JPH04281788A - Isoenzyme and its mixture of gamma-glutamyltranspeptidase - Google Patents

Abstract

PURPOSE:To provide the title novel isoenzyme for e.g. medicines, produced by Bacillus cells, capable of hydrolyzing D-gamma-glutamyl-p-nitroanilide, thus having such activity as to convert gamma-glutamyl groups into amino acids such as D- glutamic acid. CONSTITUTION:Bacteria belonging to Bacillus (e.g. Bacillus licheniformis A35) is inoculated into a 1% glucose-meat extract medium and put to shaking culture at 30 deg.C for 22hr followed by centrifugation of the resultant culture solution to remove microbial cells followed by addition of acetone at -30 deg.C and collection the precipitate produced, which is, in turn, suspended in a buffer solution containing 20mM potassium phosphate (pH7.0). The suspension is then subjected to anion exchange column chromatography to effect purification, thus hydrolyzing D-gamma-glutamyl-p-nitroanilide and giving the objective gamma-glutamyltranspeptidase isoenzyme having the following characteristics: (1) activity: capable of converting the gamma-glutamyl groups in the D-gamma-glutamyl-p-nitroanilide into amino acids including D-glutamic acid or peptides; (2) optimal pH: 8.0; (3) optimal temperature for activity: 55-60 deg.C; (4) molecular weight (gel filtration technique):30000.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to γ-glutamyl transpeptidase (hereinafter referred to as γ-glutamyl transpeptidase) produced by bacteria of the genus Bacillus.
GTP) isoenzyme mixtures and isoenzymes. γ-GTP is known as an enzyme that catalyzes a reaction that hydrolyzes γ-glutamyl compounds such as glutathione and transfers the resulting γ-glutamyl group to a peptide or amino acid. In this way, γ-GTP is capable of enzymatic synthesis of various γ-glutamyl amino acids or peptides, and by γ-glutamylation of amino acids, it increases the solubility of amino acids and protects them from various amino acid degrading enzymes. Therefore, it has industrial utility value, such as being able to be used as a medicine.

0002

BACKGROUND OF THE INVENTION γ-GTP is widely present in animals, plants and microorganisms. For example, it exists in the human body, and its content in the blood is used as an indicator of liver function. Since the existence of bacterial γ-GTP was confirmed in the Gram-negative bacterium Proteus vulgaris in 1952, its presence has been reported in many Gram-negative bacteria, and its amino acid and gene sequences have not been determined. Some have. However, the productivity of conventionally known animal, plant, and microorganism cells is low, and since γ-GTP is mainly present in cell membranes, it is difficult to separate and purify it, making it unsuitable for industrial production. .

0003

[Problems to be Solved by the Invention] In view of the current situation, the inventor of the present invention aimed to produce γ-GTP at a high concentration. As part of this, the present inventor has developed a method for developing bacteria of the genus Bacillus, particularly Bacillus licheniformis A35 (Bacillus licheniformis A35).
We discovered that S. illus licheniformis A35 (hereinafter referred to as BL-A35) secretes γ-GTP extracellularly, and further purified γ-GTP.
Its properties have already been published (Asada et al.; Japanese Society of Agricultural Chemistry, Abstracts of the 1986 Conference, p. 226 (3Ea-7).
), 1988). However, the inventor has discovered that γ-GTP
As a result of intensive research on production, it was surprisingly discovered that this bacterium co-produces two new enzymes that have the same functions, i.e., isoenzyme (hereinafter referred to as isoenzyme), in addition to the well-known γ-GTP. Moreover, it has been found that these new isoenzymes have higher productivity than conventional γ-GTP (hereinafter also referred to as isoenzymes). Therefore,
The present inventor cultivated bacteria of the genus Bacillus, particularly BL-A35, to simultaneously produce a group of isoenzymes of γ-GTP. established a method to separate and purify
The present invention was completed by clarifying the physical and chemical properties of each.

