JP3492776B2 - Method for producing cis-3-hydroxy-L-proline - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to cis-3-hydroxy-L-, which is useful as a synthetic raw material for pharmaceuticals or as a food additive.
The present invention relates to a method for industrially producing proline and a novel L-proline 3-position hydroxylase useful in the method.

[0002]

2. Description of the Prior Art As a conventional method for producing cis-3-hydroxy-L-proline, a method of chemically synthesizing is known [J. Amer. Chem. Soc.]. , 84 , 39
80 (1962), ibid., 85 , 2824 (196).
3), Nature, 289 , 310 (198)
1), Journal of Organic Chemistry (J. Org. Chem.), 54 , 1866 (1989), Acta Chemica Scandin
avica), 43 , 290 (1989)].

Methods for producing cis-3-hydroxy-L-proline by directly and regio- and stereoselectively hydroxylating L-proline have hitherto been carried out by both synthetic chemical methods and methods utilizing biological functions. Not reported.

[0004]

The conventional method for producing cis-3-hydroxy-L-proline by chemical synthesis is as follows:
(1) expensive raw materials, (2) long reaction process,
(3) The separation / purification process is complicated, (4) the production efficiency is low, and the like, which is not necessarily a satisfactory industrial production method, and industrially advantageous cis-3-hydroxy-L-proline. Is required.

The object of the present invention is to industrially conveniently and advantageously oxidize L-proline, which is industrially produced at low cost from an inexpensive sugar source by fermentation, directly by microbial or enzymatic hydrolysis. It is intended to provide a method for producing cis-3-hydroxy-L-proline and an L-proline 3-hydroxylase useful for the method.

[0006]

According to the present invention, the presence of an enzyme source, a divalent iron ion and 2-ketoglutarate, which catalyzes the hydroxylation reaction of L-proline to cis-3-hydroxy-L-proline. Below, in culture or in an aqueous medium, L-proline is converted to cis-3-hydroxy-L-proline, and the produced cis-3-hydroxy-L-proline is removed from the culture or in the aqueous medium. It is possible to provide a method for producing cis-3-hydroxy-L-proline, which is characterized by collecting, and a novel L-proline 3-position hydroxylase.

The enzyme source used in the method for producing cis-3-hydroxy-L-proline of the present invention has an activity of catalyzing the reaction of hydroxylating L-proline to produce cis-3-hydroxy-L-proline. Any of microorganisms, cultures thereof, cells, treated cells, purified enzyme or crude enzyme may be used as long as it has Preferred examples of microorganisms, Streptomyces (Streptomyces) genus or Bacillus (Bacillu
s ) Microorganisms belonging to the genus. More specifically, Streptomyces Kanusu (Streptomyces c anus) ATCC 1
2647 and ATCC 12646, Streptomyces microstrip Seth sp (Streptomyces sp.) TH1, Bacillus sp. (Bac
illus sp.) TH2 and TH3, or subcultures, mutants or derivatives of these strains.

Streptomyces SP
yces sp.) TH1 is a microorganism newly isolated from the soil by the present inventors. The mycological properties of Streptomyces sp. TH1 are shown below. 1. Morphological properties Table 1 shows the morphological properties of the TH1 strain.

[0009]

[Table 1]

2. Cultural Properties The TH1 strain shows normal or vigorous growth in commonly used synthetic and natural media, and the basic hyphae are pale pink,
Shows an orange or greenish brown system. The brown or black soluble dye may be produced depending on the medium. The characteristics when cultured at 28 ° C for 14 days on various media are
It is shown in Table (1) and Table 2- (2). The color display is Colo
r Harmony Manual (Container Corporation of Americ
According to the color classification according to a).

[0011]

[Table 2]

[0012]

[Table 3]

3. Physiological Properties Table 3 shows the physiological properties of the TH1 strain. The growth temperature range is 7 days, and the other results are 28 ° C., and the results after 2 to 3 weeks are described.

[0014]

[Table 4]

4. Chemotaxonomic properties Optical isomers of diaminopimelic acid in bacterial cells; LL type or higher, morphologically, forming a spore chain consisting of 10 or more spores spirally in the aerial mycelium. Since the cell wall is type I (LL-diaminopimelic acid), this strain is classified as Streptomyces in actinomycetes.

This strain was named Streptomyces sp. TH1 and was designated as FERM BP-4399 at the Institute of Biotechnology, Agency of Industrial Science and Technology on September 1, 1993, based on the Budapest Treaty. Deposited. Bacillus sp. TH2 and TH3 are microorganisms newly isolated by the present inventors from soil. The mycological properties of both strains are shown below.

The cell size is TH2-1.5-1.8.
× 3 to 4 μm, TH3 is 1.2 to 1.5 × 3.5 to 4 μm
m, the base composition of DNA (G + Cmol%) is TH2 36.4
Both strains have the following properties except that 6% and TH3 are 37.74%. 1. Morphological properties The morphological properties of TH2 and TH3 are shown in Table 4.

