JP3347710B2 - Proline determination method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to trans-4-hydroxy- useful as a raw material for synthesizing pharmaceuticals or as a food additive.
The present invention relates to a method for industrially producing L-proline, a novel L-proline 4-position hydroxylase useful for the method, and a proline-specific highly sensitive detection method useful for clinical tests and the like.

[0002]

2. Description of the Related Art As a method for producing trans-4-hydroxy-L-proline using a microorganism, 1) using a microorganism belonging to the genus Escherichia, trans-4-hydroxy-L-proline is converted from 4-hydroxy-2-oxoglutarate. (Japanese Unexamined Patent Publication No. 3-26)
6995) 2) Direct fermentation production using molds (European Pat
ent Application EP 0 547 898 A2) 3) Using a microorganism belonging to the genus Streptomyces, L-
Production method from proline [Journal of Biological Chemistry (J. Biol. Chem.), 254]
Volume, pages 6684-6690 (1979), Biochemical and Biophysical Research Communication (Biochem. Biophys. Res. Comm.), 12
0, pages 45-51 (1984)].

In the method 1), 4-hydroxy-2
-Oxoglutaric acid is expensive and difficult to obtain, has low productivity, and has low productivity even in the method of 2). In the method of 3), the enzyme involved in the production of trans-4-hydroxy-L-proline is used. Since the activity is extremely weak, the amount of the reaction product is so small that it cannot be detected without using a radioactive compound, and industrialization is difficult using any method.

[0004] An enzyme that catalyzes the hydroxylation reaction to trans-4-hydroxy-L-proline has not been isolated and is derived from a microorganism belonging to the genus Dactylosporandium or Amycolatopsis and is trans-transformed from L-proline. -4
A method for producing trans-4-hydroxy-L-proline using an enzyme source that catalyzes a hydroxylation reaction to -hydroxy-L-proline is not known. In addition, it has been reported that L-proline 4-position hydroxylase was purified from microorganisms belonging to the above Streptomyces genus [Tetrahedron Letters, 34
Vol., Pp. 7489-7492 (1993)], but its physicochemical properties are not disclosed. In any case, L-catalyst catalyzes the reaction of hydroxylating free L-proline to produce trans-4-hydroxy-L-proline.
Proline 4-hydroxylase has not been isolated and its physicochemical properties have not been clarified.

As a method for the analysis and quantification of prolines, a colorimetric quantification method using ninhydrin is known. However, the color development sensitivity of ninhydrin to imino acid of prolines is low, and it is difficult to detect prolines with high sensitivity. Can not. As a specific detection method for imino acids of prolines,
A method is known in which prolines are NBD-ized with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) chloride under basic conditions, and the resulting proline derivative is detected by high performance liquid chromatography. Analytical Biochemistry (Anal. Bi
ochem.), 138, 390-395, 1984.
Year〕.

[0006] Ligand exchange chromatography for immobilizing an optically active amino acid as a ligand on a carrier and forming a complex between the optically active amino acid and a target amino acid via copper ions to separate optical isomers of amino acids. The method is known [Journal of Chromatography (J.
atogr.), 60, 280-283, 1971;
216, 406-412, 1981]. Based on this principle, it is known to automatically separate optical isomers of amino acids using a commercially available column for high performance liquid chromatography.

[0007]

The conventional transformer-4-
The method for producing hydroxy-L-proline is industrial because (1) the raw material is expensive, (2) there are many reaction steps, (3) the separation and purification steps are complicated, and (4) the productivity is low. The production method is not always satisfactory, and an industrially advantageous method for producing trans-4-hydroxy-L-proline is required.

With respect to the method of analyzing and quantifying prolines, the conventional method in which prolines are converted into NBD and the NBD-modified prolines are separated and detected by high performance liquid chromatography cannot separate and detect the optical isomers of prolines. .
In addition, the method cannot automatically analyze a sample and cannot process a large amount of sample. On the other hand, the use of ligand exchange chromatography can automatically separate the optical isomers of prolines, but the detection using the absorbance method has low sensitivity and makes it difficult to detect trace components. Therefore, development of a new method for automatically detecting a proline optical isomer with high sensitivity is desired.

[0009] An object of the present invention is to provide an industrially advantageous method of trans-4-hydroxy-hydroxylation by directly microbial or enzymatic hydroxylation of L-proline industrially produced from a sugar source by a fermentation method. It is possible to provide a method for producing L-proline and an L-proline 4-hydroxylase useful for the method, and to efficiently process a large number of samples using high performance liquid chromatography.
It is an object of the present invention to provide a method for separating an optical isomer of prolines present in a sample and detecting and quantifying the separated prolines specifically and with high sensitivity without disturbing other contaminating amino acids.

[0010]

According to the present invention, a hydroxylation reaction from L-proline to trans-4-hydroxy-L-proline derived from a microorganism belonging to the genus Dactylosporandium or Amycolatopsis is provided. In the presence of a catalytic enzyme source, ferrous iron and 2-ketoglutaric acid,
Converting L-proline into trans-4-hydroxy-L-proline in a culture or an aqueous medium and collecting the resulting trans-4-hydroxy-L-proline from the culture or the aqueous medium And a process for producing trans-4-hydroxy-L-proline and L-proline 4-position hydroxylase.

The enzyme source used in the present invention is derived from a microorganism belonging to the genus Dactylosporangium or Amycolatopsis and trans-4-hydroxy-L-hydroxylated L-proline. As long as it has the activity of catalyzing the reaction for producing proline, it may be any of microorganisms, cultures thereof, microbial cells, processed microbial cells, purified enzymes or crude enzymes. Preferred examples of microorganisms include Dactylosporandium sp .
ylosporangium sp.) RH1, Amycolattopsis sp. RH2, or subcultures, mutants or derivatives of these strains.

The RH1 strain is a microorganism newly isolated from a tree in Tokyo, and the RH2 strain is a microorganism newly isolated from the soil in Saitama Prefecture by the present inventors. The bacteriological properties of RH1 and RH2 are shown below. 1. Morphological properties Table 1 summarizes the morphological properties of RH1 and RH2 when cultured on various media at 28 ° C. for 14 days.

[0013]

[Table 1]

2. Cultural properties The RH1 strain shows normal or vigorous growth on commonly used synthetic and natural media, and the underlying mycelium shows an orange color. The medium may produce ocher or pale red soluble pigments. The RH2 strain shows normal or vigorous growth on commonly used synthetic and natural media, with the basal hypha showing pale yellow or brown, and the aerial hypha showing white or gray. The medium may produce a brown colored soluble dye.

The RH1 strain and the RH2 strain were cultured on various media for 2 hours.
The characteristics of growth and color when cultured at 8 ° C. for 14 days are summarized in Tables 2- (1) and 2- (2). The colors are displayed in the Color Harmony Manual (Container Corporation of A
merica).

[0016]

[Table 2]

[0017]

[Table 3]

3. Physiological properties The physiological properties of the RH1 and RH2 strains are summarized in Table 3. The growth temperature range summarizes the results after 7 days of liquid shaking cultivation, and the results at 28 ° C and 2 to 3 weeks after the others.

