JPH04281787A - Production of gamma-glutamyltranspeptidase - Google Patents

Abstract

PURPOSE:To obtain in high yield the title enzyme for e.g. medicines by aerobically incubating in a yeast extract-contg. medium gamma-- glutamyltranspeptidase-productive bacteria belonging to Bacillus and by collecting the product with the production of viscosityraising substances suppressed. CONSTITUTION:Bacteria belonging to Bacillus capable of producing gamma- glutamyltranspeptidase (gamma-GTP) [e.g. Bacillus subtilis Asahikawa (FERM P-12056)] is put to shaking culture under aerobic conditions at 30 deg.C for 3 days in a medium containing yeast extract, glucose, peptone, etc., followed by centrifugation at low temperatures to remove microbial cells, and acetone at 30 deg.C is added to the supernatant so as to come to 45vol% in the final concentration followed by centrifugation at-5 deg.C and 10000rpm and collection of protein, which is, in turn, subjected to anion exchange column chromatography and hydroxyapatite column chromatography in succession, to effect purification, thus giving the objective gamma-GTP.

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). 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, increases the solubility of amino acids by γ-glutamylation, and protects amino acids 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.

For example, H. Suzuki et al. used a genetically modified Escherichia coli strain to produce a purified enzyme that was several dozen times that of the parent strain, but its specific activity was only 3 U/mg.
Production volume is also low (H. Suzuki et al.: Bioc
hm. Biophys. Res. Commun. Vol.
．． 150 p33 (1988)). As described above, conventionally known animal and plant cells and microorganisms all have low productivity and are difficult to separate and purify because they mainly exist in cell membranes, making them unsuitable for industrial production.

[0004]Recently, the present inventor has studied bacteria of the genus Bacillus, particularly Bacillus subtilis.
B. ubtilis, hereinafter referred to as B. ubtilis. Bacillus subtilis) and Bacillus licheniformis
licheniformis, hereinafter referred to as B. licheniformis. licheniformis) produces γ-GTP at high concentrations, and has already announced it (C. Cheng, Y. Asad
aand T. Aida, Agric. Biol. Ch
em. , Vol53, 2369, 1989).

Although the productivity of these bacteria is much higher than that of conventional animal and plant cells and microorganisms, it is still insufficient, and moreover, these bacteria of the genus Bacillus produce γ-d, which is the main component of the mucilage of natto. 1. Polyglutamic acid (hereinafter referred to as γ-P)
It was difficult to separate and purify γ-GTP because the viscosity of the medium became stronger as the culture progressed, and γ-PGA itself made it difficult to separate γ-GTP. .

[0006]

Problems to be Solved by the Invention Under these circumstances, the inventors of the present application have focused on the development of bacteria belonging to the genus Bacillus, particularly B. licheniformis or B. We have conducted intensive research on a method of producing γ-GTA at higher concentrations using S. subtilis without co-producing γ-PGA. As a result, they found that under specific culture conditions, these bacteria produce particularly large amounts of γ-GTA, and do not co-produce γ-PGA.The culture conditions and separation and purification method were determined, and the γ-GTA of the present invention was determined. GT
A method for manufacturing P was completed.

[0007] According to the production method of the present invention, in addition to features such as high production concentration of γ-GTP and no co-production of γ-PGA, γ-GTP is produced outside the bacterial body, so separation and recovery is extremely easy. It is. Moreover, it has many features such as no need to add glutamic acid to the culture medium.

[0008]

[Means for Solving the Problems] In the above-mentioned situation, the method for producing γ-GTP disclosed by the present invention involves culturing Bacillus bacteria under aerobic conditions in a medium containing yeast extract, and extracting γ-GTP from the culture medium. -γ- characterized by collecting GTP
This is a method for producing GTP. Bacteria of the genus Bacillus are B. subtilis or B. Particularly preferred is one selected from any of the P. licheniformis species because of its high production concentration. B. subtilis, for example, B. subtilis. subtilis IFO3022 strain, B. subtilis IFO strain 3335, or B. subtilis IFO strain 3335, or B. subtilis IFO strain 3335. subtilus A
sahikawa (Microtechnology Research Institute No. 12056), B
．． sub. Sawa et al., and B. Licheniformis, for example, B. Licheniformis ATCC9945, B
．． licheniformis ATCC9945A, B. Licheniformis A35 (Feikoken Bacteria No. 12055) can be used.