[0018]

[Table 5]

2. Cultural properties The culture properties of TH2 and TH3 are shown in Table 5.

[0020]

[Table 6]

3. Physiological Properties The physiological properties of TH2 and TH3 are shown in Table 6- (1) and Table 6- (2).

[0022]

[Table 7]

[0023]

[Table 8]

4. Other properties are shown in Table 7, and chemotaxonomic properties are shown in Table 8.

[0025]

[Table 9]

[0026]

[Table 10]

Regarding the bacteria having the above-mentioned mycological properties, Bergey's Manual of Systemati
c Bacteriology, Vol. 2, 1986). From the above results, both of the two strains were Bacillus ( Ba
cillus) was identified as a bacterium belonging to the genus, respectively, Bacillus sp. (Bacillus sp.) TH2, Bacillus sp. (Bacillus sp.) TH3 was named, in 1993 September 1, dated on the basis of the Budapest Treaty, respectively FERM BP-4397 and FERM BP-4398 at the Institute of Biotechnology, Industrial Technology Institute
Deposited as.

The medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms,
L-proline is hydroxylated to give cis-3-hydroxy-L-
Either a natural medium or a synthetic medium may be used as long as it is a medium capable of efficiently culturing a microorganism having an activity of catalyzing a reaction for producing proline. The carbon source may be one that can be assimilated by each microorganism, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysates, acetic acid, organic acids such as propionic acid, Alcohols such as ethanol and propanol are used.

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

As the inorganic substance, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, 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 96 hours. PH during culture is 5.0 to 9.0
Hold on. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

The amount of enzyme activity in the reaction solution of these enzyme sources is determined by the amount of the substrate used, etc.
10,000,000 U / l, preferably 1,000-
It is 2,000,000 U / l. When using the microbial cells and the treated product of the microbial cells, the concentration is usually 1 to 300 g / l for wet microbial cells. The enzyme activity is expressed as 1 unit (U) which is an activity of producing 1 nmol of cis-3-hydroxy-L-proline in 1 minute under the following measurement conditions. 5
mM L-proline, 5 mM 2-ketoglutarate, 1
The enzyme preparation is added to 100 mM TES buffer solution (pH 7.0) containing mM ferrous sulfate and 5 mM L-ascorbic acid to make 100 μl in total and reacted at 35 ° C. for 10 minutes. After the reaction solution is heated at 100 ° C. for 2 minutes to stop the reaction, the cis-3-hydroxy-L-proline produced in the reaction solution is quantified using high performance liquid chromatography.

Any method can be used for the quantification as long as it can quantify cis-3-hydroxy-L-proline. For example, a post-column derivatization method or reaction solution usually using high performance liquid chromatography is usually used. The target compound in the mixture was previously prepared by using NBD chloride (7-chloro-4-nitrobenzo-2-oxa-1,3-diasol) to give NB
A method in which D-derivatization is carried out, and this is subjected to reverse-phase chromatography using high performance liquid chromatography to separate the NBD-derivatized product and then quantified using its fluorescence (excitation wavelength 503 nm, fluorescence wavelength 541 nm) (pre-column derivatization method) And so on. The detection by the precolumn derivatization method is carried out by the method of William J. Lind Blood and Robert F. Diegelman [Analytical Biochemis.
try), 138 , 390, (1984)]. The concentration of L-proline used for the hydroxylation reaction of L-proline to cis-3-hydroxy-L-proline is
It is 1 mM-2M.

Divalent iron ions are required for the reaction, and usually 1 to 100 mM is used. As divalent iron ions,
Any material can be used as long as it contains divalent iron and does not inhibit the reaction. Examples thereof include sulfides such as ferrous sulfate, chlorides such as ferrous chloride, ferrous carbonate, and organic acid salts such as citrate, lactate, and fumarate. .

Although 2-ketoglutaric acid is required for the reaction, 2-ketoglutaric acid may be added to the reaction solution, or 2-ketoglutaric acid may be added depending on the metabolic activity of the cells to be used and the processed product of the cells. A compound that can be converted to ketoglutaric acid may be used. Examples of such compounds include sugars such as glucose, glutamic acid, succinic acid and the like. These compounds may be used alone or in combination of two or more.

The aqueous medium includes water, phosphates, carbonates, acetates, borates, citrates, buffers such as Tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, and acetone. An aqueous solution containing an organic solvent such as ketones and amides such as acetamide. The reaction may be carried out in a culture of the above-mentioned microorganism having an activity of catalyzing the reaction of hydroxylating L-proline to produce cis-3-hydroxy-L-proline, or separated from the culture. It may be carried out in an aqueous medium using the microbial cells or a treated product of the microbial cells or a purified enzyme or a crude enzyme.