[0019]

[Table 4]

4. Chemical taxonomic properties The chemical taxonomic properties of the RH1 and RH2 strains are summarized in Table 4.

[0021]

[Table 5]

As described above, the RH1 strain morphologically does not form aerial hyphae, forms a rod-shaped sporangia on the underlying hyphae, and the sporangium contains 2 to 4 motile spores. In terms of chemical classification, diaminopimelic acid contained in the cell wall is 3-hydroxy-diaminopimelic acid, and arabinose and xylose are contained as reducing sugars in the whole cell hydrolyzate. It is classified into the genus Dactylosporangium .

The RH2 strain morphologically forms aerial hyphae, breaks aerial hyphae and basal hyphae, and chemically classifies meso-diaminopimelic acid as diaminopimelic acid on the cell wall, and galactose and reducing sugar as reducing sugars. That a trace amount of arabinose is contained, that arabinose and galactose are contained as reducing sugars in the whole cell hydrolyzate, that mycolic acid is not contained as a cell component,
It contains phosphatidylethanolamine as a phospholipid but does not contain phosphatidylcholine and an unknown glucosamine-containing phospholipid, and the main component of menaquinone is M
Since it is K-9 (H _4), it is classified into Amycolatopsis (Amycolatopsis) genus in actinomycetes.

The RH1 strain was used for Dactylosporangium sp. RH1 and the RH2 strain was used for Amycolatopsis sp. RH.
RH1 strain was named FERM BP-4400 on September 1, 1993, based on the Budapest Treaty.
The strain was deposited on February 22, 1994 as FERM BP-4581 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

The culture medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, and the like which can be assimilated by the microorganism.
-Proline is hydroxylated to give trans-4-hydroxy-L
-Any of a natural medium and a synthetic medium may be used as long as the medium can efficiently culture a microorganism having an activity of catalyzing a reaction for producing proline. The carbon source may be any one that can be assimilated by each microorganism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid,
Organic acids such as propionic acid and alcohols such as ethanol and propanol are used.

Examples of the nitrogen source include ammonium salts of various inorganic and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep and the like. Liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof are used.

As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 to 37 ° C, and the culturing time is usually 16 to 37 ° C.
96 hours. During the cultivation, the pH is maintained at 5.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like.

Examples of the treated cells include dried cells, freeze-dried substances, surfactant-treated substances, enzyme-treated substances, ultrasonically treated substances, mechanically milled substances, mechanical pressure-treated substances, and solvent treatments. Products, protein fractions of cells, and immobilized products of cells and processed cells. In addition, enzymes having an activity of catalyzing a hydroxylation reaction of L-proline to trans-4-hydroxy-L-proline obtained by extraction from the cells as an enzyme source, purified samples of these enzymes, and immobilized products Are also used. Examples of the enzyme source having the activity include a novel L-proline 4-position hydroxylase having the following physicochemical properties (1) to (10). (1) Action and substrate specificity It acts on free L-proline in the presence of 2-ketoglutaric acid and ferrous ion to produce trans-4-hydroxy-L-proline. (2) Optimum pH In a reaction at 30 ° C. for 20 minutes, pH 6.0 to 7.0.
Has an optimum pH. (3) pH stability By treating at 4 ° C for 24 hours, the pH is kept stable in the range of 6.5 to 10.0. (4) Optimum temperature It has an optimum temperature of 30 to 40 ° C in a reaction at pH 6.5 for 15 minutes. (5) Temperature stability Completely inactivated by treatment at pH 9.0, 50 ° C. for 30 minutes. (6) Inhibitor Inhibited by metal ions of Zn ⁺⁺ and Cu ⁺⁺ and ethylenediaminetetraacetic acid. (7) Activation Activation does not require a coenzyme.

The addition of L-ascorbic acid to the reaction solution
Accelerate the reaction. (8) Km value In a reaction solution containing 4 mM L-ascorbic acid, 2 mM ferrous sulfate and an enzyme standard in 80 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.5). The Km value for L-proline measured in the above was 0.27 mM, and the Km value for 2-ketoglutarate was
The value is 0.55 mM. (9) Molecular weight The molecular weight measured by sodium dodecyl sulfate-polyacrylamide electrophoresis was 32,000 ± 5,000 daltons, and the molecular weight measured by gel filtration was 43,80.
0 ± 5,000 daltons. (10) N-terminal amino acid sequence having an N-terminal amino acid sequence represented by SEQ ID NO: 1

[0030]

Embedded image

The amount of enzyme activity in the reaction solution of these enzyme sources is determined by the amount of substrate used and the like.
0,000,000 U / l, preferably 1000
2,000,000 U / l. The concentration of L-proline used in the reaction is 1 mM to 2M.

The reaction requires ferrous iron ion, and usually 1 to 100 mM is used. As ferrous ions,
Any substance containing divalent iron and not inhibiting the reaction can be used. Examples include sulfides such as ferrous sulfate, chlorides such as ferrous chloride, ferrous carbonate, and the like, and organic acid salts such as citrate, lactate, and fumarate. .

The reaction requires 2-ketoglutaric acid, but 2-ketoglutaric acid may be added to the reaction solution, or 2-ketoglutaric acid may be added to the reaction mixture depending on the metabolic activity of the cells used and the processed cells. A compound that can be converted to ketoglutaric acid may be used. Such compounds include saccharides such as glucose, glutamic acid, succinic acid and the like. These compounds may be used alone or in combination.

Examples of the aqueous medium include water, phosphates, carbonates, acetates, borates, citrates, buffers such as Tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, and acetone. And amides such as acetamide. The reaction may be performed in a culture of the microorganism having an activity of catalyzing the reaction of hydroxylating L-proline to produce trans-4-hydroxy-L-proline, or from the culture. The separation may be performed in an aqueous medium using the separated cells, a processed product of the cells, or a purified enzyme or a crude enzyme.

The reaction is usually carried out at a temperature of 15 to 50 ° C. and a pH of 6.
Perform at 0-9.0 for 1-96 hours. If necessary, a surfactant or an organic solvent is added during the cell treatment or the reaction. Examples of the surfactant include cationic surfactants such as polyoxyethylene stearylamine (for example, Nimeen S-215, manufactured by NOF Corporation), cetyltrimethylammonium bromide, cation FB, cation F2-40E, and sodium oleyl. Anionic surfactants such as amidosulfuric acid, Neurex TAB, and Ravisol 80; amphoteric surfactants such as polyoxyethylene sorbitan monostearate (eg, Nonion ST221); tertiary amines PB and hexadecyldimethylamine; And any one that promotes the reaction can be used. These are usually 0.1-50m
g / ml, preferably 1-20 mg / ml.

As the organic solvent, toluene, xylene,
Aliphatic alcohol, benzene, ethyl acetate and the like are used. Usually 0.1 to 50 μl / ml, preferably 1 to 2
Used at a concentration of 0 μl / ml. As a method for recovering trans-4-hydroxy-L-proline from a culture or an aqueous medium, a usual separation method such as column chromatography using an ion exchange resin or a crystallization method is used. The recovered trans-4-hydroxy-L-proline had a ¹³ C-NMR spectrum, ¹ H-NM
The structure can be confirmed by ordinary analyzing means such as R spectrum, mass spectrum, specific light intensity and the like.

