JPS6112296A - Production of l-phenylalanine - Google Patents

Abstract

PURPOSE:To produce L-phenylalanine, in high efficiency, by adding 5-benzylhydantoin to a culture liquid of Flavobacterium sp I-3 strain capable of producing L-phenylalanine from 5-benzylhydantoin. CONSTITUTION:Flavobacterium sp I-3 strain (FERM-P No.6901) capable of producing L-phenylalanine from D-, L- or DL-5-benzylhydantoin is cultured in a nutrient medium. The obtained culture liquid, bacterial cells collected from the culture liquid, or treated bacterial cells are made to react with D-, L- or DL-5- benzylhydantoin at about 20-60 deg.C and about 6-10pH. The objective L-phenylalanine important for nutritional and pharmaceutical purposes can be obtained by separating and purifying the produced L-phenylalanine by ion exchange resin treatment, etc.

Description

[Detailed description of the invention]

(Industrial Application Field J The present invention relates to a method for producing 1.-7 enylalanine from ID-, L- or DL-5-benzylhydantoin.

[Prior art 1] L-'phenylalanine is one of the essential amino acids and is a substance of nutritional and pharmaceutical importance. 5-benzylhydantoin, which is a raw material for the method of the present invention, is a substance that is easily synthesized by organic synthetic chemistry.
- As a method for producing L-phenylalanine from penzylhyguntoin, Azu, Flavobacterium Ia.
Flavobacterium amino --ge
A method is known for producing L-phenylalanine by allowing FERM-P 3133 to act on 5-benzylhyf7'j-yne (Japanese Patent Publication No. 54-22).
74). (Objective of the Invention) The present invention provides a method for efficiently producing D-, L- or DL-5-benzylhydantoin mustard-phenylalanine by an enzymatic method. (Structure and Effects of the Invention) The present inventors have developed an extremely efficient method for converting 0-0L- or DL-5-benzylhydantoin into L-phenylalanine (Flavobacterium th. I-3 (Flavobacteriumllp.
I-33'i was successfully isolated from soil and the present invention was completed. Therefore, 1 the present invention is a culture of Flavobacterium sp. L-7enyl Ylanin is obtained by allowing the bacterial cells obtained or a substance treated with the bacterial cells to act on D-0L- or DL-5-penzylhyguntoin, and collecting the produced L-7enyl Ylanin. This is the manufacturing method. 7 Lab Bugterium sp. used in wood invention 1-
:3 Bacterium is a new fungal species newly isolated by Kiwata & et al., and its mycological properties are as follows. In addition, 1 new Sui species Flavobacterium sp. 1nee 31idl'
It has been deposited as gRM-P6901 (Fiber Science and Technology Research Institute No. 6901). lal Morphology Cell shape and size: Bacillus, 0.4-0.9 X 1
゜0~2.0μ Cell A-form: None Motility: Yes 0 periflagellates 1 bubble: Not formed Durham staining: Negative acid-fastness: None fbl Growth status in each medium (1) Broth agar plate culture: Growth Growth is moderate, gold-rimmed, slightly ridged, 0 surface is faded, opaque, dull brown, glossy (2) Meat juice agar slant culture 2 Growth is moderate, filamentous, dark brown, glossy (3) Meat juice liquid culture: No film formation, precipitate formation, inner layer turbidity, ring formation along the tube wall (4) Meat juice gelatin puncture culture: Liquefies. (5) Litmus mill:
+I Thomas will return the money. ial to liquefy Physiological properties (1) Reduction of nitrate: Reducing.・ (2) Denitrification reaction: Negative +31 MR test: Negative 141VP test: Negative (5) Indole production: Negative: 6) Hydrogen sulfide production: Negative (7) Starch hydrolysis: Negative (8) Citric acid Usage: Co-f-medium, Simon's medium, Christensen's medium (
Use it even if it's off. 19) Utilization of inorganic nitrogen source: 6t [salt and ammonium salt gold are used. (11 color pigment formation: 11.11 that does not generate water-soluble pigments)
Urease: negative / 1z oxidase: weakly positive) 13) Catalase: positive (14)lt * range: 'at pH 6-937°C'
15) Attitude towards oxygen: bloody, anaerobic +161 t
J-F test dioxidative 117) Production of acid and gas from affl: As described above, ■-3 Mycological and beach characteristics of bacteria include (1) Bacillus 12) Motility, (3) Durham negative, (4) Colonies are yellow, (5) catalase positive, (6)
Anaerobic/facultative, (7) Gradually decomposes glucose oxidatively. etc. are set back. Based on these properties, B er g @ 7'B Man u alof D
eterminative Bacteriology
When searching based on the 8th edition, it is determined that the I-3 bacterium belongs to Flavobacterium CFlavobactrium) 1fi4. However, even if you search the history for species included in the genus Labobacterium, the above Ber