As the treated cells, dried cells, freeze-dried products, surfactant and / or organic solvent treated products, enzyme treated products, ultrasonic treated products, mechanically ground treated products, solvent treated products , Protein fractions of bacterial cells, immobilized products of bacterial cells and treated cells, and the like. Further, enzymes having hydroxylase activity extracted from the cells, purified preparations of these enzymes, immobilized products and the like are also used. The reaction is usually at a temperature of 15-50.
C., pH 6.0 to 9.0 for 1 to 96 hours. If necessary, a surfactant or an organic solvent is added during the cell treatment or reaction. As the surfactant, polyoxyethylene stearylamine (for example, Nimein S-21
5, manufactured by NOF CORPORATION, etc., cetyltrimethylammonium bromide, cation FB, cation F2-40E
Cationic surfactants such as, sodium oleyl amide sulfate, Nurex TAB, anionic surfactants such as Ravisol 80, polyoxyethylene sorbitan monostearate (for example, nonion ST221)
And amphoteric surfactants such as PB, hexadecyldimethylamine, and the like, and any one that accelerates the reaction can be used. These are usually 0.
It is used at a concentration of 1 to 50 mg / ml, preferably 1 to 20 mg / ml.

As the organic solvent, toluene, xylene,
Aliphatic alcohol, benzene, ethyl acetate, etc. are used. Usually 0.1 to 50 μl / ml, preferably 1 to 20 μ
Used at a concentration of l / ml. As a method for recovering cis-3-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 cis-3-hydroxy-L-proline had ¹³ C-NMR spectrum, ¹ H-NMR spectrum,
The structure can be confirmed by usual analytical means such as mass spectrum and specific rotation.

Next, a method for obtaining the L-proline 3-position hydroxylase of the present invention will be described. The enzyme cultivates a microorganism having an ability to produce L-proline 3-hydroxylase, and causes L-proline 3-hydroxylase to be produced and accumulated in the culture. Obtained by collecting the enzyme. As long as it is a microorganism capable of producing L-proline 3-position hydroxylase, any microorganism such as a wild strain, a subculture thereof, a mutant or a derivative can be used. Suitable examples include Streptomyces (Streptomyces) genus or Bacillus (Bacillus)
Microorganisms belonging to the genus and having the ability to produce L-proline 3-hydroxylase are mentioned. Streptomyces Kanusu that specifically described above (Streptomyces c anus) AT
CC 12647 and ATCC 12646, Streptomyces sp cell (Streptomyces sp.) TH1, Bacillus sp. (Ba
Cillus sp.) TH2 and TH3, or subcultures, mutants or derivatives of these strains.

The medium for culturing such a microorganism contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism, and efficiently cultivates the microorganism having the ability to produce L-proline 3-position hydroxylase. Either a natural medium or a synthetic medium may be used as long as it can be carried out in a desired manner. The carbon source may be one that can be assimilated by each microorganism, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysates, acetic acid, organic acids such as propionic acid, ethanol,
Alcohols such as propanol are used.

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

As the inorganic substance, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, 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 96 hours.

During the culture, the pH is maintained at 5.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. During the culture, L-proline may be added if necessary.

In order to isolate and purify the enzyme from the culture broth, conventional enzyme isolation and purification methods may be used. For example, a cell-free extract is obtained by centrifuging the culture solution to collect the cells, and then ultrasonically crushing or mechanically crushing using French press, Mantongaurin, Dynomill or the like. The supernatant after centrifugation is salted out with ammonium sulfate, anion exchange chromatography such as DEAE (diethylaminoethyl) -sepharose, hydrophobic chromatography such as butyl cellulose and phenyl cellulose, gel filtration using a molecular sieve, etc. The purified enzyme preparation is obtained by performing an electrophoresis method such as electric focusing. The physicochemical characteristics of the obtained enzyme preparation can be specified by a usual enzymatic method.

The L-proline 3-position hydroxylase thus obtained has the following physicochemical properties (1) to (11). (1) Action and substrate specificity It acts on free L-proline in the presence of 2-ketoglutarate and divalent iron ion to give cis-3-hydroxy-L.
-Produces proline. (2) Optimum pH 30 ° C., 20 minutes reaction, pH 6.5-7.5
It has an optimum pH. (3) pH stability It is kept stable in the pH range of 6.5 to 8.0 by treatment at 4 ° C for 23 hours. (4) Optimum temperature It has an optimum temperature of 35 to 40 ° C in a reaction at pH 7.0 for 15 minutes. (5) Temperature stability It is inactivated by treatment at pH 7.5 at 50 ° C for 30 minutes. (6) Inhibitors Zn ⁺⁺ , Cu ⁺⁺ , Co ⁺⁺ and Ba ⁺⁺ metal ions and EDT
Inhibited by A. (7) Activation No coenzyme is required for activation. The addition of L-ascorbic acid to the reaction solution accelerates the reaction. (8) 5 mM in TES buffer (pH 7.0) with Km value of 100 mM
K for L-proline measured in a reaction solution containing L-ascorbic acid, 1 mM ferrous sulfate and enzyme preparation
The m value is 0.49 mM and the Km value for 2-ketoglutaric acid is 0.11 mM. (9) Isoelectric point fast system (Phast system; made by Pharmacia)
The isoelectric point measured in 4.3 is 4.3. (10) Molecular weight: 35,000 ± 5,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (11) N-terminal amino acid sequence It has an N-terminal amino acid represented by SEQ ID NO: 1.