[0004] Furthermore, the above-mentioned Bacillus licheniformis A
No. 35 has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM p-12055. Conventionally, it was not known that bacteria produce γ-GTP isoenzyme. As a result of the present invention, for the first time, bacteria, especially bacteria of the genus Bacillus, especially BL-A35,
- It has been revealed that GTP isoenzymes are produced.

[0005]

[Means for Solving the Problems] In the above-mentioned situation, the enzyme of the present invention is a γ-glutamyl transpeptidase produced by a bacterium of the genus Bacillus, and has the following properties: (a) substrate specificity; Has the activity of hydrolyzing glutamyl-p-nitroanilide and transferring the γ-glutamyl group in the compound to an amino acid or peptide containing D-glutamic acid; (b) Optimal pH; optimum pH for γ-glutamyl group transfer reaction; Appropriate pH
is 8.8; (c) the optimum temperature for action; the optimum temperature for the γ-glutamyl group transfer reaction is 55-60°C; and (d) the molecular weight as determined by gel filtration is 30,000; Glutamyl transpeptidase isoenzyme [A]
and γ-glutamyl transpeptidase isoproduced by the same bacterium of the genus Bacillus, sharing the above-mentioned physicochemical properties (a), (b), and (c), and having a molecular weight of 35,000 as determined by gel filtration. It is enzyme [B].

Bacteria that produce isoenzymes with such physical and chemical properties must belong to the genus Bacillus. As mentioned above, other bacteria, such as Gram-negative bacteria, are unable to produce enzymes with such properties, especially isoenzymes. Furthermore, bacteria of the genus Bacillus are BL-A3
5 is preferable. The novel isoenzymes [A] and [B] may be used individually, or they may be mixed and used as a mixture. Alternatively, a γ-GTP isoenzyme [C] with a molecular weight of 40,000, which is produced by the same bacterium of the genus Bacillus and has already been published, may be further mixed and used. Isoenzyme [C] has the above-mentioned physicochemical properties (a), (b), and (c) of isoenzyme [A] and [
B], but it is an enzyme with a large molecular weight of 40,000. The combination of isoenzymes is not particularly limited, and may include a mixture of isoenzyme [A] and isoenzyme [C], isoenzyme [B] and isoenzyme [B], and isoenzyme [B] and isoenzyme [C].
mixture with isoenzyme [A], isoenzyme [B]
It may also be a mixture of [C] and three isoenzymes.

[0007] The mixing ratio of these isoenzyme mixtures is not particularly limited. The ratio may be the same as the bacteria are produced, or the isoenzyme may be separated and mixed at an appropriate ratio. Further, the isoenzyme mixture may be a mixture of purified isoenzymes or may be an unpurified mixture. Furthermore, enzymes other than γ-GTP may be present in the mixture.

Substrate specificity and optimal pH of these isoenzymes
and the optimum temperature for action are the same, so in many cases a mixture can be used. In this case, a mixture containing three isoenzymes has high enzyme productivity and can be used economically. However, since each isoenzyme has a slightly different thermostability, it is often possible to use isoenzyme [A] or [B] alone to increase the effectiveness of the use, taking advantage of their heat resistance properties. For example, isoenzyme [A] has the highest thermal stability and retains 90% of its activity after a one-hour thermal stability test at 55° C., so it can be used particularly effectively when used at high temperatures. In addition, isoenzyme [B] has the lowest thermal stability, for example, it loses 50% of its activity in 1 hour at 50% concentration, so it has the characteristic that its activity can be easily sealed by low-temperature treatment after the desired reaction is completed. be. isoenzyme [A] and [B
] can have both characteristics as well as economy. A mixture containing isoenzymes [B] and [C] can have both thermal stability and economic efficiency. The mixture containing isoenzymes [B] and [C] can be processed at low temperatures and economically.