To quantitatively analyze a sample containing prolines, including trans-4-hydroxy-L-proline produced by the present invention, the following method (hereinafter referred to as a post-column derivatization method). It can be carried out. When analyzing a sample containing prolines using high-performance liquid chromatography, the components contained are separated and eluted with a column. After mixing a buffer containing a chelating agent, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) was added. chl
or NBD chloride) solution, and NBD-modified prolines generated in the reaction solution are detected specifically and with high sensitivity by the fluorescence of the NBD-modified proline.

In the present invention, prolines include proline such as D-proline and L-proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline and trans-4-hydroxy-D-proline. Proline, cis-4-hydroxy-D-proline, trans-3-hydroxy-L-proline, cis-3-hydroxy-L-
Proline, trans-3-hydroxy-D-proline,
And hydroxyproline such as cis-3-hydroxy-D-proline.

The sample may be any sample as long as it contains the above-mentioned prolines alone or in combination of two or more thereof, and a sample derived from a living body such as blood, saliva, urine, or tissue fluid is used. In order to efficiently process a large number of samples using high-performance liquid chromatography and to separate various isomers of prolines present in the samples, an optically active amino acid is immobilized as a ligand on a carrier and analyzed. A so-called ligand exchange chromatography method for separating optical isomers of amino acids by forming a complex with the target amino acid via copper ions is used. A column for high performance liquid chromatography based on this principle is commercially available, for example, MCI GELCRS10W manufactured by Mitsubishi Kasei Kogyo Co., Ltd.
(DLAA), TSKgel Enantio L1 manufactured by Tosoh Corporation, SUMCHIRAL OA5000 manufactured by Sumika Chemical Analysis Service Co., Ltd., and preferably SUMCHIRAL OA5000 manufactured by Sumika Chemical Analysis Service Co., Ltd.
HIRAL OA5000 can be used. The separation conditions may be in accordance with the use conditions specified for the product.

The detection of prolines by NBD derivatization is performed by the following method. First, prolines are separated and eluted using ligand exchange chromatography. Next, a buffer solution is added to the eluate in the liquid sending line to make the pH of the eluate alkaline. At this time, if a buffer solution is added to the eluate, precipitation occurs, and the solution sending line becomes clogged, making measurement substantially impossible. As a result of intensive studies from the viewpoint that the cause is that the hydroxide of copper ions in the eluate forms an insoluble precipitate, a buffer solution in which the metal chelating agent was dissolved in advance was sent. It has been found that if mixed in the line, the line will not be clogged.
After mixing the buffer solution in which the metal chelating agent has been dissolved with the eluate in the liquid sending line, the NBD chloride solution is further mixed in the liquid sending line, and then the prolines in the eluate are mixed in the liquid sending line. The proline is detected by measuring the fluorescence of the NBD-proline.

Any metal chelating agent may be used as long as it is used as a masking agent for capturing copper ions.
Ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (β-aminoethyl ether) tetraacetic acid (EG
TA), cyclohexanediaminetetraacetic acid (CDTA), hydroxyethylethylenediaminetriacetic acid (HEDTA),
Diethylenetriaminepentaacetic acid (DTPA), dihydroxyethylglycine (2-HxG), nitrilotriacetic acid (NTA)
And the like ^, bipyridyls such as 2,2 ^, -bipyridyl ^, and phenanthroline derivatives such as 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline. Carboxylic acids, particularly preferably ethylenediaminetetraacetic acid (E
DTA). The concentration of the metal chelate may be determined depending on the concentration of copper sulfate in the eluate and the mixing ratio of the buffer to the eluate, and is usually used at a concentration of 1 to 100 mM, preferably 10 to 50 mM.

Any buffer may be used as long as it does not react with NBD chloride.
For example, phosphate buffers such as 1 potassium phosphate / borax buffer, 1 potassium phosphate / sodium phosphate buffer, disodium phosphate / sodium hydroxide buffer, hydrochloric acid / borax buffer, boric acid / carbonic acid Boric acid buffers such as sodium buffer, boric acid / borax buffer, boric acid / sodium hydroxide buffer, borax / sodium hydroxide buffer, borax / sodium carbonate buffer, sodium bicarbonate / sodium hydroxide buffer Buffer, tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) buffer, tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer, 3-N-cyclohexylamino -2-hydroxypropanesulfonic acid (CAPSO) buffer,
2- (cyclohexylamino) ethanesulfonic acid (CHES)
Buffer, 2- (N-morpholino) ethanesulfonic acid (ME
S) Buffers and the like can be mentioned, but preferably a borate buffer, particularly preferably a borate / sodium hydroxide buffer is used.
The pH of the buffer is determined by mixing the eluate on the liquid feed line.
Should be determined to be 8.5 to 10.5, preferably 9 to 10. The concentration of the buffer is 0.1-0.6M, preferably 0.2-0.4M. The mixing speed may be determined so that the concentration of the buffer solution after mixing is 5 to 200 mM, preferably 10 to 100 mM.

As a solvent for dissolving NBD chloride, any solvent can be used as long as it can dissolve NBD chloride. For example, alcohols such as methanol, ethanol and propanol, ketones such as acetone, nitriles such as acetonitrile, and dimethylform Examples include amides such as amides, esters such as ethyl acetate, and dimethyl sulfoxide. Preferably, methanol is used.
Although the concentration of NBD chloride is determined by the mixing speed, it is usually 0.1 to 5.0 g / l, preferably 0.2 to 1.0 g / l.
The concentration is 0 g / l. The mixing speed may be such that the concentration of NBD chloride after mixing is usually from 0.02 to 1.0 g / l, preferably from 0.05 to 0.5 g / l.

After the NBD chloride is mixed, the NBD derivatization reaction is carried out by directly heating the liquid sending line. The reaction temperature is from 30 to 80C, preferably from 40 to 70C. The reaction time is 0.5 to 10 minutes, preferably 1 to
5 minutes. After the reaction, the reaction solution is guided to an ultraviolet detector or a fluorescence detector as it is, and the detection is performed by measuring the ultraviolet absorption or the fluorescence intensity. The ultraviolet wavelength and the fluorescent wavelength used may be those specific to each derivative. According to the above method, a very small amount of prolines on the order of picomoles can be separated and detected as their optical isomers. In addition, the detection sensitivity by NBD derivatization of other amino acids other than prolines is as low as about 1/50 or less of that of prolines, and it is hardly hindered by unnatural amino acids. Although it is possible, it is possible to almost completely eliminate the interference caused by the amino acids by subjecting the sample to o -phthalaldehyde in advance and converting the primary amino acids in the sample to o -phthalaldehyde.