Examples of the present invention will be shown below.

[0046]

【Example】

Example 1. Production of cis-3-hydroxy-L-proline SR3 medium [glucose 1.0%, soluble starch 1.
0%, yeast extract 0.5%, tryptone 0.5%,
Meat extract 0.3% and magnesium phosphate 0.05
% Of which was adjusted to pH 7.2 with 6N NaOH] was dispensed into test tubes (diameter 25 mm x 200 mm) in 10 ml aliquots and sterilized at 120 ° C for 20 minutes. Add HT to this medium
Agar plate medium [soluble starch 1%, NZ amine 0.2
%, Yeast extract 0.1%, meat extract 0.1% and agar 1.5%, and cultivated in a medium sterilized at 120 ° C for 20 minutes after adjusting the pH to 7.2 with 6N NaOH]. ( Streptomyces sp.) TH1
Was inoculated with 1 platinum loop, shake-cultured at 28 ° C. for 2 days, and used as a seed culture. The seed culture was further dispensed into a 2-liter Erlenmeyer flask, inoculated into 200 ml of SR3 medium sterilized at 120 ° C. for 20 minutes, and shake-cultured at 28 ° C. for 2 days.
This culture solution was dispensed into a 5-liter jar fermenter, and a 2-liter Df3 medium [soluble starch 5%, corn steep liquor 3.0%, monopotassium phosphate was used.
It contains 0.05%, magnesium sulfate heptahydrate 0.05% and calcium carbonate 0.5%, pH 7 with 6N NaOH.
Aseptically inoculated into the medium adjusted to 0], 700 rpm,
The cells were cultured at 28 ° C for 2 days under the condition of 1 vvm. The pH during culture was adjusted. 15,000 of the obtained culture solution
× g, centrifuge at 4 ° C for 10 minutes to remove wet cells and culture medium 1
Obtained 75 g per liter. The wet cells were washed with physiological saline at 4 ° C, centrifuged, and stored frozen at -80 ° C until use.

1.0 g of the obtained wet cells was added to 10 ml of the reaction solution (a) [5 mM L-proline, 5 mM α-ketoglutarate, 5 mM L-ascorbic acid and 1 mM.
100 mM TES buffer containing ferrous sulfate (pH
In 1.5), a nymein solution (4 g of nymein S-215 (manufactured by NOF CORPORATION) dissolved in 10 ml of xylene) was added at 1.4% (v / v)] and reacted at 30 ° C. for 5 hours. It was

After the reaction, the bacterial cells were removed by centrifugation from the bacterial cell reaction solution, and the cis-3-hydroxy-L-proline produced in the supernatant was analyzed. The analysis was performed by high performance liquid chromatography using a post-column derivatization method under the following conditions. For detection, elute the target compound on the column,
On-line NBD derivatization was performed using fluorescence of NBD derivatized products. High Performance Liquid Chromatography Analysis Conditions [1] Equipment: 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 Detection Vessel RF-550A [2] Column used: SUMCHIRAL OA5000 (Sumitomo Chemical Analysis Center Co., Ltd.) (diameter 4.5 mm × 250 mm) [3] Analysis conditions: 1) Mobile phase: 1 mM copper sulfate aqueous solution 2) Same as above Flow rate: 1.0 ml / Min 3) Column temperature: 38 ° C 4) Buffer: 0.3M Borate buffer (pH 9.6) 25mM EDTA 5) Same as above Flow rate: 0.2 ml / min 6) NBD chloride solution: 0.5g / l Methanol solution 7) Same as above Flow rate: 0.5 ml / min 8) Reaction temperature: 60 ° C. 9) Reaction time: Approximately 3 minutes 10) Detection wavelength: Excitation wavelength 503 nm Fluorescence wavelength 541 nm 11) Sample: 10 μl As a result, reaction solution Cis-3-hydroxy -L- proline 910μM (119mg / l) was produced in.

Example 2. Purification of cis-3-hydroxy-L-proline 100 g of the wet microbial cell obtained in Example 1 was suspended in 1 liter of reaction solution (a) and stirred in a 2 liter beaker while stirring,
The reaction was carried out at 30 ° C. for 5 hours. After the reaction, cis-3-hydroxy-L-proline produced in the supernatant obtained by centrifuging the bacterial cells from the bacterial cell reaction solution was analyzed. As a result, 809 μM (106 mg / l) cis- was added in the reaction solution.
3-Hydroxy-L-proline had been produced.