[0009] As the medium containing the yeast extract used in the present invention, for example, a commercially available glucose-yeast extract medium (referred to as GY medium) containing 1% glucose and 1% yeast extract and having a pH value of 7.0 can be used. Preferably, the medium does not contain any other nutritional sources other than yeast extract and carbohydrates. Carbohydrates as an energy source do not necessarily have to be glucose. For example, sugars including sucrose, fructose, mannitol, maltose, etc. can be used alone or in combination, or molasses, blackstrap molasses, etc. can also be used.

[0010] Yeast extract is a water-soluble extract of autolyzed yeast cells that is dried and powdered, and commercially available yeast extracts such as Kyokuto Seiyaku Yeast Extract can be used. When the amount of yeast extract is 1.0 to 2.0% based on the medium, it is appropriate to achieve the highest production concentration of γ-GTP. 1
% or more than 2.0%, the production concentration gradually decreases. Yeast extract is a well-known culture medium component.

However, the conventional usage method is to add 0.05 to 0.1% to chemically synthesized media for the purpose of replenishing vitamins and nucleic acid substances or promoting growth; Even when yeast extract is used as a nitrogen source, the usual amount is 0.5% or less. Blending more than 1% is usually not done. In any medium using other nutrient sources instead of yeast extract, the amount of γ-GTP produced is small, or γ-PGA is co-produced, making separation and purification not easy.

[0012] These media may be used as solid media solidified with agar, agarose, etc., or may be used as liquid media. Cultivation must be carried out under aerobic conditions. However, it is not necessary to introduce a large amount of air, and in a liquid medium, a culture method under normal aerobic culture conditions such as shaking culture is sufficient. By culturing in this manner at a normal temperature, for example around 30° C., for several days, γ-GTP is accumulated in the medium at a high concentration.

The method for producing γ-GTP of the present invention has two major features. One of them is γ-GTP as mentioned above.
is produced in large quantities. Ordinary glucose-bouillon medium (referred to as GB medium), casamino acid medium (referred to as CA medium), bouillon medium (referred to as B medium) or Tr.
Compared to the case of using ypticase soy broth medium (referred to as TSB medium), the production amount can be several times to ten times higher.

Another feature of the present invention is that the generated γ-G
The TP separation and purification process can be performed extremely easily. The reason for this is that in the medium used in the present invention, Bacillus bacteria produce γ-GTP extracellularly as well as γ-GTP.
- Since PGA is not co-produced, the culture solution has almost no viscosity, the production of protease and other enzyme proteins is also suppressed, and the composition of the culture solution after culturing is simple. For example, Proteus mirabi
lis) using R. 1 in the method of Nakayama et al.
According to the production method of the present invention, a purification step that required one step can be performed in only three steps.

For example, after culturing Bacillus bacteria in GY medium, the bacterial cells are removed by centrifugation at a low temperature, and acetone at -30°C is added to the supernatant to a final concentration of 45% by volume. Proteins can be collected by centrifugation at -5°C and 10,000 rpm. These proteins are 20mM
Suspend in potassium phosphate buffer (pH 7). To separate γ-GTP from this, depending on the required degree of purification,
This is carried out in the following 1, 2 or 3 steps.

First, DEAE-Cellulofine
The above acetone precipitated suspension is subjected to anion column chromatography packed with A-500 and eluted with a 20 mM potassium phosphate buffer (pH 7) with a sodium chloride concentration gradient. For example, when the concentration of sodium chloride is changed linearly from 0.15M to 0.3M and eluted, the cultured bacteria become B.
lich. In the case of A35, the sodium chloride concentration is 0.
20-0.23M range (referred to as category A) and 0.24
γ-GTP elutes in the range of -0.25M (referred to as section B). Class A and Class B have different molecular weights of γ.
- GTP isoenzyme, and when purification is not required, these two categories may be used separately or in combination.

