JPS58134997A - Preparation of gamma-glutamyl-alpha-amino-n-butyryl-glycine - Google Patents

Abstract

PURPOSE:To prepare ophthalmic acid useful as an ophthalmic drug, in high efficiency, by reacting gamma-glutamyl-alpha-amino-n-butyric acid with glycine in the presence of glutathione synthetase at a pH within a specific range. CONSTITUTION:gamma-Glutamyl-alpha-amino-n-butyric acid, glycine and glutathione synthetase are dissolved in an aqueous medium, and maintained at 4-10pH and about 10-70 deg.C. The gamma-glutamyl-alpha-amino-n-butyryl-glycine (ophthalmic acid) accumulated in the aqueous solution is separated and purified with ion exchange resin, etc. The glutathione synthetase may be those obtained from an animal source such as pigeon liver, pig liver, etc., a vegetable source such as bean embryo, wheat embryo, etc. or a microbial source such as Saccharomyces cerevisiae ATCC7752, etc.

Description

Detailed Description of the Invention The present invention relates to γ-glutamyl-α-amino n-butyryl-glycyl 7 (1-glutamyl-2-amin
The present invention relates to a method for producing o-n-butyryl-glycine (hereinafter referred to as "ophthalmic acid").

Ofsalmic acid (op-1ha1mi4 acid)
exists on the surface of the eyeball, and it has been suggested that it can be used as an eye drug.

The present inventors conducted research to develop a simpler production method for such ophthalmic acid, and found that γ-glutamyl-α-7minor n-butyric acid (γ-glutamic acid)
yl-α-amino-n-butyricaci
d+ (hereinafter referred to as rGABJ) and glycine (glycin
e) and glutathione synthetase (glutathj)
one 5 synthetase, 6 + 3 + 2
+3 γ5.

-L -glutamyl -L -cy@tetne
: Glycineljgase (ADP)
) in aqueous solution due to the action of ? It has been found that ophthalmic acid can be efficiently obtained by carrying out the reaction at a pH in the range of 4 to 10 O.

That is, the present invention is a method for producing ophthalmic acid, which is characterized by maintaining an aqueous solution containing GAB and glycine in the pH range of 4 to IO in the presence of glutathione synthetase.

As glutathione synthetase, not only purified enzyme preparations but also crude preparations containing components other than these enzymes can be used. There is also glutachio. That is, substances known to have this enzyme activity, for example, those of animal origin such as pigeon liver and pig liver, those of plant origin such as bean germ and wheat germ, Suckermyces cerevisiae ATCC 7752, Suckermyces Suckermyces spp., such as Suckersbergensis ATCC 9080, Candig+13--Theilis ATCC 162
39 such as Candida spp., Schizosatsukap Myces bombe IAM4779 and other spp., Torulopsis spp. such as Torulopsis parsatiris NRRLY-6652, and other N. Pseudomonas species such as mothership IF, Pseudomonas aeruginosa ATCCI 0145,
Corynebacterium spp. such as Phrynebacterium equi ATCC 6939, Staphyroptucus spp. such as Staphyrcoticus aureus ATCC 4012, Escherichia spp. such as Escherichia@coli ATCC 11246, Enterobacter spp. such as Enteribacter erogenes ATCC 13048 , Proteus vulgaris F
E RM-P 4796, Proteus mirabilis I
Puteus genus such as F03849, Flucaligenes genus such as Alcaligenes faecalis ATCC 8750, Bacillus genus such as Bacillus subtilis ATCC 13952, Bacillus Φ plebis ATCC 8185, Brevibacterium genus such as Brevibacterium ammoniagenes ATCC 6B71, 7gp Bacterium radiobacter ATCC 47 18 7 groups of Pacterium, such as
Furthropacter spp. such as Arthrobacter simplex ATCC 6946, Microfuccus spp.
Those of Erwinian origin such as Levinia herbicola ATCC21434, Mucoa javanicus ATCC152
Mpua genus such as 42, Rhizopus Φdelmar IF047
75, etc., 7 Supergillus species such as Aspergillus oryzae ATCC16@240, Penicillium species such as Henicillium luteum ATCC 9644, those originating from molds such as Neurospora fistulae ATCC 9277, Streptomyces fradia ATCC 10746, etc. Any of those originating from actinomycetes such as Streptomyces can be used.

When using animal-derived glutathione synthase, the tissue can be crushed and used as is, or the enzyme protein can be purified by ammonium sulfate fractionation, cellulose column chromatography, biogel column chromatography, etc. Use it as such. When using microbial origin, it may be prepared as follows.

A method for obtaining a microbial culture or bacterial cells does not require the use of a specific method, and may be cultured in a conventional medium using a conventional method. As glutathione synthetase,
The culture may be used as it is, or the bacterial cells, washed bacterial cells (lyophilized bacterial cells, acetone-dried bacterial cells, bacterial cells contacted with toluene, surfactant, etc., bacterial cells treated with lysozyme, super Microbial cells exposed to sound waves, mechanically disrupted microbial cells, etc.) or enzyme-protein fractions having glutathione synthetase activity obtained from these microbial cells or processed microbial cells by conventional enzyme fractionation methods; immobilized bacterial cells,
Any insolubilized product of treated bacterial cells can be used.