Next, a method for obtaining the L-proline 4-position hydroxylase of the present invention will be described. The enzyme cultivates a microorganism having the ability to produce L-proline 4-hydroxylase, generates and accumulates L-proline 4-hydroxylase in the culture, and removes L-proline 4-hydroxylase from the culture. Obtained by collecting the enzyme. As long as it is a microorganism capable of producing L-proline 4-position hydroxylase, any microorganism, such as a wild-type strain, a subculture thereof, a mutant, or a derivative thereof, can be used. Preferable examples include microorganisms belonging to the genus Dactylosporangium and producing L-proline 4-position hydroxylase. Specifically, Dactylosporandium Sp.
ylosporangium sp.) RH1 (FERM BP-440)
0) or subcultures, mutants, derivatives and the like of these strains.

The medium for culturing such a microorganism contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be utilized by the microorganism.
Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture a microorganism having an ability to produce L-proline 4-position hydroxylase. The carbon source may be any one that can be assimilated by each microorganism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, and organic acids such as propionic acid. Alcohols such as ethanol and propanol are used.

Examples of the nitrogen source include ammonium, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., ammonium salts of various inorganic and organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn corn, and the like. Chip liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof are used.

As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 to 37 ° C, and the culturing time is usually 16 to 37 ° C.
96 hours.

During the cultivation, the pH is kept at 5.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like. During culturing, L-proline may be added as necessary. The fact that L-proline 4-position hydroxylase is produced in the cells obtained by culturing in this way means that the aqueous medium suitable for the enzyme reaction in the culture or containing the cells or the treated cells By adding L-proline, ferrous iron ion and 2-ketoglutaric acid therein, and adding a surfactant or an organic solvent as necessary, L-proline can be trans-
It can be known by converting to 4-hydroxy-L-proline.

Examples of the aqueous medium include water, phosphates, carbonates, acetates, borates, citrates, buffers such as Tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, and acetone. And amides such as acetamide. The reaction can be performed as described above.

The trans-4-hydroxy-L-proline produced as a result of the reaction can be recovered and its structure can be confirmed in the same manner as described above. The production of trans-4-hydroxy-L-proline is confirmed by this reaction, and the activity of L-proline 4-hydroxylase, which is confirmed to be produced in the cells, is measured by the following method. Can be. The enzyme activity was measured under the following measurement conditions.
The activity of producing 1 nmol of trans-4-hydroxy-L-proline per minute is expressed as 1 unit (U).

An 80 mM MES buffer containing 4 mM L-proline, 8 mM 2-ketoglutaric acid, 2 mM ferrous sulfate and 4 mM L-ascorbic acid (pH
6.5) Add enzyme preparation to a total of 250 μl,
React at 0 ° C for 20 minutes. After the reaction solution is heated at 100 ° C. for 2 minutes to stop the reaction, trans-4-hydroxy-L-proline formed in the reaction solution is quantified by using high performance liquid chromatography.

For the determination, trans-4-hydroxy-L-
Any method may be used as long as proline can be quantified.For example, a post-column derivatization method usually using high performance liquid chromatography or an NBD derivatization of a target compound in a reaction solution in advance is used. A method in which an NBD derivative is separated by reversed-phase chromatography using high-performance liquid chromatography and then quantified using its fluorescence (excitation wavelength: 503 nm, fluorescence wavelength: 541 nm) (precolumn derivatization method).

The detection by the precolumn derivatization method was performed according to the method of William J. Lindblad and Robert F. Diegelman [William J. Lindb.
lad and Robert F. Diegelmann, Analytical Biochemistry, 13
8, 390-395, 1984]. However, in this method, prolines can be specifically detected, but not all optical isomers can be distinguished.

In the measurement of the activity during enzyme purification, etc.,
Since various compounds are used in the form of a buffer or the like, the above two methods can be used depending on the situation. In order to isolate and purify the enzyme from the culture solution, a general method for isolating and purifying the enzyme may be used. For example, the culture solution is collected by centrifugation, washed, and sufficiently washed, and then the cells are disrupted by an ultrasonic cell disruptor, a French press, a Manton-Gaurin homogenizer, a Dynomill, or the like to obtain a cell-free extract. The supernatant obtained after centrifugation is subjected to salting out with ammonium sulfate or the like, anion exchange chromatography such as diethylaminoethyl (DEAE) -Sepharose, hydrophobic chromatography such as butyl sepharose or phenyl sepharose, or dye affinity chromatography such as red agarose. And a gel filtration method using a molecular sieve or an electrophoresis method such as isoelectric focusing to obtain a purified enzyme preparation. The physicochemical characteristics of the obtained enzyme preparation can be specified by ordinary enzymatic techniques.

The L-proline 4-hydroxylase thus obtained has the following physicochemical properties (1) to (10). (1) Action and substrate specificity It acts on free L-proline in the presence of 2-ketoglutaric acid and ferrous ion to produce trans-4-hydroxy-L-proline. (2) Optimum pH In the method for measuring the activity of L-proline 4-position hydroxylase, the buffer component in the reaction solution was adjusted to pH 3.5 to 5.5.
Is sodium acetate buffer, pH 5.5-6.5 is MES
Buffer solution, pH 7.0 to 7.5 is TES buffer [N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid], pH 8.0 to 9.0 is TAPS buffer [N
-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid], pH 9.5 to 11.0 is CAPS
As a result of substitution reaction with an O buffer (3-N-cyclohexylamino-2-hydroxypropane-sulfonic acid), the optimum pH was 6.0 to 7.0. (3) pH stability This enzyme was used in a 50 mM buffer [pH 3.5 to 5.5 was a sodium acetate buffer, pH 5.5 to 6.5 was a MES buffer, and pH 7.0 to 7.5 was a TES buffer. PH 8.0
9.0 is TAPS buffer, pH 9.5 to 11.0 is CA
PSO buffer], 2 mM dithiothreitol (DTT)
And 20% (v / v) glycerol at 4 ° C, 2
Hold for 4 hours. After the retention, the activity is measured. pH 6.5-
Enzyme retained in 10.0 range is 90% of the activity before retention
It has the above activity, and its activity is kept stable in the pH range of 6.5 to 10.0. (4) Optimal temperature In the method for measuring L-proline 4-position hydroxylase activity,
As a result of measuring the activity at various temperatures, pH 6.5, 1
It has an optimum temperature of 30 to 40 ° C. in the reaction for 5 minutes. (5) Temperature stability The enzyme is completely inactivated by treating it at pH 9.0, 50 ° C. for 30 minutes. (6) Inhibitor This enzyme activity is determined by the metal ions of Zn ⁺⁺ and Cu ⁺⁺ and ED
Inhibited by TA. In the L-proline 4-hydroxylase activity measurement method, when Cu ⁺⁺ and Zn ⁺⁺ ions were added at a concentration of 1 mM, respectively, and the activity was measured, the activity was reduced to 13% and 6%, respectively, when no addition was made. Similarly, EDT
When A was added at 5 mM and the activity was measured, no activity could be detected. (7) Activation No activation of this enzyme by various metal ions and coenzymes was observed, and no activation of this enzyme requires a coenzyme. Addition of ascorbic acid to the reaction solution promotes the reaction. (8) Km value The Km value for L-proline measured in a reaction solution containing 80 mM MES buffer, pH 6.5, 4 mM L-ascorbic acid, 2 mM ferrous sulfate and an enzyme standard was 0.27 mM, The Km value for 2-ketoglutaric acid was 0.55 mM. (9) Molecular weight The molecular weight was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (polyacrylamide gel PAGEL NP manufactured by ATTO).
U-12.5L and Bio-Rad molecular weight standard SD
S-PAGE Molecular Weight Standard, Broad Range) was calculated to be 32,000 ± 5,000 daltons. Gel filtration using high performance liquid chromatography (column G-3000SW, 21.5 m
mx 60 cm, using a molecular weight marker for HPLC manufactured by Oriental East Co., Ltd. as a standard) to obtain 43,800 ± 5,000 daltons. (10) N-terminal amino acid sequence As a result of analysis using Protein sequencer model PPSQ-10 manufactured by Shimadzu Corporation, the N-terminal amino acid sequence of the present enzyme protein was determined as follows.