After adjusting the pH of the reaction solution supernatant to 4.5, the ion exchange resin DIAION SK1B (NH ₄
⁺ Type, manufactured by Mitsubishi Kasei Co.) 200 ml. Cis-3
The fraction containing -hydroxy-L-proline was concentrated under reduced pressure and then passed through 20 ml of ion exchange resin Diaion PA412 (OH ^- type, manufactured by Mitsubishi Kasei Co., Ltd.). The fraction containing cis-3-hydroxy-L-proline was concentrated under reduced pressure and then the pH was adjusted 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. , A miscellaneous primary amino acid was converted to o-phthalaldehyde. Sepa beads SP207
(Manufactured by Mitsubishi Kasei Co., Ltd.), and passed through 10 ml of o-phthalaldehyde-modified primary amino acid and cis-3-hydroxy-
L-proline was isolated. Cis-3-hydroxy-L-
Fraction containing proline was concentrated under reduced pressure and then 20 ml of ion exchange resin Diaion PA412 (OH ^- type, manufactured by Mitsubishi Kasei)
After passing through the column, a fraction containing cis-3-hydroxy-L-proline was obtained. This fraction was concentrated and dried to obtain 68 mg of white crystals of cis-3-hydroxy-L-proline (yield 63.
%).

The physicochemical properties of the above compound are as follows:
Is. Specific rotation: [α]_D ^{twenty one}= −93.4 ° (c = 0.50
3, H₂O) FAB-Mass spectrum: 132 (M + H)^{+ 13} C-NMR spectrum (D₂O 2, 125 MHz) ppm:
33.9, 44.5, 68.3, 71.6, 171.3¹ H-NMR spectrum (D₂O, 500MHz) ppm:
2.18 (1H), 2.27 (1H), 3.52 (1
H), 3.62 (1H), 4.18 (1H), 4.77
(1H) Specific rotation of the above, molecular weight measured by mass spectrum,¹H
-NMR spectrum, ¹³C-NMR spectrum etc.
The analysis results are shown in the literature [The Journal of Biolo
Zical Chemistry (J. Biol. Chem.),2411
300 (1966)] [Journal of Antibiotics
Tix (J. Antibiotics),45, 824 (199
2)] and a chemical compound synthesized according to the method described in the literature.
Adult cis-3-hydroxy-L-proline [Liebigs
  Annalen Der Chemie (Liebigs Ann. Chem.),
1881, (1979)] [Tetrahedron (Tetrahed
ron),42, 2421 (1986)].

Example 3. Production of cis-3-hydroxy-L-proline Streptomyces canus AT
CC 12647 and ATCC 12646, Bacillus sp. (Baci
llus sp.) TH2 and TH3 were used to hydroxylate L-proline. SR3 medium [glucose 1.0%,
Soluble starch 1.0%, yeast extract 0.5%, tryptone 0.5%, meat extract 0.3% and magnesium phosphate 0.05%, and a medium adjusted to pH 7.2 with 6N NaOH] was tested. 10 ml each was dispensed into a tube (diameter 25 mm × 200 mm) and sterilized at 120 ° C. for 20 minutes. One platinum loop of each strain grown on the HT agar plate medium was inoculated into this medium, shake-cultured at 28 ° C. for 1 day, and used as a seed culture.

On the other hand, Df4 medium [glycerol 2.5
%, Glucose 2.5%, soybean meal 1.
A test tube (diameter 25 mm × 200 mm) containing 5%, 1 potassium phosphate 0.05%, magnesium sulfate heptahydrate 0.05%, calcium carbonate 0.5% and adjusted to pH 7.0 with 6N NaOH. ) And 10 ml each, and sterilized at 120 ° C. for 20 minutes. 1 ml of the seed culture was aseptically inoculated into this medium, and the mixture was shake-cultured at 28 ° C. for 1 day. 2 ml of the obtained culture solution was added to 15,000 × g, 10
Centrifuge at 4 ° C for minutes. The obtained bacterial cells are treated with 80 mM TE
After washing with S buffer (pH 7.5), it was centrifuged. The obtained wet cells were suspended in 1 ml of the reaction solution (a) and reacted at 30 ° C. for 3 hours.

As a result, in the reaction solution, 242 μM,
202 μM, 318 μM and 141 μM cis-3-hydroxy-L-proline had been produced.

Example 4. Formation of cis-3-hydroxy-L-proline Bacillus sp. TH2 and TH
3 was added to Df2 medium (soluble starch 5%, dry yeast 1.%, added with 1 g / l of L-proline instead of Df4 medium).
5%, 1 potassium phosphate 0.05%, magnesium sulfate heptahydrate 0.05%, calcium carbonate 0.5%, and a medium adjusted to pH 7.0 with 6N NaOH]
In the same manner as in Example 3, the seed culture was performed for 1 day and the Df2 medium was used for 1 day. As a result, TH2 strain was 745μ in the culture supernatant.
M (97.6 mg / l), TH3 strain with 327 μM (4
2.8 mg / l) of cis-3-hydroxy-L-proline had been produced.

Example 5 Isolation and purification of L-proline 3-hydroxylase (1) Preparation of cell-free extract After dissolving 30 g of the frozen cells obtained in Example 1 , 200 ml of buffer solution (A) [1 mM Dithiothreitol (DTT),
0.2 mM EDTA, 0.1% (v / v) Tween 20 (Tw
een 20) and 10% (v / v) glycerol 2
The suspension was suspended in 0 mM piperazine buffer (pH was adjusted to 5.3 with 6N HCl) under ice cooling. The bacterial cells in the suspension were ultrasonically disrupted under ice cooling and centrifuged at 4 ° C., 30,000 × g 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. Acid treatment After adjusting the pH of the supernatant obtained in the above step to 4.5 with 6N hydrochloric acid, the resulting precipitate was centrifuged at 15,000 g for 30 minutes. It was removed and the supernatant was obtained.