In gθy's mnual 8th edition, there is no description of bacterial species that match bacteria 1-3. According to the description in Bergey's Manual K 8th edition, among the 12m species belonging to the 7 labobacterium genus,
The motile species is Flavobacterium ligense (F.
rigen+ie + , Flavobacterium indoltheticum (It'-indoltheticum)
) + 7 Labobacterium tyrenicum (F 1 t
irrenicum) and 7 Labobacterium e devorans (F-dsvorans).However, 7 Labobacterium regenset is clearly different because ■-3kan does not grow at 37°C.7 Labobacterium・It differs from Indolceticum in its ability to hydrolyze starch and reduce sucrose and D-glucose. They differ in the ability to produce acid from sugar and maltose and the ability to produce nitrates.Furthermore, although it is not described in the 8th edition of Bergey's Manual, L- Flavobacterium aminogenes (F. amino8snegl), which has been described as having an enzyme that produces 7-enylalanine.
It differs from i' E RM-P 3133 in terms of motility, availability of citric acid in Cordy's medium, and availability of acetic acid source. Based on the above, I-3 bacteria was recognized as a new m4ft belonging to 7Labobacterium 4, and 7Labobacterium sp-3 (F'lavobacterium 8p I
-3). As a medium for culturing the microorganisms used in the present invention, a conventional medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, an amino acid hydantoin compound such as 5-benzylhydantoin can be used. As a carbon source, any carbon source that can be used by the I-3 bacteria can be used, such as glucose, lactose, sucrose, and maltose. Suitable examples include esteric acids such as dextrin, glycerine, sorbitol, and rtI alcohols. Nitrogen sources include nitrogen, such as ammonium salt, ammonium phosphate, ammonium phosphate, ammonium carbonate, etc. (ammonium salt, ammonium fumarate, ammonium citrate, Organic acid ammonium salts, etc. can be used.For example, meat extract, yeast processing, sugar, tea liquor, casein hydrolyzate, IJ, etc.It is also possible to use any natural nitrogen source. In many cases, nitrogen sources can serve as both a nitrogen source and a VC carbon source. Inorganic salts include, for example, ferrous nitrate, magnesium sulfate, mannomene sulfate, and phosphoric acid. - Potassium, disodium phosphate, sodium chloride, chloride + 1 um, etc. can be used as appropriate.
Higher activity can be obtained by adding amino acid hydantoin compounds such as -(indol-3-yl-methyl)hydantoin to the culture medium. As for culture. It can be carried out using conventional methods. For example, the pH of the medium
5-8. Preferably adjusted to 6 to 7.5. inoculated with kC. After 20-33℃. Preferably 26
~30°C. Chivalry. Stir for 16 to 72 hours. The culture solution obtained in this way is. It may be used as a source for fermentation as it is in the conversion reaction to nilalanine, D-, L- or DI,-5-benzylhydanodoin.゛Also a crude fermentation standard. Of course, purified enzyme preparations are also available. Cells collected from the culture solution or processed products of the cells [for example, freeze-dried cells, acetone-dried cells, acetone-dried cells. Ground bacterial cells. Washed bacterial cells. Autolysed product of bacterial cells. Ultrasonicated bacterial cells. Bacterial cell extract, etc.] can also be used as an enzyme source. Furthermore. Bacterial body J) Or is it a tooth body treatment? ．． For example, carrageenan gel method. F) by a known method such as the polyacrylamide method
It can also be used in a quaternary form. D-, L-again! , -f DL-5-benzylhydantoin f I, -71 nyl r The reaction for converting to lanine is as follows:
D-, L-', 7- can be carried out in an aqueous medium using DL-5-bendifrehydantoin and ff-1- of the above 1i\'A source. The reaction is carried out at a temperature of 20-6
0℃. Preferably, the temperature is 30-45°C and the pH is 6-10, preferably 8-9. l) -, I, -also, MaDL-
Concentration of 5-benzylhydantoin Jild, io, t%~
It can be used as a 30 width boat. In Q& using a high concentration of substrate, the ::ni' is completely 1 and does not dissolve in the solvent and becomes an insoluble substance, and C does not interfere with the upward progression of ;f in this reaction. Furthermore, Enzyme Source Toji-C1! (2) When using standardized bacterial cells, a continuous method may be used in which the purified bacterial cells are packed into a column and then poured into a solution containing a substrate. In addition, in the method of the present invention, a surfactant (for example, cetyl I-IJ methyl ammonium bromide,
triton x-ioo, etc.], dimethyl sulfoxide, etc.),