[0057]

Embedded image

Examples of the present invention will be described below.

[0059]

【Example】

Example 1 trans-4-hydroxy-L
-Production of proline SR3 medium [glucose 1.0%, soluble starch 1.0
%, A yeast extract 0.5%, a tryptone 0.5%, a meat extract 0.3% and a magnesium phosphate 0.05%, and adjusted to pH 7.2 with 6N NaOH] in a test tube (diameter 25 mm × 200 mm). ), And sterilized at 120 ° C. for 20 minutes. HT agar plate medium [soluble starch 1%, NZ amine 0.2%, yeast extract 0.1%, meat extract 0.1% and agar 1.5
%, Adjusted to pH 7.2 with 6N NaOH,
Dactylosporangium sp. R grown on a medium sterilized for 20 min.
One loopful of H1 was inoculated, shake-cultured at 28 ° C. for 2 days, and used as a seed culture.

On the other hand, a Df1 medium [soluble starch 5%, soybean meal 1.5%, monopotassium phosphate 0.05%,
A medium containing 0.05% of magnesium sulfate heptahydrate and 0.5% of calcium carbonate and adjusted to pH 7.0 with 6N NaOH] into a test tube (diameter 25 mm × 200 mm).
The mixture was dispensed in ml and sterilized at 120 ° C. for 20 minutes. This culture medium was aseptically inoculated with 1 ml of a seed culture and cultured with shaking at 28 ° C. for 2 days. The obtained culture solution was 7,000 × g,
Centrifuged at 4 ° C. for 10 minutes. 80 m of the obtained cells
After washing with MTES buffer (pH 7.5), the mixture was centrifuged. 150 mg of the obtained wet cells was added to 1.5 ml of a reaction solution [80 mM TES buffer (pH 7.5) containing 4 mM L-proline, 8 mM α-ketoglutaric acid, 4 mM L-ascorbic acid and 2 mM ferrous sulfate.
1.4% of a Nimeen solution (4 g of Nimeen S-215 (manufactured by NOF CORPORATION) in 10 ml of xylene)
(V / v) added solution], and reacted at 30 ° C. for 2 hours.

After the reaction, the hydroxyproline produced in the supernatant from which the cells were removed from the cell reaction solution by centrifugation was analyzed. The analysis was performed by high performance liquid chromatography under the following conditions. Detection is performed by eluting the target compound on the column, derivatizing it with NBD on the liquid sending line,
This was performed by measuring the fluorescence of the derivatized product.

High-performance liquid chromatography analysis conditions [1] Apparatus: Shimadzu high-performance liquid chromatography chromatopack CR6A system controller SCL-6B auto-injector SIL-6B liquid pump LC-6A column oven CTO-6A chemical reaction tank CRB- 6A Fluorescence detector RF-550A [2] Column used: SUMC manufactured by Sumika Chemical Analysis Service, Ltd.
HIRAL OA5000 (diameter 4.5 mm x 250 mm) [3] Analysis conditions: 1) Mobile phase: 1 mM aqueous copper sulfate solution 2) Same flow rate: 1.0 ml / min 3) Column temperature: 38 ° C 4) Buffer: 0.3 M Borate buffer, pH 9.6 25 mM ethylenediaminetetraacetic acid 5) Same as above: 0.2 ml / min 6) NBD chloride solution: 0.5 g / l methanol solution 7) Same as above: 0.5 ml / min 8) Reaction temperature: 9) Reaction time: about 3 minutes 10) Detection wavelength: Excitation wavelength 503 nm Fluorescence wavelength 541 nm 11) Sample: 10 μl As a result, 249 μM (32.6 mg / l) in the reaction solution
It was confirmed that trans-4-hydroxy-L-proline was produced.

Example 2 trans-4-hydroxy-L
-Purification of proline The seed culture in Example 1 was performed using a 2 l Erlenmeyer flask into which 200 ml of the SR3 medium was dispensed. This seed culture was aseptically inoculated into 2 l of Df1 medium sterilized at 120 ° C. for 20 minutes after dispensing in advance into a 5 l jar fermenter, and cultured at 28 ° C. for 2 days at 700 rpm and 1 vvm. The pH during the culture was not adjusted. The obtained culture was centrifuged at 7,000 × g for 10 minutes at 4 ° C. to obtain 75 g of wet cells per liter of culture. The wet cells are washed with physiological saline at 4 ° C., centrifuged and used at −8 until use.
Stored frozen at 0 ° C. 400 g of the wet cells were suspended in 2 l of the reaction solution described in Example 1, and the mixture was reacted in a 3 l beaker at 30 ° C. for 4 hours with stirring.

After the reaction, hydroxyproline produced in the supernatant obtained by centrifuging and removing the cells from the cell reaction solution was analyzed. As a result, 480 μM (63.
(0 mg / l) of trans-4-hydroxy-L-proline was confirmed to have been produced. The reaction solution supernatant was
After adjusting to H4.5, the mixture was passed through a column filled with 200 ml of ion-exchange resin Diaion SK1B (NH ₄ ⁺ type, manufactured by Mitsubishi Kasei Corporation). Trans-4-hydroxy-L
-The fraction containing proline was concentrated under reduced pressure, and then passed through a column packed with 20 ml of ion exchange resin DIAION PA412 (OH ^- type, manufactured by Mitsubishi Kasei Corporation). After concentrating the fraction containing trans-4-hydroxy-L-proline under reduced pressure, the pH was increased to 9.6.
Then, a 10% volume o -phthalaldehyde solution (0.075 g / ml ethanol solution) and a 2% volume β-mercaptoethanol solution (10% v / v aqueous solution) were added, and the mixture was kept at 60 ° C. for 5 minutes. The primary amino acid was converted to o -phthalaldehyde. This mixed solution was passed through a column filled with 10 ml of Sepabeads SP207 (manufactured by Mitsubishi Kasei Co., Ltd.) to separate o -phthalaldehyde-converted primary amino acids from trans-4-hydroxy-L-proline. After concentrating the fraction containing trans-4-hydroxy-L-proline under reduced pressure, the ion-exchange resin
The solution was passed through a column packed with 20 ml of PA412 (OH ^- type, manufactured by Mitsubishi Kasei Corporation) to obtain a fraction containing trans-4-hydroxy-L-proline. This fraction is concentrated and dried, and
78 mg of white crystals of -hydroxy-L-proline were obtained (yield 62%). As a result of analyzing this white crystal, ¹³ C-N
MR spectra, ¹ H-NMR spectra, mass spectra, specific rotation or the like, trans-4-hydroxy -L-
It was the same as Proline standard product (Nacalai Tesque, Inc.).