(2) -2. Resource Q column chromatography (I) The supernatant obtained in the above step was equilibrated with the buffer solution (A), and a Resource Q column manufactured by Pharmacia (RESOURCE ^™ Q, 6 ml) was used.
Passed through. After washing with buffer (A), the fraction containing the enzyme was eluted with a linear concentration gradient of sodium chloride between 0 and 0.3 M prepared in buffer (A).

(2) -3. Resource Q column chromatography (II) The active fraction obtained in the above step was added to the buffer solution (B) [1 mM DT
T, 0.2 mM EDTA and 10% (v / v) glycerol in 20 mM TES buffer (pH adjusted to 7.5)] and then diluted 3-fold, and equilibrated with buffer (B) manufactured by Pharmacia Resource Q column (RESOURCE ^TM Q, 1ml)
Passed through. After washing with buffer (B), the fraction containing the enzyme was eluted using a linear concentration gradient of sodium chloride between 0 and 0.3 M prepared in buffer (B).

(2) -4. Phenyl Sepharose Chromatography A phenyl sepharose column equilibrated with a buffer solution (B) containing 2M sodium chloride was prepared by adding and dissolving 2M sodium chloride to the active fraction obtained in the above step. (Phenyl Sepharose HP HiLoa
d, 1 ml). After washing with a buffer solution (B) containing 2M sodium chloride, a fraction containing the enzyme was eluted with a buffer solution (B) containing 0.1% (v / v) Tween 20.

(2) -5. Resource Q Column Chromatography (III) The active fraction obtained in the above step was desalted using a Pharmacia PD-10 column equilibrated with the buffer solution (A), and then buffered. liquid
It was passed through a Resource Q column (RESOURCE ^™ Q, 1 ml) manufactured by Pharmacia Co., which was equilibrated with (A). After washing with buffer (A), the fraction containing the enzyme was eluted with a linear concentration gradient of sodium chloride between 0 and 0.3 M prepared in buffer (A).

(2) -6. Resource Q Column Chromatography (IV) The active fraction obtained in the above step was treated with a buffer solution (B) containing 0.1% (v / v) Tween 20. After diluting 3 times,
Pharmacia resource Q equilibrated with buffer (B)
It was passed through a column (RESOURCE ^™ Q, 1 ml). Buffer solution (B)
After washing with, the fraction containing the enzyme was eluted using a linear concentration gradient of sodium chloride in the buffer solution (B) between 0 and 0.3M. The outline of isolation and purification of L-proline 3-hydroxylase is summarized in Table 9.

[0063]

[Table 11]

Example 6. Properties of L-proline 3-position hydroxylase (1) Analysis by electrophoresis The purified enzyme preparation obtained in Example 5 was subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis (polyacrylamide PAGEL NPU-12.5L manufactured by ATTO). And Bio-Rad SDS-PAGE Molecular Weight Stan
dard Broad Range). As a result, it was revealed that this enzyme is composed of a substantially uniform subunit having a molecular weight of about 35,000 ± 5,000. (2) Properties relating to enzymatic reaction By using the following reaction solutions, a substrate omission test (omission test) and an additive test of reaction components are carried out to obtain an essential compound and an accelerating compound for the L-proline 3-hydroxylase reaction. , Inhibitory compounds were investigated.

The basic reaction solution composition was 100 mM TES buffer solution (pH 7.0) containing 5 mM L-proline, 5 mM 2-ketoglutarate, 1 mM ferrous sulfate, 5 mM L-ascorbic acid and enzyme preparation. Then, the total liquid volume was 100 μl. The reaction is started by addition of enzyme and reacted at 35 ° C. for 10 minutes. 100 reaction mixture
After the reaction was stopped by heating at ℃ for 2 minutes, the cis-3-hydroxy-L-proline produced in the reaction solution was quantified by high performance liquid chromatography by the 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) o −
Add 16 μl of ethanol solution of phthalaldehyde and add 6
It was left at 0 ° C. for 30 seconds. Further, 50 μl of an ethanol solution of 2% (w / v) NBD chloride was added, and the mixture was kept at 60 ° C. for 4 hours.
Reacted for 0 minutes. After the reaction was stopped by adding 30 μl of 1N hydrochloric acid, the precipitate was removed by centrifugation and filter filtration, and analyzed by high performance liquid chromatography to quantify the cis-3-hydroxy-L-proline produced. High performance liquid chromatography was performed under the following conditions. 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
As a result, L-proline, 2-ketoglutarate, and Fe ⁺⁺ ion are essential for the L-proline 3-hydroxylase reaction, and L-ascorbic acid promotes the reaction, and Zn ⁺⁺ , C
Addition of u ⁺⁺ , Co ⁺⁺ and Ba ⁺⁺ ions and EDTA inhibited the reaction. The results are shown in Table 10.