[0009] The term "isoenzyme" as used herein refers to a group of enzymes that exist in the same biological species and catalyze the same reaction, but have different enzymatic chemical properties. As mentioned above, γ-G
The existence of TP isoenzymes has not been previously known in bacteria, and their existence has been made known for the first time by the present invention. Regarding the novelty of the γ-GTP isoenzyme according to the present invention, it differs in substrate specificity from γ-GTP conventionally known for Gram-negative bacteria such as Proteus or Escherichia or animal cells. It is clear from this. That is, all of the isoenzymes according to the present invention are D-γ-glutamyl-
p-nitroanilide (hereinafter referred to as D-γ-GpNA)
Alternatively, it has an activity of transferring to D-glutamine and D-glutamic acid using L-γ-glutamyl-p-nitroanilide (hereinafter referred to as L-γ-GpNA) as a γ-glutamyl group donor. However, the γ-GTP produced by Gram-negative bacteria or animal cells mentioned above is D
- Shows no activity towards either glutamine or D-glutamic acid. Furthermore, it does not have any activity toward D-γ-GpNA, and cannot perform a rearrangement reaction using D-γ-GpNA as a donor.

The known isoenzyme [C] and the new isoenzymes [A] and [B] contained in the isoenzyme mixture of the present invention are clearly distinguished in molecular weight. The molecular weight of the former is 40,000, and the molecular weights of the latter two are 30,000 and 35,000, respectively. Furthermore, as shown in Table 1 and Table 5 of Examples, the relative rearrangement activity with respect to amino acids and heat resistance properties are different. (Method for measuring physicochemical properties) The physicochemical properties of the γ-GTP isoenzyme according to the present invention are measured as follows.

(a) Substrate specificity The present isoenzyme has the function of transferring a γ-glutamyl group to an amino acid, and various amino acids are selected as active substrates, and the transfer activity toward these is expressed as a relative value. That is, D- or L-γ-GpNA was used as the γ-glutamyl group donor, glycylglycine (hereinafter referred to as Gly-Gly) was selected as the representative active substrate, and the γ-glutamyl group transfer activity against this was determined. It is expressed as 100, and transfer activity toward other amino acids is expressed as a relative value.

Specifically, D- or L-γ-GpNA
0.2 ml of 1 mM solution, 0.3 ml of 20 mM solution of Gly-Gly (or other amino acid) and Tris-
3-5 μg of γ-GTP was added to a mixture of 1.3 ml of 50 mM hydrochloric acid buffer (pH 8.0), and γ-GTP was incubated at 37°C.
Transfers glutamyl group. After a predetermined period of time, acetic acid 3
．． The reaction is stopped by adding 1.0 ml of a 5N aqueous solution, and the absorbance of the reaction solution at 410 nm is measured to determine the amount of p-nitrosoaniline (ε=8800) liberated by the rearrangement reaction. The amount of p-nitrosoaniline produced directly indicates the transfer activity of the enzyme, but when expressed in activity units, the production of 1 μmol of p-nitrosoaniline per minute is considered to be 1 unit (U), and the following It can be calculated using the formula.

[0013] U/ml=absorbance (410 nm)*0.
003 * 106/8800 * 0.2 * 20 (b) Optimum pH The p at which the transfer activity for the above Gly-Gly is maximized
Say the H value. (c) Suitable temperature for action Similarly to pH 8.0, this refers to the temperature at which the transfer activity for Gly-Gly is maximized.

(d) Average molecular weight Values measured by gel filtration using Sephadex G-100 are shown. Molecular weight standards include albumin (B
ovine serum), alcohol dehydrogenase (Y
east), β-amylase (Sweet pota
to), carboxylic acid dehydrogenase (Bovine Ery
throcytes), cytochrome C (Horse
Use standard proteins such as (γ-
Method for producing GTP isoenzyme)