[For example, pyridoxal phosphate, etc., inorganic ions (m) such as manganese ions, magnesium ions, etc.] can be added to shorten the reaction time or increase the amount of L-phenylalanine accumulated. It may be valid. Thus, 1 to 100 hours after the start of the reaction, the provided D-, L
-Also, 1dDL~5-benzylhydantoin is a complete KL-
It is converted to phenylalanine and accumulated in one reaction solution. The produced L-phenylalanine (by ordinary crystallization method,
Separation and purification can be achieved by appropriately using ion exchange (supporting) lipids and other known methods. . Hereinafter, the method of the present invention will be explained in detail with reference to Examples. Incidentally, the stationary liquid chromatography of L-phenylalanine in the Examples was carried out by the Tahi-o-Hussey method using Hiroco/Stock metsucentiloides p-60. Confirmation of L-phenylalanine was carried out by completely comparing the specific optical rotation NMR, IR spectrum, etc. of the crystal L-element analysis value obtained from one reaction solution with that of the standard product. Example 11 11) Culture of inoculum Glucose 5%, ammonium heptamalate 0.1%, potassium phosphate 0.3<, dipotassium phosphate 0°7x, magnesium sulfur heptahydrate o, oi %, DL-5-+(
A medium containing 0.2% of hydantoin was adjusted to pH 7.0. Put 50 Wt of the medium into a 500-Yosaka flask. Sterilization was performed at 120°C for 10 minutes. In addition, Flavobacterium sp.
- Inoculate one platinum loop of 3 bacteria (Feikoken Bacteria No. 69o1),
24 hour rise at 30℃ (14Q rpm, amplitude 7C)
1+3 culture was performed. (2) Main culture dextrin 2%, ammonium chloride 0.4%. Yeast extract 0.1%, peptone 0.1%, phosphoric acid -
Potassium 0.3%, dipotassium phosphate 0.7%, magnesium sulfate 7' hydrate 0.01%, @ferric acid heptahydrate 0.001%, manganese sulfate o, ooi sulfur, DL-
5-(indol-3-ylmethyl)hydantoin 0
．． A medium containing 5% was adjusted to pH 7.0, and the medium containing 5%
0- in a 500-Yosaka flask and heated at 120℃ for 10
Sterilized for minutes. 0.5 of the seed culture solution obtained in (1)
4 was inoculated and cultured at 30°C for 24 hours. (3) 0L-5-benzylhydantoin 1.
(Add 5y and hydroxide) Adjust the liquid to pH 8.5 with IIium.
After adjusting the temperature, the reaction was carried out at 37°C for 16 hours. After the end of the reaction, L-phenylalanine 1゜3y- was produced and accumulated in the reaction solution (conversion rate: 98%).The reaction solution was sterilized by centrifugation, and then concentrated to extract L-phenylalanine. 1.39'i of crude crystals were obtained. The crude crystals were dissolved in a small amount of aqueous ammonia, neutralized with acetic acid to crystallize, and left in the refrigerator overnight, and the crystals were collected by filtration to obtain L. Obtained 1.1 crystals of -7 enylalanine. Yield: 84 curses [α
) D = -33,8° (C = 2. Water) Example 2 Example 1 - (11, (21) The bacterial cells were collected by centrifugation from the main culture solution 5° obtained in the same manner as in 21. It was suspended with water to obtain 104. On the other hand, DL-5-benzylhydantoin 57 and heptyltrimethylammonium bromide 5m9i2
About 15% of sodium oxide was dissolved in N7, the pH was adjusted to pH 8.5 with 2N hydrochloric acid, and water was added to make the solution 40%. Mix both, maintain the liquid at pH 8.5, and add 3
The reaction was carried out at 7°C for 16 hours. After the completion of the reaction, 4.37 L-phenylalanine was produced and accumulated in the reaction solution (conversion rate = 97%). Example 3 DL-5-1 indol-3-yl-methyl]hydantoin' Example 1-tl) and (2) were used using the same medium composition as in Example 1-tl) and (2) except that DL-5-benzylhydantoin was used. The cells were cultured in the same manner as +11 and +21. 200 TrJ of the culture medium a and D[
, -5-benzylhydantoin solution 40- were mixed, and the mixture was reacted at 37° C. for 16 hours while the liquid was maintained at pH 8.5. After the completion of the reaction, 4.159 L-phenylalanine was produced and accumulated in the reaction solution (conversion rate = 93 degrees).

Claims (1)

[Claims] D-, L- or DL-5-benzylhydantoin to L
-Flavobacterium sp. I-3 (Flavobacterium sp.
erium sp. L- characterized by collecting
Method for producing phenylalanine.