Example 3 trans-4-hydroxy-L
-Production of Proline The SR3 medium was dispensed into test tubes (diameter 25 mm x 200 mm) in 10 ml portions and sterilized at 120 ° C for 20 minutes. A platinum loop of Amycolatopsis sp. RH2 grown on an HT agar plate medium was inoculated into this medium, shake-cultured at 28 ° C. for 2 days, and used as a seed culture solution.

On the other hand, a Df1 medium was placed in a test tube (25 mm in diameter).
x 200 mm) at 120 ° C, 2
Sterilized for 0 minutes. This culture medium was aseptically inoculated with 1 ml of a seed culture and cultured with shaking at 28 ° C. for 2 days. The resulting culture was centrifuged at 7,000 × g for 10 minutes at 4 ° C. The obtained cells were added to a 100 mM TES buffer (pH
After washing in 7.5), centrifugation was performed. Obtained wet cells 10
0 mg was added to 1 ml of the reaction solution [5 mM L-proline, 5 mM
100 mM containing mM α-keto-glutaric acid, 5 mM L-ascorbic acid and 1 mM ferrous sulfate
A Nimeen solution (4 g of Nimeen S-215 (manufactured by NOF Corporation) dissolved in 10 ml of xylene) was added to TES buffer (pH 7.5) at 1.4% (v / v), and suspended at 30 ° C. A time reaction was performed.

After the reaction, hydroxyproline produced in the supernatant obtained by removing the cells from the cell reaction solution by centrifugation was analyzed. Analysis and detection were performed according to Example 1. As a result, 25 μM (3.3 mg
/ L) was confirmed to produce trans-4-hydroxy-L-proline.

Example 4 trans-4-hydroxy-L
-Purification of proline Seed culture in Example 3 was performed using a 2 l Erlenmeyer flask into which 200 ml of SR3 medium was dispensed. This seed culture was aseptically inoculated into 2 l of Df1 medium sterilized at 120 ° C. for 20 minutes after dispensing in advance into a 5 l jar fermenter, and cultured at 28 ° C. for 2 days at 700 rpm and 1 vvm. The pH during the culture was not adjusted. The obtained culture was centrifuged at 7,000 × g for 10 minutes at 4 ° C. to obtain 75 g of wet cells per liter of culture.
400 g of the wet cells were suspended in 2 liters of the reaction solution described in Example 3, and reacted in a 3 liter beaker at 30 ° C. for 4 hours with stirring.

After adjusting the pH of the reaction solution supernatant to 4.5, the ion exchange resin Diaion SK1B (NH ₄ ⁺ type,
The mixture was passed through a column filled with 200 ml (manufactured by Mitsubishi Chemical Corporation). The fraction containing trans-4-hydroxy-L-proline was concentrated under reduced pressure, and then ion-exchange resin Diaion PA412 (O
(H ^- type, manufactured by Mitsubishi Kasei Corporation) was passed through a column packed with 20 ml. The fraction containing trans-4-hydroxy-L-proline was concentrated under reduced pressure to pH 9.6, and then a 10% volume o -phthalaldehyde solution (0.075 g / ml ethanol solution) and a 2% volume β- A mercaptoethanol solution (10% v / v aqueous solution) was added, and the mixture was kept at 60 ° C. for 5 minutes to convert the primary amino acid into o -phthalaldehyde. This mixture was passed through a column filled with 10 ml of Sepabeads SP207 (manufactured by Mitsubishi Kasei Corporation) to separate o -phthalaldehyde-modified primary amino acids and trans-4-hydroxy-L-proline. The fraction containing trans-4-hydroxy-L-proline was concentrated under reduced pressure, and again passed through a column filled with 20 ml of ion exchange resin DIAION PA412 (OH ^- type, manufactured by Mitsubishi Kasei Co., Ltd.).
A fraction containing -hydroxy-L-proline was obtained. This fraction was concentrated and dried to obtain 4.8 mg of trans-4-hydroxy-L-proline as white crystals (yield: 62%).

As a result of analyzing the white crystals, ¹³ C-NMR
Spectrum, ¹ H-NMR spectrum, mass spectrum, specific rotation, etc.
-It was consistent with Proline Standard (Nacalai Tesque, Inc.).

Example 5 Isolation and Purification of L-Proline 4-Hydroxylase (1) Preparation of Cell-Free Extract After thawing 600 g of the frozen cells obtained in Example 2, 3 l of buffer A [2 mM DTT 0.2 mM EDTA and 2
The suspension was suspended in 50 mM TAPS buffer (pH 9.0) containing 0% (v / v) glycerol under ice-cooling. The suspension is dyno-milled (DYNO-MILL, WILLY A BACHOFEN MASCHINENFAB
RIK, BASEL, Switzerland) to disrupt the cells. This treated solution was centrifuged at 6,500 × g at 4 ° C. for 30 minutes to obtain a supernatant. All subsequent operations were performed under ice cooling or at 4 ° C. (2) Isolation and purification by column chromatography (2) -1 Stream line The supernatant obtained in the above step was equilibrated with buffer A
The mixture was passed through a Pharmacia streamline (STREAMLINE ^™ ) filled with 300 ml of the EAE adsorbent, and the fraction containing L-proline 4-position hydroxylase was eluted with a buffer A containing 0.3 M saline.

(2) -2 DEAE Sepharose Column Chromatography The active fraction obtained in the above step was diluted 3-fold with buffer A, and then equilibrated with buffer A beforehand. DEAE Sepharose column (5 cm × 15 cm) Passed through the tower. After washing the column with buffer A, the fraction containing the enzyme was eluted with a linear concentration gradient of saline from 0 to 0.3 M made in buffer A.

(2) -3 Butyl Sepharose Column Chromatography The active fraction obtained in the above step was dissolved by adding sodium chloride to a concentration of 3 M and butyl sepharose previously equilibrated with buffer A containing 3 M sodium chloride. Column (Butyl Sepharose 4
Fast Flow, 2.6 cm x 13 cm). The enzyme was used in four kinds of buffer solutions having different salt concentrations of buffer A containing 3 M salt, buffer A containing 1.98 M salt, buffer A containing 0.99 M salt, and buffer A alone. It eluted step by step from high to low.

(2) -4 Phenyl Sepharose Column Chromatography The active fraction obtained in the above step was dissolved by adding sodium chloride to a concentration of 3 M, and phenyl sepharose previously equilibrated with buffer A containing 3 M sodium chloride. Column (Phenyl Sepharose)
HP HiLoad 16/10, 1.6cm x 10cm). 3M
After washing with buffer A containing saline, the fraction containing the enzyme was eluted with buffer A.