[0066]

[Table 12]

(3) Optimal pH In the method for measuring the activity of L-proline 3-hydroxylase, the buffer solution component in the reaction solution was adjusted to MES [2
-(N-morpholino) ethanesulfonic acid] buffer, pH
6.5 to 7.5 are PIPES [piperazine-N, N'-bis (2-ethanesulfonic acid) buffer solution, pH 7.0 to 8.0
Was replaced with TES buffer solution, and pH 8.0-9.0 was replaced with TAPS [N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid] buffer solution.
At 5 to 7.5, 80% or more of the maximum activity was shown. The results are shown in Table 11.

[0068]

[Table 13]

(4) The enzyme solution in the pH stability buffer (B) was added to a 100 mM buffer [pH 5.
5-6.5 is MES buffer, pH 6.1-7.5 is PIPES
Buffer solution, pH 7.0-8.0 is TES buffer solution, pH 8.0.
0-9.0 is TAPS buffer] and diluted 3 times, 4 ℃, 23
After holding for a period of time, the activity was measured. pH 6.5-8.0
The enzyme retained in the above range had an activity of 80% or more of the activity before the retention, and the activity was kept stable in the range of pH 6.5 to 8.0. (5) Optimum temperature L-proline 3-position hydroxylase activity measurement, 15
As a result of performing the reaction for 15 minutes in the temperature range of -50 ° C, 35
It exhibited 80% or more of the maximum activity at -40 ° C. The results are shown in Table 12.

[0070]

[Table 14]

(6) Temperature stability The present enzyme was added to the buffer (B) at a temperature range of 0 to 60 ° C. for 30 days.
The enzyme was found to be 50
It was completely deactivated by the treatment at 30 ° C for 30 minutes. (7) Km value 100 mM TES buffer solution (pH 7.0), K for L-proline measured in a reaction solution containing 1 mM ferrous sulfate, 5 mM L-ascorbic acid and enzyme preparation
m value is 0.49 mM, Km for 2-ketoglutarate
The value was 0.11 mM. (8) Isoelectric point fast system (Phast system; made by Pharmacia)
As a result of measuring the isoelectric point with, the isoelectric point was 4.3. (9) N-terminal amino acid sequence Protein sequencer model PPSQ-10 (manufactured by Shimadzu Corporation)
The following results were obtained as a result of analyzing the N-terminal amino acid sequence of this enzyme protein using.

[0072]

[Chemical 1]

Example 7. Hydroxylation of L-proline Using the purified enzyme preparation obtained in Example 5, L-proline was hydroxylated. 20 mM 2-ketoglutarate,
200 mM TES buffer (pH 7.0) containing 20 mM L-proline, 5 mM L-ascorbic acid, 1 mM ferrous sulfate and 106 U of purified enzyme preparation 10.
The reaction was carried out with 0 μl at 35 ° C. for 30 minutes. As a result, the reaction solution contained 18 mM (2.4 g / l) of cis-
3-Hydroxy-L-proline was produced.

[0074]

[Sequence list]

[0075] SEQ ID NO: 1 Length of sequence: 29 SEQ type: amino acid Topology: linear sequence type: peptide Origin Organism: Streptomyces sp (Streptomyces
sp.) Strain name: TH1 (FERM BP-4399) Sequence Met Arg Ser His Ile Leu Gly Arg Ile Glu Leu Asp Gln Glu Arg Leu Gly 1 5 10 15 Arg Asp Leu Glu Tyr Leu Ala Thr Val Pro Thr Val 20 25

[0076]

INDUSTRIAL APPLICABILITY According to the present invention, L-proline industrially produced from a sugar source by a fermentation method is directly microbially or enzymatically hydroxylated to industrially advantageously produce cis-3-. A method for producing hydroxy-L-proline and a novel L-proline 3-position hydroxylase useful in the method can be provided.

─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Yoichi Uozaki 3-9-11 Nakamachi, Machida City, Tokyo (56) References Biochem. J. , Vol. 206 (1982), p. 499-503 Biochem. Biophys. Res. Commun. , Vol. 76, No. 2 (1977), p. 275-281 Appl. Environ. Micr obiol. , Vol. 62, No. 6 (1996), p. 1903-1907 J.I. Bacteriol. , Vol. 179, No. 18 (1997), p. 5677-5683 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 13/00-13/24 C12N 9/00-9/99 CA / REGISTRY (STN) BIOSIS / WPI (DIALOG) PubMed

Claims (10)