If purification is required, each fraction is refractionated using the following column chromatography packed with hydroxyapatite HTP. That is, the γ-GTP fraction obtained in the previous step is applied to a chromatography column and eluted with a concentration gradient potassium phosphate buffer. When elution is carried out by linearly changing the concentration from 20mM to 0.4M, for example, the B. lich. When eluting section A of A35 culture solution,
γ-GTP is eluted in a range of 50-100mM (referred to as division A-1) and a range of 110-150mM (referred to as division A-2). Category A-1 and Category A-
2 are γ-GTP isoenzymes with different molecular weights.

That is, B. licheniformis A35 produces three isoenzymes. The aforementioned section B can be similarly purified (referred to as section B'). These divisions A-1, A-2 and B' can be used alone or in combination.

[0019] Practically sufficient purification is achieved through the above steps, but when purifying to a higher degree, each section is
It can be purified by gel chromatography packed with Sephdex G-100. With this method, at least 30U
/mg of purified γ-GTP can be obtained.

[0020]

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples. Prior to the Examples, a method for measuring the production amount of γ-GTP will be explained. Method for measuring γ-GTP production;
The production amount was evaluated based on the ability of the culture solution after separating the bacterial cells to transfer the γ-glutamyl group to an amino acid. Specifically, 0.2 ml of a 1 mM solution of L-γ-glutamyl-p-nitroanilide (hereinafter referred to as L-γ-GpNA) was used as a γ-glutamyl group donor, and glycylglycine (hereinafter referred to as Gly-GpNA) was used as an amino acid. Gly) 20mM solution and 0.3ml of Tris-HCl buffer 50mM (pH
8.0) Add 0.2 ml of the culture solution whose γ-GTP concentration is to be measured to 1.3 ml of the mixed solution, and hold at 37° C. for 30 minutes to transfer the γ-glutamyl group.

Thereafter, 1.0 ml of 3.5N acetic acid aqueous solution was added to stop the reaction, and the absorbance of the reaction solution at 410 nm was measured to determine whether p-nitrosoaniline (ε
=8800). From the amount of p-nitrosoaniline produced, the γ-GTP concentration is calculated as activity units per ml of culture solution using the following formula. U/ml=absorbance (410nm)*0.003*1
06/8800*0.2*20

Examples 1 to 6. As a preculture, 5 ml of GB medium with the following composition was placed in each of six test tubes, and after sterilization, each strain was inoculated with 1ase of the strains listed in Table 1, and incubated at 30°C for 2 hours.
The culture was incubated with shaking for 2 hours. glucose 1% peptone
1% bouillon
1% sodium chloride 0
．． 5% pH
7.0

Next, six test tubes containing 10 ml of sterilized GY medium containing 1% glucose and 1% yeast extract and having a pH value of 7.0 were prepared, and each test tube was filled with 0.0 ml of the precultured culture solution. 2 ml was inoculated and the main culture was performed at 30°C for 3 days with shaking. As a result, the concentrations of γ-GTP accumulated in the main culture medium were as shown in Table 1, and no production of γ-PGA was observed in either case.

Comparative Examples 1 to 6. Add the same G as the pre-culture to the main culture solution.
Examples 1-6 were repeated except that B medium was used. The results are shown in Table 1.

