysis of Z-Gly-Hyp in the crude enzyme solution, that of a genus Xanthomonas belonging strain had been produced observed.

On the other hand, the actinase did not hydrolyze any of the synthetic peptides.

Test Example 2 Test of hydrolytic activity in gelatin

Gelatin (Wako Pure Chemical Industries, Ltd.) for the preparation of a 0.5% (w / v) gelatin solution in 50 mM bis-Tris-HCl buffer (pH 6.5). 50 μl of the Reference Example 1 prepared crude enzyme solution became 50 μl of this gelatin solution, the mixture is sufficient stirred and reacted at 37 ° C for 24 hours. Subsequently wur the 4 .mu.l of 1 N hydrochloric acid of the reaction solution continues to complete the implementation. By measuring the free Amino acid concentration in this reaction solution was the Activity in hydrolysis of gelatin, d. h. who the Amino acids from gelatin releasing activity, be Right. The test of free amino acid concentration was according to a phenylthiocarbamoyl (PTC) amino acid analysis (Waters Co.). The results were as Ratio of the amount of a released amino acid (the Hydrolysis with hydrochloric acid is indicated as 100% taken) to the amount of one in the starting material of the gelatin containing amino acid expressed.

As a comparison test, 50 μl of a liquid, obtained by suspending the actinase in 50 mM bis-Tris-HCl. Buffer (pH 6.5, containing 200 mM sodium chloride) were added (100,000 tyrosine units were added per g Gelatin added).  

The test results are shown in Table 4.

As can be seen from Table 4, all showed Raw enzyme solutions made by the microorganisms were belonging to the genus Xanthomonas and in used in this test, a strong activity in the Release of hydroxyproline and proline from gelatin. On the other hand, although a release of hydroxyproline and Proline was observed by the actinase, those were through they obtained comparatively much lower.

Test Example 3

The Xanthomonas species JCM No. 3857 (FERM-BP-4475) was using a medium containing gelatin as the main stick contains, as shown in Table 5, 72nd Bred for hours at 30 ° C with shaking. After that were the cells by centrifugation (15,000 rpm, 20 minutes) removed the culture supernatant with "Sep-Pak-Cartridges" (C18, Waters Co.) to treat hydrophobic contaminants remove, and the concentrations of free amino acids using a PTC amino acid analysis system (Waters Co.).

The test results are shown in Table 6.

As shown in Table 6, the content was free hydroxyproline in the culture broth 2.49 g / l, and 73.3% of the hydroxyproline contained in the protein were in the medium recovered as free hydroxyproline. So it was shown that Amino acids also by a semifermentative method Use of the microorganism made of gelatin who you can.  

Test Example 4

Xanthomonas maltophilia NA-62 (FERM-BP-4479) using two liquid media A and B, the Gelatin as the main source of nitrogen as shown in Table 7 72 hours at 30 ° C with shaking ge breeds. Thereafter, the cells were centrifuged (15,000 rpm, 20 minutes), hydrophobic impurity with "Sep-Pak-Cartridges" (C18, Waters Co.) from the Culture supernatant removed and the concentrations of the free Amino acids using a PTC amino acid analysis system (Waters Co.).

The test results are shown in Table 8, in the amount of each amino acid extracted from the culture broth acid is stated to be 100% pure Hydrolysis with hydrochloric acid refers. Further, too the proportion of hydroxyproline in the total amount of amino acids in the culture broth.

As shown in Table 8, hydroxyproline by the semitermentative method using the Microorganism not only with a high yield, but also obtained with a high total content of amino acids become. Therefore, efficient production of hydroxy Proline can be expected by this invention.

Test Example 5

Xanthomonas maltophilia JCM No. 3807 (FERM-BP-4474) was made using a liquid medium, the gelatin as the main source of nitrogen, as shown in Table 9, containing 72 hours shaking at 30 ° C with shaking. The cells were then centrifuged (15,000 rpm, 20 minutes), hydrophobic impurities with "sepa- Pak Cartridges "(C18, Waters Co.) from the culture supernatant removed, and the concentrations of free amino acids using a PTC amino acid analysis system (Waters  Co.).

The test results are shown in Table 10, in the amount of each amino acid extracted from the culture broth acid is stated to be 100% pure Hydrolysis with hydrochloric acid refers. Further, too the proportion of hydroxyproline in the total amount of amino acids in the culture broth.

As shown in Table 10, hydroxyproline by the semitermentative method using the Microorganism not only with a high yield, but also obtained with a high total content of amino acids become. Therefore, efficient production of hydroxy Proline can be expected by this invention.

