JPS59132889A - Preparation of l-alanine dehydrogenase - Google Patents

Abstract

PURPOSE:To prepare L-alanine dehydrogenase efficiently, by cultivating a bacterium belonging to the genus Streptomyces, capable of producing L-alanine dehydrogenase in a DL-alanine-containing medium. CONSTITUTION:A bacterium such as Streptomyces griseoluteus, Streptomyces hygroscopicus, etc. belonging to the genus Streptomyces, capable of producing L-alanine dehydrogenase is cultivated in a medium containing 0.1-10wt%, preferably 0.5-2wt% DL-alanine, crude L-alanine dehydrogenase is collected from the culture mold, it is treated with a metal chelating agent such as ethylenediaminetetra-acetic acid, alpha,alpha'-dipyridyl, o-phenanthroline, etc., and peptidase existing with it is removed.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to L-alanine dehydrogenase (L-alanine dehydrogenase) belonging to the genus Streptomyces.
en-ase (EC'1.4.1.1), hereinafter A
The present invention relates to a method for producing AJDH, which comprises culturing a strain capable of producing AJDH (referred to as NDH) in a medium containing DL-alanine, and collecting AβDH from the resulting culture.

AβDH is L-alanine + NAD" + 'H20!
Catalyze the reaction of pyruvate, NH3++ NADH+ H+, quantify L-alanine, or act with various peptidases using a peptide containing L-alanine as a substrate.
It is important as an enzyme used in a method for measuring peptidase activity by measuring monoalanine released from it using AβDH. Furthermore, in recent years, this enzyme has been attracting attention as an enzyme used in enzyme reactors such as the synthesis of L-alanine by acting on this enzyme in the presence of pyruvic acid, ammonium salts, and NADH.

Conventional AβDH producing bacteria include Bacillus cereus [Journal of Bacteriology (J.

Bacteriol, ) vol. 76, p. 578, 195
8th year], Bacillus subtilis [Biochim, Bioph
ys, Acta) Vol. 96, p. 248, 1965), Bacillus sphaericus [European Journal of Biochemistry (Eur, J. B.
iocheIll, ) Volume 100, Page 29, 1
979], Thermus thermophilus [Biochimica Ebiophysica Acta (BiochiIIIl,
Biophys.

^cta) Vol. 615, p. 34, 1980], Halobacterium salinariium [Vol. 570, p. 1 of the same magazine,
1979], Streptomyces clavuligerus [
Archives Office Microbiology (Ar
ch, Mic-robiol, ) vol. 125, 13
7, 1980] is known. A from the above strains
j! To obtain DH, conventional carbon sources, nitrogen sources,
The cells are cultured in a medium containing an inorganic salt and D-alanine or L-alanine added as an inducer.

Although this method has been used, it has not been fully satisfactory in terms of AβDH productivity, price, etc.

The present inventors searched for strains that produce AβDH and repeatedly studied methods for producing them, and found that when a strain belonging to the genus Streptomyces is cultured in a medium containing DL-alanine as an inducer, AβDH accumulates in large amounts. I found out. DL-alanine is extremely inexpensive compared to monoalanine or D-alanine, and has great industrial value.

Furthermore, the present inventors purified jlDH from the obtained culture using ethylenediaminetetraacetic acid (hereinafter referred to as II!).
It was discovered that mixed peptidases can be easily removed by using a metal chelating agent (called DTA), and as a result, Aj! We succeeded in obtaining DH in high yield. Peptidase must not be mixed when AJDH is used as one of the enzymes for measuring peptidase activity.

The strain belonging to the genus Streptomyces used in the present invention is Streptomyces griseortelus (Streptomyces griseortelus).
omyces griseoluteus), Streptomyces hygeloscopicus (S, hygros)
copicus),.

Streptomyces albolonges (S, albol)
ongus), Streptomyces limosus (S,
rimo-SUS), Streptomyces phaeochromogenes (S, phaeochromogen)
es), Streptomyces ridicus (S, I
ydicus), Streptomyces caespitosus (S, caespitosus) % Streptomyces coelicolor (S, coeLicolor)
, Streptomyces roseus (S, roseus
), Streptomyces alpus (S, albu
s), Streptomyces oligochromogenes (3,
01ivochro-mogenes)-, Streptomyces ruber (S, ru-ber), Streptomyces griseus (S, gr-iseus)
or Streptomyces melanosporea (S, me
lanosporea). Among these, Streptomyces griseolterus (
S. griseoluteus ) IAM 006
0, Streptomyces hygeloscopicus (S, hy
groscopei-cus) IFO3934, Streptomyces rimosus (S, rimosus) I
FO3226 or Streptomyces phaeochromogenes (S, phaeochromogenes)
It is IFO3149.

The above strain was cultured in a medium containing DL-alanine, and /IDHI with the conventionally known Bacillus sphaericus bacteria was obtained.
D properties were compared. (Table 1) Table 1 From Table 1, the usefulness of the method of the present invention can be seen.