Next, the method for producing the γ-GTP group of the present invention will be described. It is manufactured by culturing Bacillus bacteria and using a fermentation method. Bacteria of the genus Bacillus are Bacillus liheniformis A35 (Bacillus).
S licheniformis A35) is γ-G
It is particularly preferable because it has characteristics such as high productivity of the TP group, co-production of three isoenzymes, and production outside the bacterial cells, so there is no need to add glutamic acid to the culture medium. Regarding the same bacterium, C. Cheng, Y. Asada and T. Aida is Agr・Biol. Chem. Magazine No. 5
It is described in Volume 3, page 2369 (1989). The culture method is not particularly limited, but for example, sterilized G
Bacillus liheniformis A35 was inoculated into B medium,
Culture with shaking at 30-37°C for 20-22 hours. This culture was used as a seed culture, and glucose-bouillon medium (GB medium), bouillon medium (B medium), glucose-yeast extract medium (GY medium), casamino acid medium (CA medium), peptone medium (P medium), TSB By inoculating 0.1-10% into a medium or a medium such as M medium and culturing with shaking at 30°C for several days, γ-GTP group is produced and accumulated in the medium. This medium is centrifuged at 10,000 rpm for 10 minutes, acetone at -30°C is added to the supernatant to give a final concentration of 45% by volume, and the protein is precipitated by standing at +5°C for 1-2 hours. . The protein is collected by centrifugation at 10,000 rpm for 10-15 minutes and suspended in 20 mM potassium phosphate buffer (pH 7.0). Since the obtained precipitate contains many proteins, it is purified as follows.

(Method for purifying γ-GTP isoenzyme) First,
Pre-resolve proteins by anion column chromatography. DEAE-Cellulofine A-5
The acetone precipitate suspension was applied to 0.00 and eluted with a sodium chloride concentration gradient of 20 mM potassium phosphate buffer (pH 7). When the sodium chloride concentration is changed linearly from 0.15M to 0.3M and elution is carried out, the sodium chloride concentration becomes 0.
A weak smooth protein elution curve was obtained in the range of 0.20-0.23M (referred to as section A), then 0.24-
An elution section is obtained which is represented by a strong sharp curve in the 0.25M range (referred to as section B). The latter category B is conventional γ-GTP (ie, known isoenzyme [C]), and γ-GTP has been collected and purified from this category. Category A did not provide a clear elution curve using the same separation column, and the presence of isoenzymes was unexpected, so it was thought to be another protein and was left alone.

As a result of the present invention, it has been found that isoenzymes [A] and [B] exist in category A. In other words, separation is insufficient in section A, and isoenzymes [A] and [B] cannot be collected from it, but when section A is re-fractionated using the next hydroxyapatite column, a clear elution fraction is obtained.
Moreover, as a result of such purification, the specific activity of the isoenzyme is unexpectedly high. Specifically, Section A is applied to a hydroxyapatite HTP column and eluted with a concentration gradient potassium phosphate buffer. When elution is performed by linearly changing the concentration from 20mM to 0.4M, category A is 50-100mM.
and can be divided into two sharp fractions in the range 110-150mM. The former fraction (Category A-1
) contains isoenzyme [A], and the latter fraction (referred to as segment A-2) contains isoenzyme [B].

For further purification of these fractions, S
By gel chromatography on an ephadex G-100 column. That is, each fraction is separately applied to the column and eluted with 20mM potassium phosphate buffer.

[0019]

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples. Example 1. 1% glucose-meat extract medium (
After sterilization, Bacillus liheniformis A35 was inoculated with 1ase and cultured with shaking at 30°C for 22 hours. This culture solution was used as a seed culture, and the following glucose-yeast extract medium (GB medium) was prepared: Yeast extract 1% Glucose 1% pH 7.0200m
Inoculate 6 ml into a 1 liter flask containing 30
℃ for 3 days with shaking, then cooled to +5℃,
The cells were removed by centrifugation at 0,000 rpm for 10 minutes. Add -30°C acetone to the medium from which bacterial cells have been removed to a final concentration of 45% by volume, and leave it at +5°C for 2 hours.
Proteins were collected by centrifugation at 0,000 rpm for 15 minutes and added to 20 mM potassium phosphate buffer (pH 7.0).
It was suspended in 400ml. The production amount of the γ-GTP total isoenzyme mixture determined from the production amount of p-nitrosoaniline described above was 2.03 units (U) per ml of the medium.