Comparative Examples 7 to 12. Examples 1 to 6 were repeated except that the following CA medium containing casamino acids was used in the main culture solution instead of yeast extract. The results are shown in Table 1. Casamino acids 1% glucose
1% pH
7.0

Comparative Example 13~
18. Examples 1 to 6 were repeated except that the following medium B containing bouillon was used instead of yeast extract as the main culture solution. The results are shown in Table 1. Bouillon 1% glucose 1% pH
7.0

Comparative Example 19
~24. Examples 1 to 6 were repeated except that the following peptone medium (referred to as P medium) containing peptone was used in the main culture solution instead of yeast extract. The results are shown in Table 1. Peptone 1% glucose 1% pH
7.0

Comparative Example 25
~30. Examples 1 to 6 were repeated except that TSB medium having the following composition was used as the main culture solution. The results are shown in Table 1. Trypticase Soy broth 1% glucose 1% KNO3
0.5% pH
7.4

[0029]
Comparative Examples 31-36. Examples 1 to 6 were repeated except that M medium having the following composition was used alone as the main culture solution. The results are shown in Table 1. However, in this medium, although the production concentration of γ-GTP was relatively high, a large amount of γ-PGA was produced in addition to γ-GTP. Glucose 3%NH4Cl
1%K2HPO4
0.15%MgSO4
・7H2O 0.035%Mn
SO4 ・2-6H2O 0.005
%CaCO3 3%
pH 7.2

Comparative Examples 37-42. Examples 1 to 6 were repeated except that the main culture solution was the GM medium prepared by adding 1% L-glutamic acid to the M medium described above. The results are shown in Table 1. However, in this medium, although the production concentration of γ-GTP was high, a large amount of γ-PGA was produced in addition to γ-GTP.

Example 7. B. to cultured bacteria. The operations of Examples 1 to 6 were repeated using A35 strain of Licheniformis. At this time, the production concentration of γ-GTP when changing the concentration of yeast extract in GY medium in the range of 1% to 4% is shown in Figure 1.
It was shown to.

Example 8. B. to cultured bacteria. The operations of Examples 1 to 6 were repeated using A35 strain of Licheniformis. At this time, instead of glucose in GY medium, sucrose, fructose,
Maltose or mannitol was used, respectively. The production concentrations of γ-GTP in these media are shown in FIG. 2.

[0033]

[Table 1]

[0034]

[Effect] By the production method of the present invention, it has become possible to produce γ-GTP at a high concentration. Furthermore, according to the production method of the present invention, the generated γ-GTP can be separated and purified extremely easily. As a result, the economic benefits obtained are immeasurable.

Bacillus subtilis Asahika
Bacillus licheniformis A35 has been deposited with the National Institute of Microbiological Technology, Agency of Industrial Science and Technology as FEM Microbial Deposit No. 12066 (FERM P-12066), and Bacillus licheniformis A35 has been deposited with FEM Bacterial Deposit No. 12055 (FERM P-1). 1
2055).

[Brief explanation of the drawing]

FIG. 1 shows the relationship between the concentration of yeast extract in the medium and the concentration of γ-GTP production.

FIG. 2 shows the relationship between the type of carbohydrate in the medium and the concentration of γ-GTP produced.

Claims (5)

[Claims] Claim 1: Bacillus bacteria capable of producing γ-glutamyl transpeptidase are cultured under aerobic conditions in a medium containing yeast extract, and γ-glutamyl transpeptidase is collected from the culture medium. A method for producing γ-glutamyl transpeptidase. 2. The bacterium belonging to the genus Bacillus is Bacillus subtilis or Bacillus licheniformis.
2. The method for producing γ-glutamyl transpeptidase according to claim 1, wherein the γ-glutamyl transpeptidase is a bacterium of the same type. 3. The method for producing γ-glutamyl transpeptidase according to claim 1, wherein the medium is a medium to which no other nutrient source is added other than yeast extract and carbohydrates. 4. The method for producing γ-glutamyl transpeptidase according to claim 3, wherein the amount of yeast extract is 1% or more. Claim 5: Bacillus subtilis IFO strain 3022, Bacillus subtilis IFO
3335 strain, Bacillus subtilis Asahikawa (February International Laboratories No. 12056), Bacillus liheniformis ATCC9945, Bacillus liheniformis ATCC9945A, and Bacillus liheniformis A35 (February International Laboratories No. 12055). γ according to claim 1 characterized in
- A method for producing glutamyl transpeptidase.