As the medium used in the method of the present invention, either a synthetic medium or a natural medium can be used as long as it contains various carbon sources, nitrogen sources, inorganic salts, and DL-alanine. The culture form may be either solid culture or liquid culture, but since the present enzyme is an intracellular enzyme, liquid culture is preferred because it is convenient for bacterial collection.

The amount of DL-alanine added to the medium is 0.1 to 10%, more preferably 0.5 to 2%. Table 2 shows the effect of adding oL-alanine when Streptomyces phaeochromogenes strain IP03149 was cultured in a nutrient medium consisting of malt extract, yeast extract, and inorganic salts.

Table 2 Describing the culture conditions for the bacterial strain used in the present invention, the culture temperature may be any temperature within the range where the bacteria can grow/IDH can be produced, but preferably 25 to
The temperature is 35°C. pu is usually carried out in the range of 6-8. The culture time may be selected so that the enzyme titer reaches its maximum, and is usually 24 to 48 hours.

In order to collect A11DH from the culture obtained as described above, the bacterial cells are first disrupted since this enzyme exists within the bacterial cells. That is, only the bacterial cells are collected from the culture as is, preferably by filtration or centrifugation, and the bacterial cells are disrupted by a mechanical method such as ultrasonication or alumina grinding. Thereafter, solid matter is removed by filtration or centrifugation to obtain a crude enzyme solution, and the enzyme solution is further subjected to appropriate combinations of known purification methods such as salting out, organic solvent precipitation, adsorption chromatography, ion exchange chromatography, and gel filtration. A purified AJDH specimen is thereby obtained.

Here, as described above, the present inventors found that by using a metal chelating agent as a means of purification, contaminating peptidases can be easily removed. Examples of the metal chelating agent include EDTA, α, α-dipyridyl, and 0-phenanthroline, which need only be added to the enzyme solution at any point during the purification step. Addition concentration is 0.1-101, more preferably 0.5-2m
It is M.

Streptomyces phaeochromogenes lPO314
Cultured bacterial cells of 9 strains were mixed with 20mM containing 1mM RDTA.
) A crude enzyme extract was obtained by suspending the enzyme in Lis-HCl buffer and crushing it with Cellmil (manufactured by Edmund Vuiller). Add ammonium sulfate to this solution to 50% saturation to precipitate Al1Df, dissolve the precipitate in the same buffer as above, gel filtrate with Sephadex G-25 (manufactured by Pharmacia), and then DBAE-cellulose ( Braun Co. column chromatography was performed to measure M!DH activity and peptidase activity at each stage.
Table 3 shows a comparison with the case where the same procedure as above was performed without adding DTA. The peptidase activity was measured using L, a known reagent for measuring leucine aminopeptidase.
APC-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

As shown in Table 3, it can be seen that peptidase is easily removed by adding EDTA.

If no additive is used, a more complicated purification process is required.

By using the above method, a significant amount of Aj! can be obtained using extremely inexpensive medium raw materials! DH could be accumulated and peptidase-free AβDH could be obtained in good yield through simple operations.

Next, a method for measuring the activity and physicochemical properties of AβDH obtained as described above will be described.

The enzymatic activity of AβDH is produced by reacting the enzyme with L-alanine and nicotinamide adenine dinucleotide (hereinafter referred to as NAD+) as substrates. (340nm
It is calculated by measuring the increase in absorbance at . That is, 0.1M glycine-NaOH buffer (p
H10,0) 0.87me, 21mM NAD" 0
．． 05me, 0.5M L-alanine 0.06- and enzyme solution 0.02- are mixed and reacted at 25°C, and the increase in absorbance at 340 nm for 1 minute after the start of the reaction is measured. As a control, operate in the same manner as above using water instead of the L-alanine solution, and subtract the obtained absorbance from the above measured value as a blank value, and determine the amount of produced NADH from that value. Enzyme activity is expressed as 1 μmol NA per minute.
The amount of enzyme that produces DH was defined as 1 unit.

Streptomyces phaeochromogenes lPO31
The physicochemical properties of AβDH obtained from 49 strains are as follows.

Tl) Effect This enzyme catalyzes the reaction shown in the following formula.

L-alanine ten NAD++ H20 4 pyruvate ten NH3” + NADH+ H” (2)
Substrate specificity As shown in Table 4, this enzyme has little or no effect on amino acids other than L-alanine. It does not act on D-amino acids including D-alanine. When NADP+ is used as a coenzyme, it exhibits only about 0.6% activity compared to NAD+.

Table 4 (3) Optimal pH When the reaction was carried out at 25° C. for 1 minute at each p++ of pH 7 to 11, the optimum value was pi (around 10).