(2) -5 Dye Affinity Column Chromatography The active fraction obtained in the above step was separated from Pharmacia's PD-10.
After desalting using a column, Reactive Red 120 column (Sigma Reacti) previously equilibrated with buffer A
ve red 120, 1 cm x 12.7 cm). After washing with buffer A, the enzyme-containing fraction was eluted using a linear gradient of 0 to 1.5 M saline prepared in buffer A.

(2) -6 Resource Q Column Chromatography The active fraction obtained in the above step was treated with buffer B [2 mM DTT,
0.1% (v / v) Tween 20 and 20% (v / v)
50 mM TAPS buffer containing glycerol (pH
8.0)], desalting was performed using a Pharmacia PD-10 column equilibrated in the above, and then a resource Q column (Pharmacia RESOURCE ^™ Q,
1 ml). 0 to 0.2 prepared in buffer B
Elution was carried out using a linear gradient of saline up to M.

Table 5 summarizes the isolation and purification of L-proline 4-position hydroxylase.

[0079]

[Table 6]

Example 6 Properties of L-proline 4-position hydroxylase (1) Analysis by electrophoresis The purified sample obtained in Example 5 was subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis (polyacrylamide manufactured by ATTO). Gel PAGEL NPU-12.5L and Bio-Rad molecular weight standard SDS-PAGE Molecular Weight Stan
dard, Broad Range). As a result, it was revealed that the present enzyme was composed of substantially uniform subunits having a molecular weight of about 32,000 ± 5,000 daltons. (2) Properties related to enzymatic reaction By using the following reaction mixture, a substrate omission test (omission test) and an additive test of the reaction components were carried out to obtain an essential compound and a promoting compound for the L-proline 4-position hydroxylase reaction. And inhibitory compounds were studied.

The basic composition of the reaction solution was 4 mM L-proline, 8 mM 2-ketoglutaric acid, 2 mM ferrous sulfate, 4 mM L-ascorbic acid, 2 m catalase.
An 80 mM TES buffer (pH 7.5) containing g / ml and an enzyme standard was used, and the total volume was 500 μl. The reaction was started by the addition of the enzyme and was carried out at 30 ° C. for 15 minutes. The reaction was stopped by heating the reaction solution at 100 ° C. for 2 minutes. The amount of trans-4-hydroxy-L-proline formed in the reaction solution was quantified by a precolumn derivatization method. To 100 μl of the reaction solution, 100 μl of 0.3 M borate buffer (pH 10.7), 4 μl of 10% (v / v) mercaptoethanol aqueous solution and 5% (w / v)
16 μl of an o -phthalaldehyde ethanol solution was added and left at 60 ° C. for 30 seconds. 2% (w / v) NBD
Add 50 μl of an ethanol solution of chloride, and add
The reaction was performed for 40 minutes. After stopping the reaction by adding 30 μl of 1N hydrochloric acid, the precipitate was removed by centrifugation and filtration with a filter, and analyzed by high performance liquid chromatography.
The produced trans-4-hydroxy-L-proline was quantified.

High performance liquid chromatography is performed under the following conditions:
I went in the matter. Mobile phase: 10 mM citric acid (pH 4.0) / Methanol
= 3/1 (v / v) Flow rate: 1 ml / min Column: YMC Pack ODS AQ-312 (YMC, 6 × 15
0 mm) Column temperature: 50 ° C. Detection: fluorescence detection, excitation wavelength 503 nm, fluorescence wavelength 541
nm As a result, L-proline 4-position hydroxylase reaction
Proline, 2-ketoglutaric acid, Fe⁺⁺Ion is essential
L-ascorbic acid accelerates the reaction, ⁺⁺, Cu⁺⁺
Addition of ions and EDTA inhibited the reaction.

The results are shown in Table 6.

[0084]

[Table 7]

(3) Optimum pH In the method for measuring the activity of L-proline 4-position hydroxylase, the buffer components in the reaction solution components were pH 3.5 to 5.5: sodium acetate buffer, pH 5.5 to 5.5. 6.5 is MES buffer, pH 7.0 to 7.5 is TES buffer, pH 8.0 to
9.0 is TAPS buffer, pH 9.5 to 11.0 is CA
As a result of substitution reaction with a PSO buffer, the pH was 6.0 to 6.0.
At 7.0, the activity was 90% or more of the highest activity. The results are shown in Table 7.

[0086]

[Table 8]

(4) pH stability The present enzyme was used in a 50 mM buffer [pH 3.5 to 5.5 was a sodium acetate buffer, pH 5.5 to 6.5 was a MES buffer, and pH 7.0 to 7.5 was a buffer. TES buffer, pH 8.0-
9.0 is TAPS buffer, pH 9.5 to 11.0 is CA
PSO buffer], 2 mM DTT and 20% (v /
v) The activity was measured after keeping at 4 ° C. for 24 hours in the presence of glycerol. The enzyme retained in the pH range of 6.5 to 10.0 has an activity of 90% or more of the activity before retention,
The activity was kept stable in the pH range of 6.5 to 10.0. (5) Optimal temperature In the method for measuring the activity of L-proline 4-position hydroxylase, 1
As a result of performing the reaction in a temperature range of 5 to 50 ° C. for 15 minutes, 3
The activity was 90% or more of the maximum activity at 0 to 40 ° C. The results are shown in Table 8.

[0088]

[Table 9]

(6) Temperature stability This enzyme was prepared by adding 2 mM DTT, 0.1% (v / v) Tween2
50 containing 0 and 20% (v / v) glycerol
After being kept in a temperature range of 0 to 60 ° C. for 30 minutes in a mM TAPS buffer (pH 9.0), the activity was measured. As a result, the enzyme was completely inactivated by treatment at 50 ° C. for 30 minutes. (7) N-terminal amino acid sequence As a result of analyzing the N-terminal amino acid sequence of the present enzyme protein using Shimadzu Corporation Protein sequencer model PPSQ-10,
The following results were obtained.

[0090]

Embedded image

Example 7 Hydroxidation of L-proline L-proline was hydroxylated using the purified enzyme preparation obtained in Example 5. 20 mM L-proline, 20 m
M 2-ketoglutaric acid, 5 mM L-ascorbic acid,
50 μl of 200 mM MES buffer (pH 6.5) containing 2 mM ferrous sulfate and 90 U of the purified enzyme preparation
The reaction was carried out at 35 ° C. for 30 minutes. As a result, 12.9 mM (1.7 g / l) of trans-
4-Hydroxy-L-proline was formed.