Example 2. Add 2 DEAE-Cellulofine A-500 to a chromatography tube with a diameter of 3.6 cm.
A chromatographic separation column was prepared by packing 0 cm. This column was washed with 400 ml of 0.25 hydrochloric acid and 800 ml of deionized water.
ml, 0.25N sodium oxide aqueous solution, deionized water 8
00 ml of the γ-GTP total isoenzyme mixture obtained in Example 1 was added to the column.
40 ml of M potassium phosphate buffer (pH 7.0) suspension was provided. Then, the sodium chloride concentration was adjusted to 0.15-0.
Add 1,000 ml of 20mM potassium phosphate buffer (pH 7.0) with a linear concentration gradient of 3M to 30ml/
The γ-GTP isoenzyme was eluted by flowing at a flow rate of h to obtain Section A and Section B. The elution curve is shown in Figure 1.

Example 3. Hydroxyapatite HTP was swollen with 20 mM potassium phosphate buffer (pH 7.0), degassed in a batch method, and placed in a chromatography tube with a diameter of 4 cm.
A chromatographic separation column was prepared by packing 0 cm. This column was mixed with 20mM potassium phosphate buffer (pH 7.0) for 40 minutes.
After equilibration with 0 ml, the section A obtained in Example 2 was added to 100 ml of 20 mM potassium phosphate buffer (pH 7.0).
Washed with l. This column was then mixed with 20mM-0.4
Elution was performed with a linear concentration gradient of M potassium phosphate buffer (pH 7.0) to obtain the sections A-1 and A-2. The elution curve is shown in Figure 2. As a result of calculating each category from the production amount of p-nitrosoaniline mentioned above, the amount of γ-GTP isoenzyme was 0.26 units (U) per ml in terms of culture medium, respectively.
It was 0.49 units (U).

Example 4. Example 3 was repeated for Division B obtained in Example 2, and the isoenzyme [C] contained in Division B was
was purified. This amount is 0.2 per ml in terms of culture medium.
It was 8 units (U).

Examples 5 to 7. A chromatographic separation column was prepared by filling a chromatographic tube with a diameter of 2.0 cm with 30 cm of Sephadex G-100. Add this column to 20mM
After equilibration with 400 ml of potassium phosphate buffer (pH 7.0), Section A-1 and Section A obtained in Examples 3 and 4
-2 and Category B were respectively applied and eluted with 20mM potassium phosphate buffer (pH 7.0), isoenzyme [A], [
B] and [C] were purified.

Examples 8-10.1mM D-γ-Gp
NA solution 0.2ml, 20mM Gly-Gly solution 0
．． 3 ml and 50 mM Tris-HCl buffer (p
H8.0) 1.3 ml of the purified isoenzymes [A], [B] and [C] obtained in Examples 5 to 7 were added to the mixed solution.
．． 2 ml of each was added and reacted at 37°C for 30 minutes. Thereafter, 1.0 ml of a 3.5N acetic acid aqueous solution was added to stop the reaction, and the transfer activity of the enzyme was determined from the absorbance of the reaction solution at 410 nm, and this value was set as 100.

Examples 11-13. Examples 8 to 10 using the amino acids listed in Table 1 instead of Gly-Gly
repeated. The relative transfer activity of these amino acids to Gly-Gly is also listed in Table 1. As is clear from this table, isoenzymes [A], [B] and [C] have similar relative transposition activities.

Examples 14-16. Table 1 also shows the results of repeating Examples 8 to 10 using L-γ-GpNA instead of D-γ-GpNA.

Examples 17-19. Examples 11 to 13 as L
The results of repeated experiments using -γ-GpNA are also listed in Table 1. Even if L-γ-GpNA is used, isoenzyme [A], [B
] and [C] have similar relative transposition activities.