According to the invention described above poorly degradable proteins, for example collagen, casein and prolamine, and polypeptides or oligo derived therefrom peptides hydrolyzed efficiently under mild conditions and High value amino acids, including proline and hydro xyproline, produced so far only by chemical treatments, such as an acidic one Hydrolysis was possible. Furthermore, hydroxyproline (trans-4-) Hydroxy-L-proline) with this invention from those difficult degradable proteins or peptides particularly efficient and be selectively produced.

Especially amino acids, for example proline and Hydroxyproline prepared by the process according to the invention can be made as raw materials for the Production of medicines useful.

Microorganisms with which tests were carried out Name of the trunk No. Xanthomonas maltophilia NA-62 FERM BP-4479 Xanthomonas maltophilia JCM No. 3807 (FERM-BP-4474) Xanthomonasart JCM No. 3857 (FERM-BP-4475)

Composition of the liquid medium meat extract 5 (g / l) peptone 10 yeast extract 3 sodium chloride 3 glucose 10 Primary potassium phosphate 3 magnesium sulfate 5 PH value 7.2

Table 3

Hydrolytic activity of synthesized peptides

Table 4

Activity in the release of amino acids from gelatin

Composition of the liquid medium meat extract 5 (g / l) gelatin 50 yeast extract 5 sodium chloride 3 glucose 10 Primary potassium phosphate 3 magnesium sulfate 5 PH value 7.2

Table 6

Activity in the hydrolysis of gelatin in semipermentation

Table 7

Composition of the liquid medium

Table 8

Hydrolysis of gelatin by semifermentation

The yield of each amino acid was calculated as assuming that the value of hydrolysis by chlorine water substance acid is 100%.

Traces: Despite the evidence of small amounts, a quantitative determination was impossible.
-: not established

Composition of the liquid medium meat extract 5 g (l) gelatin 10 yeast extract 5 sodium chloride 3 glucose 10 Primary potassium phosphate 3 magnesium sulfate 5 PH value 7.2

Hydrolysis of gelatin by semifermentation Free amino acid Yield (%) hydroxyproline 86 aspartic acid 8th glutamic acid 2 serine - glycine - histidine 20 arginine 3 threonine - alanine 1 proline - tyrosine 3 valine - methionine - isoleucine 3 leucine - phenylalanine 15 lysine 1 Content (%) of hydroxyproline compared to the total amount of amino acids 81

The yield of each amino acid was calculated as assuming that the value of hydrolysis by chlorine water substance acid is 100%.

Claims (10)

1. A process for the preparation of an amino acid, in you look for a protein or peptide that is difficult to break down and contains proline and / or hydroxyproline residues, cells a microorganism of the genus Xanthomonas containing the Ability to hydrolyze the protein or peptide, or a treated product of the cells with said Ability to interact and the amino acid produced wins. 2. The method of claim 1, wherein the cells of the Microorganism of the genus Xanthomonas by breeding the Microorganism in a medium containing the protein or peptide contains, on the protein or peptide interact. 3. The method of claim 1, wherein the cells of the Microorganism of the genus Xanthomonas or the treated Product of the cells by contacting the cells or the treated product of the cells with the protein or Peptide in an aqueous medium to the protein or peptide act. 4. The method according to any one of claims 1 to 3, in the microorganism is a microorganism of the species Xanthomonas maltophilia with the corresponding ability or Xanthomonas JCM 3857 (Ferm-BP-4475). 5. The method according to any one of claims 1 to 3, at the microorganism Xanthomonas maltophilia NA-62 (FERM. BP-4479) or JCM 3807 (FERM-BP-4474). 6. The method according to any one of claims 1 to 5, at the treated product of the cells by destruction The product obtained from the cells or an enzyme is that the product obtained by the destruction of the cells was obtained and a heavy hydrolysis ability degradable protein or peptide possesses. 7. The method according to any one of claims 1 to 6, at  the hardly degradable protein collagen, casein or Prolamin is. 8. The method according to any one of claims 1 to 6, at the poorly degradable peptide is a polypeptide or oligo peptide is formed by partial hydrolysis of collagen, Casein or prolamine is obtained. 9. The method according to any one of claims 1 to 8, wherein the amino acid hydroxyproline, proline or a mixture of Is amino acids. 10. Process for the preparation of trans-4-hydroxy-L- proline, which refers to a protein or peptide that difficult to degrade and proline and / or hydroxyproline contains cells of a microorganism of the genus Xanthomonas, which possesses the ability, the protein or To hydrolyze peptide, or a treated product of Cells with the said ability to act and the generated trans-4-hydroxy-L-proline wins.