(57) [Claims] 1. A belonging to Streptomyces (Streptomyces) genus or Bacillus (Bacillus) genus and culture of a microorganism which catalyzes the hydroxylation reaction L- proline at the 3-position of the position of L- proline, the culture The bacterial cell isolated from the above, a treated product of the bacterial cell, an enzyme source selected from purified enzyme and crude enzyme, divalent iron ion, 2-ketoglutaric acid and L-proline are allowed to exist in an aqueous medium, and L-proline is cis. −
Cis-3-Hydroxy-L, characterized in that it is converted into 3-hydroxy-L-proline and the resulting cis-3-hydroxy-L-proline is collected from the aqueous medium.
-Proline production method. 2. The microorganism is Streptomyces canus ATCC12647 or ATCC12646, Streptomyces sp.
1 (FERMBP-4399), Bacillus s
p.) TH2 (FERM BP-4397) or TH3 (FERM BP-4398)
The method according to claim 1, wherein 3. The method according to claim 1, wherein the conversion of L-proline to cis-3-hydroxy-L-proline is carried out in a culture medium of a microorganism. 4. The production method according to claim 1, wherein the enzyme source is L-proline 3-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-ketoglutarate and divalent iron ion to give cis-3-hydroxy-L.
-Produces proline. (2) Optimum pH It has an optimum pH of pH 6.5 to 7.5 in a reaction at 30 ° C. for 20 minutes. (3) pH stability It is kept stable in the pH range of 6.5 to 8.0 by treatment at 4 ° C for 23 hours. (4) Optimum temperature In the reaction at pH 7.0 for 15 minutes, the optimum temperature is 35 to 40 ° C. (5) Temperature stability It is inactivated by treatment at pH 7.5, 50 ° C. for 30 minutes. (6) Inhibitors Inhibited by metal ions of Zn ⁺⁺ , Cu ⁺⁺ , Co ⁺⁺ and Ba ⁺⁺ and ethylenediaminetetraacetic acid. (7) Activation No coenzyme is required for activation. The addition of L-ascorbic acid to the reaction solution accelerates the reaction. (8) N-tris (hydroxymethyl) methyl-having a Km value of 100 mM
The Km value for L-proline measured in a reaction solution containing 5 mM L-ascorbic acid, 1 mM ferrous sulfate and the enzyme preparation in 2-aminoethane sulfonate buffer (pH 7.0) was 0.49 mM. And the Km value for 2-ketoglutaric acid is 0.11 mM. (9) Isoelectric point The isoelectric point is 4.3. (10) Molecular weight: 35,000 ± 5,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 5. L showing the physicochemical properties of the following (1) to (10)
-Proline 3-hydroxylase. (1) Action and substrate specificity It acts on free L-proline in the presence of 2-ketoglutarate and divalent iron ion to give cis-3-hydroxy-L.
-Produces proline. (2) Optimum pH It has an optimum pH of pH 6.5 to 7.5 in a reaction at 30 ° C. for 20 minutes. (3) pH stability It is kept stable in the pH range of 6.5 to 8.0 by treatment at 4 ° C for 23 hours. (4) Optimum temperature In the reaction at pH 7.0 for 15 minutes, the optimum temperature is 35 to 40 ° C. (5) Temperature stability It is inactivated by treatment at pH 7.5, 50 ° C. for 30 minutes. (6) Inhibitors Inhibited by metal ions of Zn ⁺⁺ , Cu ⁺⁺ , Co ⁺⁺ and Ba ⁺⁺ and ethylenediaminetetraacetic acid. (7) Activation No coenzyme is required for activation. The addition of L-ascorbic acid to the reaction solution accelerates the reaction. (8) N-tris (hydroxymethyl) methyl-having a Km value of 100 mM
The Km value for L-proline measured in a reaction solution containing 5 mM L-ascorbic acid, 1 mM ferrous sulfate and the enzyme preparation in 2-aminoethane sulfonate buffer (pH 7.0) was 0.49 mM. And the Km value for 2-ketoglutaric acid is 0.11 mM. (9) Isoelectric point The isoelectric point is 4.3. (10) Molecular weight: 35,000 ± 5,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 6. Streptomyces (Streptomyces) belonging to the genus or Bacillus (Bacillus) genus, and culturing a microorganism having an ability to produce 3-hydroxylase enzyme L- proline, cultures in L- proline 3-position A method for producing L-proline 3-position hydroxylase, which comprises collecting and accumulating hydroxylase and collecting L-proline 3-position hydroxylase from the culture. 7. A microorganism having a Streptomyces (Streptomyces) genus or Bacillus (Bacillus) belonging to the genus, and ability to produce 3-hydroxylase enzyme L- proline, Streptomyces Kanusu (Streptomyces canus) ATCC
12647 and ATCC12646, Streptomyces sp. TH1 (FERM BP-4399), and Bacillus sp. TH2 (FERM BP-439)
7. The method for producing L-proline 3-position hydroxylase according to claim 6, which is a microorganism selected from 7) and TH3 (FERM BP-4398). 8. Streptomyces sp. TH1 (F) having the ability to hydroxylate L-proline to produce cis-3-hydroxy-L-proline.
ERM BP-4399). 9. An active Bacillus sp. TH2 (FER) having the ability to hydroxylate L-proline to produce cis-3-hydroxy-L-proline.
M BP-4397). 10. L-proline is hydroxylated to give cis-3-.
Bacillus sp. TH3 (FERM BP-439 having the ability to produce hydroxy-L-proline
8).