[0028]

[Table 1]

Examples 20-22. The reactions of Examples 14 to 16 were carried out at different pH values, and the results are shown in FIG. Isoenzymes [A], [B] and [C] all have a pH of 8.
8 showed maximum activity. However, the pH values were adjusted using a phosphate buffer in the pH 7-8 range, a Tris-hydrochloric acid buffer in the pH 8-9 range, and a sodium carbonate buffer in the pH 9-10 range. Examples 23-25. The results of the reactions of Examples 14 to 16 performed at different temperatures are shown in FIG. 4.
Both [B] and [C] exhibit maximum activity in the temperature range of 55 to 60°C. Examples 26-29. In order to check the heat resistance, each isoenzyme was kept at various temperatures for 24 hours and then tested in Examples 14 to 1.
6 reactions were performed. The results are shown in Figure 5.
] has high heat resistance, isoenzyme [B] has the lowest heat resistance, and isoenzyme [C] has the lowest heat resistance. Examples 30-32. Sephad each isoenzyme
By the gel filtration method using ex G-100, the above-mentioned albumin, alcohol dehydrogenase,
As a result of measurement using β-amylase, carboxylic acid dehydrogenase, and cytochrome C, isoenzyme [A] 30,000
, same [B] 35,000, same [C] 40,000.

[0030]

[Effects of the Invention] According to the present invention, it has been known for the first time that bacteria, particularly bacteria of the genus Bacillus, produce γ-GTP isoenzyme. By producing a mixture including these novel γ-GTP isoenzymes, the productivity of γ-GTP has been dramatically increased, and it has become possible to use it economically. Also,
Utilizing the heat-resistant properties of the new isoenzyme, the range of uses for γ-GTP has expanded, including use at high temperatures and low-temperature inactivation.

[Brief explanation of the drawing]

[Figure 1] Figure 1 shows DEAE-Cellulofine A
Figure 2 shows the elution curve of γ-GTP using a -500 chromatographic separation column.

FIG. 2 shows an elution curve of γ-GTP isoenzyme contained in section A using a hydroxyapatite HTP chromatographic separation column.

FIG. 3 shows the pH activity curve of γ-GTP isoenzyme.

FIG. 4 shows the temperature activity curve of γ-GTP isozyme.

FIG. 5 shows a thermostable activity curve of γ-GTP isoenzyme.

Claims (5)

[Claims] Claim 1: A γ-glutamyl transpeptidase produced by Bacillus layer bacteria, which has the following properties: (a)
Substrate specificity; has the activity of hydrolyzing D-γ-glutamyl-p-nitroanilide and transferring the γ-glutamyl group in the compound to an amino acid or peptide containing D-glutamic acid; (b) optimum pH; Optimal pH for γ-glutamyl group transfer reaction
is 8.8; (c) the optimum temperature for action; the optimum temperature for the γ-glutamyl group transfer reaction is 55-60°C; and (d) the molecular weight as determined by gel filtration is 30,000; Glutamyl transpeptidase isoenzyme [A]
. Claim 2: A γ-glutamyl transpeptidase produced by bacteria belonging to the genus Bacillus, which shares the physical and chemical properties (a), (b) and (c) described in claim 1, and has a molecular weight determined by gel filtration method. isoenzyme of γ-glutamyl transpeptidase [B] having a value of 35,000. 3. Bacillus licheniformis A35 is a bacterium belonging to the genus Bacillus.
The γ-glutamyl transpeptidase isoenzyme [A] or isoenzyme [B] according to claim 1 or 2, wherein the γ-glutamyl transpeptidase isoenzyme [A] or isoenzyme [B] is S. formis A35). 4. A mixture of the isoenzyme [A] according to claim 1 and the isoenzyme [B] according to claim 2. 5. γ- produced by the same bacterium of the genus Bacillus as the isoenzyme [A] according to claim 1, the isoenzyme [B] according to claim 2, or the isoenzyme mixture according to claim 4; Mixture of glutamyl transpeptidase with other isoenzymes.