To generate ophthalmic acid from GAB and glycine, dissolve GAB, glycine, and glutathione synthetase in an aqueous solvent, and create a solution! From O? a suitable temperature in the range of 1C, and a suitable p in the range of pHA et al.
Just keep it at H for a while. Favorable results may be obtained by adding antioxidants, surfactants, etc. to the aqueous solution. Also, during the reaction, if necessary, the raw material G can be added to the reaction solution.
AB and/or glycine may be additionally added. Although it is not necessary to stir the reaction solution particularly strongly, the reaction solution may be stirred appropriately if necessary.

The ophthalmic acid produced and accumulated in aqueous solution or in liquid can be collected by a conventional method such as using an ion exchange resin.

Example 1
(■ Culture: per 14 glucose 10f1MgSO4
-7H, 100, 2f% KHIPO, I Of.

NaNH4HP0. 1 Of, citric acid-'I hydrate 7.02 μL-threonine 26QL-leucine 5゜qsL-bourine 25Q%l, -fulginine 50■,L
- Histidine 10, Thiamine 1. Medium 2Q containing CJmg and peptone 10F, pH 8, or-adjusted
Od Zuzu 5 was placed in 5 bottles of 7 rasf and sterilized by heating. This was inoculated with Pluterus mirapilis IF03849, which had been precultured in boudin medium, and 28CC-(
It was cultured with shaking for 36 hours. On the other hand, culture medium 25 having the same composition as above was placed in a 100-liter fermentor, and after sterilization, the culture solution from the 5 flasks described above was placed therein. Culture was carried out under aerobic conditions at 28c for 24 hours. The thus obtained culture solution was subjected to continuous centrifugal treatment at 20 OO rpm to obtain bacterial cells with a wet weight of 750 f. This was stored frozen at -20C.

Preparation of glutathione synthetase 1) Cell-free extract Thaw 750 tons of frozen bacterial cells and add 0.01 M 9-acid buffer (pH 7, containing 1 g of 0.6 mM MgCl).

The total volume was adjusted to 4 tons, and the cell membrane was disrupted using "Dyno Mill", and after centrifugation, 3.8 g of cell-free extract was obtained.

2) Ammonium sulfate fractionation After fractionating the cell-free extract with 40-80% ammonium sulfate, 0.
OIM! Dialysis was performed at 4C against a phosphate buffer.

3) Approximately 10% of the total protein to exclude protamine sulfate basic protein and nucleic acids.
% of protamine sulfate was added, and the precipitate formed was removed by centrifugation.

4) DEAR-cellulose column treated fraction This supernatant was diluted with 0.01 M'J acid buffer (pH 710, 5mM
A DEAE-cellulose column equilibrated with MgCl containing → was used to carry out black-spotted flyfishing. 0.05
M NaCl solution (1) Formation reaction of ophthalmic acid A reaction solution having the composition shown in Table 3 was kept at 30C for 48 hours.

The reaction yield was 49% relative to GAB, and ophthalsic acid was produced. (The generated ophthalsic acid was quantified using an amino acid analyzer.) Table 3 Reaction solution composition Tris-HCl buffer 100mMGA
8 20mM sodium adenosine triphosphate 40mM glycine
20mM Magnesium Chloride 20mM, /l/l'f
Off, /7 Yakeo *Total amount 50T111 *Adjust the final enzyme concentration of the reaction, l 2 unit/-■
Isolation and identification of ophthalmic acid The reaction solution obtained by the above reaction was
J (I (type) resin, r Dowex I X 8 J (
cH, coo-type) 4111111 to isolate ophthalmic acid. The isolated yield was 43.7%.

The obtained ophthalmic acid was identified by NMR.

Furthermore, it was confirmed that glutamic acid, glycine, and α-amino-n-butyric acid were produced by hydrolyzing the isolated crystals of ophthalmic acid.

Example 2 Glucose 10 t/l, KH, Po, 2 t
/l.

MgSO4・7H901t/Ls peptone 1t
/l.

Yeast extract 10 f/l (pH 7.0 (KOH neutralized)
) was placed in a flask with a shoulder (500 TILl).
sterilized at 1151:' for 15 minutes, left to cool, inoculated with one type of each of the microorganisms shown in Table 4, and sterilized at 31C' for 15 minutes.
The culture was incubated with shaking for 4 hours. The resulting culture fluids were centrifuged to collect bacterial cells, disrupted by sonication, and further centrifuged to obtain cell-free extracts.

Using each extract as an enzyme solution, the amount of ophthalmic acid produced by the method of Example 1 was determined using an amino acid analyzer.

Table 4 Enthylbacter erogenes ATCC130485
,6 Proteus mirapilis IFO384911,5
Proteus plregalis FERM-P 479 S
Society, 8-yx-4 Monas r-
tLJf/+ ATCC101453,2 Bacillus・
Subtilis ATCC13fj 52
2.5 Erwinia herrevirola ATCC2
14348,8 Corynebacterium equi ATCC
69392,0 Escherichia coli ATCC1124
6,6,8 Alcaligenes faecalis ATCC87
504,2 Brevibacterium ammoniagenes A
TCC68?1 2.5 Micrococcus rhizodicticus ATCC46983,2 Arthrobacter-φ simplex ATCC69462,4 Patent applicant Ajinomoto Co., Inc.

Claims (1)

[Claims] , -g/k 31 i, ni-,. -ami, -n-buj
Rick acid (γ-glutamyl-α-amino
An aqueous solution containing glutathione synthetase (butyric acid) and glycine is
utathionesynthetase), the pH is maintained in the range of 4 to 1o.
-Glutamyl-α-7 minnow n-butyryl! Method for producing glycine.