(Figure 1) (41 pH Stability When the residual activity was measured after treatment at 25°C for 13 hours at each pH of 4 to 11, it was found to be stable in the range of p)l 7 to 9. (Fig. 2) (5) Optimum Temperature When pi (10, 0, and 1 minute reaction was performed at each temperature of 20 to 60°C, the activity was maximum at around 60°C. (Fig. 3) (6) Thermal stability When the residual activity was measured at pH 7, 0 and 20 minutes at various temperatures from 0 to 65°C, it was found to be stable below 45°C. (Figure 4) (7) Inhibitor and When various agents listed in Table 5 were added as activators and the activity was measured, this enzyme was found to be activating Cu'', Pb2+ metal ions and PCMB.
The activity was inhibited by SH inhibitors such as (parachloromercuribenzoic acid). It is also activated by metal chelating agents such as Eori and S■ protecting reagents such as dithiothreitol.

(Margins below) Table 5 (8) Substrate affinity The Miberellis constant (Km value) for this enzyme, alanine, and NAD+ at pH 10, 0, and 25°C.
Each? , IX 10-3 M, 3.6X 10-
It was 5M.

(9) Molecular weight: According to the gel filtration method using Sephadex G-200, the molecular weight is approximately 240,000, and from the results of 5O5-disk gel electrophoresis, it is composed of a single subunit with a molecular weight of approximately 39,000. I understand.

Example 1 Malt extract 0.3%, yeast extract 0.2%, DL-alanine 1%, KH2PO40.1%, MCI 0.05%
, MgSO4・7H200,05%, FeSO4・7H
Medium 1 with a composition of 200,001% (pH 7.2)
00m1! One platinum loop of Streptomyces rimosus IFo 3226 strain was inoculated into a 500 m capacity Sakalo flask containing 500 m of the flask, and cultured with shaking at 30°C for 48 hours to obtain a seed culture solution.

Next, 300ml of medium 2000-ml with the same composition as above was added.
The seed culture solution was inoculated into a 00-volume Jar Fermentor and cultured with aeration at 30°C for 48 hours. Centrifuge the culture solution to collect the bacterial cells and add 20mM containing 1mM EDTA.
M) After suspending in Lis-HCl buffer (pH 7.0), the cells were disrupted for 10 minutes using a cell mill. This bacterial cell extract was centrifuged to remove solid matter, and ammonium sulfate was added to the resulting supernatant to 50% saturation to precipitate AβDH. The precipitate was collected by centrifugation, dissolved in the same buffer, and then desalted by gel filtration using Sephadex G-25. The obtained enzyme solution was diluted with the same buffer solution (pH 7.5
) and eluted with the same buffer (pH 7.5) containing 0.4) I KCI. The eluate fractions containing enzyme activity were collected and concentrated by ultrafiltration, and then dialyzed using the same buffer (pH 7.0) as an external solution. The dialysate was adsorbed onto a blue Sepharose column equilibrated with the same buffer (pH 7,0), and 0.3 M
AADH was eluted with the same buffer (pH 7.0) containing MCI. The active fractions were collected, concentrated by ultrafiltration, desalted, and then lyophilized to a specific activity of 5. 260 mg of Aj2DH powder of Ou/mg was obtained.

Example 2 Streptomyces phaeochromogenes lPO314
9 strains were operated in the same manner as in Example 1, and the specific activity was 6.
155 mg of 5 u/mg A#DH powder was obtained.

[Brief explanation of the drawing]

Figure 1 shows AADH obtained from Streptomyces phaeochromogenes IPo 3149 strain by the method of the present invention.
FIG. 2 is a diagram showing the optimum pH stability, FIG. 3 is a diagram showing the optimum temperature, and FIG. 4 is a diagram showing thermal stability. Patent application - Person Amano Pharmaceutical Co., Ltd. Figure 1 pH Figure 2 Me 3 5 7 9 1
1pl+ Figure 3 20 30 4150 60-4 Degrees (0C) Figure 4 Temperature (0c)

Claims (1)

[Claims] (11) Production of mono-alanine dehydrogenase, which is characterized by culturing mono-alanine dehydrogenase-producing bacteria belonging to the genus Streptomyces in a medium containing DL-alanine, and collecting mono-alanine dehydrogenase from the culture. (2) L-alanine dehydrogenase-producing bacteria belonging to the genus Streptomyces include Streptomyces griseoltellus, Streptomyces higeroscopicus, Streptomyces albolonges, Streptomyces limosus, Streptomyces phaeochromogenes, and Streptomyces spp. claims that are Streptomyces caespitosus, Streptomyces coelicolor, Streptomyces roseus, Streptomyces alpus, Streptomyces olipochromogenes, Streptomyces ruber, Streptomyces griseus or Streptomyces melanosporea The method for producing L-alanine dehydrogenase according to item 1. +3) The amount of DL-alanine added to the medium is 0.1 to
10% of the method for producing alanine dehydrogenase according to claim 1. (4) The production of mono-alanine dehydrogenase according to claim 1, which comprises an operation of removing mixed peptidase using a metal chelating agent when collecting the mono-alanine dehydrogenase from a culture. Law. (5) The method for producing L-alanine dehydrogenase according to claim 4, wherein the gold JfE chelating agent is ethylenediaminetetraacetic acid, α, α9-dipyridyl, or 0-phenanthroline.