Example 8 Separation and detection of prolines High-performance liquid chromatography shown in FIG. 1 [Chromatopack: CR6A, system controller: SCL-6B, auto injector: SIL-6B, liquid pump: LC-6A, column oven : CTO-6A, Chemical reaction tank: CRB-6A, Fluorescence detector:
RF-550A (all manufactured by Shimadzu Corporation) Column used: SUMCHIRA
LOA5000 (diameter 4.5 mm × 250 mm; manufactured by Sumika Chemical Analysis Service Co., Ltd.)] from the sample injection port to L-proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-D-proline, cis-4-hydroxy- 10 μl of a standard aqueous solution containing 0.01 g / l each of L-proline and D-proline was injected. The following conditions [mobile phase: 1
The proline is separated from the column with an aqueous solution of mM mM copper sulfate, mobile phase flow rate: 1.0 ml / min, column temperature: 38 ° C.], and then 25 mM EDTA-containing 0.3 M borate buffer (pH 9.0).
6) was mixed at a flow rate of 0.2 ml, and an ethanol solution of NBD chloride (0.5 g / l) was further added at a flow rate of 0.5 ml.
Per minute and heated at 60 ° C. for 3 minutes. During sending 50
The NBD derivative of prolines was detected by irradiating excitation light of 3 nm and measuring fluorescence at 541 nm.

As a result of analyzing the title standard solution under the above conditions, all the optical isomers of proline and hydroxyproline contained in the standard sample could be clearly separated and detected as separate peaks. The results are shown in Table 9 and FIG.

[0094]

[Table 10]

Example 9 Separation and detection of prolines 0.000 of trans-4-hydroxy-L-proline
From 1 g / l (7.6 pmol contained in 10 μl) to 0.1 g
/ L (containing 7.6 nmol) in a stepwise manner to prepare an aqueous solution having a constant concentration, and trans-4- according to the method of Example 8.
Hydroxy-L-proline was measured separately. Fig. 3 shows the results.
And FIG.

According to FIG. 3 and FIG.
Hydroxy-L-proline was detectable up to 0.0001 g / l, and the quantification was confirmed between 0.0001 g / l and 0.1 g / l. This result was obtained from the trans-4 obtained by the automatic analysis method without using the NBD obtained in Reference Example 1.
The value was 10 times the detection sensitivity of -hydroxy-L-proline.

Example 10 Separation and Detection of Prolines Each of the 22 amino acids shown in Table 10 was treated with 0.1% each.
An aqueous solution having a concentration of 01 g / l was prepared and analyzed by the NBD method according to the method of Example 8. The results are shown in Table 10.

[0098]

[Table 11]

According to Table 10, when the detection sensitivity of trans-4-hydroxy-L-proline is 100,
The detection sensitivity of the other 20 amino acids other than proline was 2 or less in each case. That is, it was shown that the specific detection sensitivity of trans-4-hydroxy-L-proline to other amino acids was 50 times higher than that of other amino acids, but this was due to the automatic analysis without using NBD obtained in Reference Example 2. The value was significantly higher than the specific detection sensitivity obtained by the method.

Reference Example 1 High-performance liquid chromatography shown in FIG. 5 [Chromatopack: CR6A, system controller: SCL-6B, auto injector: SIL-6B, liquid sending pump: LC-6A, column oven: CTO-6A, UV-VIS detector: SPD-6AV (or more,
Column used: SUMCHIRAL OA5000 (4.5 mm diameter x 250)
mm; manufactured by Sumika Chemical Analysis Service Co., Ltd.)], and inject 10 μl of the sample used in Example 9 under the following conditions (mobile phase: 1 mM aqueous solution of copper sulfate, mobile phase flow rate: 1.2).
ml / min, column temperature: 35 ° C.], and prolines were detected using ultraviolet absorption at 254 nm.

As a result, the detection limit was 0.001 g / dl.

Reference Example 2 The sample used in Example 9 was measured by the method of Reference Example 1. The results are shown in Table 10. According to Table 10, the transformer
Assuming that the detection sensitivity of 4-hydroxy-L-proline was 100, the detection sensitivity of 20 other amino acids other than proline was 45 to 170. That is, the specific detection sensitivity of trans-4-hydroxy-L-proline to other amino acids was 0.45 to 1.7 times.

[0103]

Industrial Applicability According to the present invention, L-4-proline industrially produced from a sugar source by fermentation is directly microbially or enzymatically hydroxylated, so that trans-4-hydroxy is advantageously produced industrially. A method for producing hydroxy-L-proline, L-proline 4-position hydroxylase useful for the production method,
Further, it is possible to provide an automatic analysis method having high detection sensitivity and capable of specific detection and quantification of prolines.

[0104]

[Sequence List] SEQ ID NO: 1 Sequence length: 27 Sequence type: amino acid Tropology: Linear Sequence type: Peptide Origin: Organism name: Dactylosporangium sp. Strain name: RH1 ( FERM BP-4400) Sequence: Met Leu Thr Pro Thr Glu Leu Lys Gln Tyr Arg Glu Ala Gly Tyr Leu Leu 1 5 10 15 Ile Glu Asp Gly Leu Gly Pro Arg Arg Val 20 25

[Brief description of the drawings]

Fig. 1 High-performance liquid chromatography system configuration using NBD

FIG. 2 High-performance liquid chromatography of prolines using NBD

[Explanation of symbols]

 (1) trans-4-hydroxy-L-proline (2) cis-4-hydroxy-D-proline (3) L-proline (4) cis-4-hydroxy-L-proline (5) D-proline

FIG. 3 is a calibration curve from 7.6 pmol to 305 pmol obtained by an automatic analysis method of prolines using NBD.

FIG. 4 is a calibration curve from 153 pmol to 7600 pmol obtained by an automatic analysis method of prolines using NBD.

FIG. 5 is a high-performance liquid chromatography system configuration that does not use NBD.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 1/14 C12R 1: 645 C12R 1: 645) (31) Claimed priority number Japanese Patent Application No. 6-60377 (32) Priority date 1994 March 30, 1994 (3.3.30) (33) Countries claiming priority Japan (JP) (56) References JP-A-1-81665 (JP, A) JP-A-61-80051 (JP, A) JP-A-60-235057 (JP, A) JP-A-2-91566 (JP, A) JP-A-3-266995 (JP, A) Sci. Food. Agri c. , 1992, 59, 217-226. Chromatogr. A, 1993, 653, 229-234 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 30/88 G01N 30/06 G01N 30/26 C12N 1/14 C12P 13/24 C12N 1/14 C12R 1: 645

Claims (4)

(57) [Claims] 1. A method for analyzing a sample containing prolines using high performance liquid chromatography, comprising the steps of: [1] adding o-phthalic aldehyde to the sample,
Step of converting a primary amino acid into o-phthalic aldehyde [2] Step of treating the sample obtained in Step [1] with a ligand exchange column [3] Inclusion of a metal chelating agent in an eluate in a liquid sending line step Ru is mixed buffer was [4] proline in the eluate and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) is reacted with a chloride, generated NBD proline A method for detecting and quantifying prolines, which comprises a step of detecting and quantifying the compounds. 2. A buffer containing a metal chelating agent is mixed into the eluate, the pH of the eluate is adjusted to 8.5 to 10.5, and an NBD chloride solution is mixed into the eluate. The method according to claim 1, wherein the liquid is maintained at 40 to 80C. 3. The method according to claim 1, wherein the buffer is a borate buffer. 4. The method according to claim 1, wherein the metal chelating agent is ethylenediaminetetraacetic acid.