JP3188576B2 - Bacterial strain producing 6-phosphogluconate dehydrogenase and method for mass-producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a 6-phosphogluconate dehydrogenase (hereinafter referred to as a 6-phosphogluconate dehydrogenase) which specifically acts on nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD).
PGDH (abbreviated as PGDH) and a method for mass-producing these bacterial strains.

[0002]

2. Description of the Related Art In recent years, enzymes have attracted attention for their excellent properties such as the stability of their reactions, their specific binding to substrates, and the ease of optical quantification. It is widely used as a catalyst. Above all, 6PGDH
Is particularly suitable for glucose-6-phosphate dehydrogenase (hereinafter G6P
Its usefulness has been pointed out in that high measurement sensitivity can be obtained when glucose 6-phosphate (hereinafter abbreviated as G6P) is quantified in the presence of DH (abbreviated as DH). That is, in the case of such G6P measurement, generally, reduced NAD (hereinafter abbreviated as NADH) or reduced nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADPH) generated by the reaction of G6PDH or 6PGDH. Absorbance at 340 nm of nitroblue tetrazolium (hereinafter referred to as NBT) using diaphorase or phenazine methosulfate (hereinafter abbreviated as PMS) or a derivative thereof with NAD (P) H as a substrate. Abbreviations), 2,6-dichlorophenolindophenol (hereinafter abbreviated as DCPIP), etc., can be reduced in color and measured in the visible region.

[0003] When such various analytes via G6P or 6PG are measured in the visible region, diaphorase has a high specificity for NADH and can be applied to PMS, so that it acts specifically on NAD. It is desirable to use 6PGDH. 6PGDH has a coenzyme
Those specifically reacting with nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP) (European Journal of Biochemistry, Volume 1, 170)
Page (1967)), acting on both NAD and NADP (Agricultural and Biological
Chemistry (Agricultural and Biological Chemistr
y), 46, 391 (1982)), those acting only on NAD (FMBS Microbiology Letters, 52)
Vol., Pp. 199 (1988)). This N
6PGDH acting only on AD is a methanol assimilating bacterium, Methyrobacillus ( Methyrobacillus).
flagellatum ).

[0004]

[Problems to be Solved by the Invention] Methyrobacillus f.
lagellatum ) KT1 strain is 6PG highly specific for NAD
It is known that DH is produced in a small amount.
DH was very unstable and was not practical because it was deactivated by 90% when stored at 4 ° C. for 3 days. Therefore, it has been impossible to actually apply this enzyme to various reagents for measurement. In addition, the enzyme content in the cells was very small, so that the enzyme could not be obtained efficiently.

For these reasons, there has been a strong demand for a means for efficiently obtaining 6PGDH which acts specifically on NAD and has excellent stability. The present invention has been made in view of the above circumstances, and provides a novel bacterial strain which specifically acts on NAD and produces 6PGDH with excellent stability, and a method for mass-producing these bacterial strains. It is intended to be.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems.
Leuconostoc lactis SHO producing GDH
47 (FERM P-13970) and Leuconostot
Ku Lactis SHO54 (FERM P-1397
1) is provided.

[0007] The present invention also provides a method for mass-producing the bacterial strain, wherein the bacterial strain is cultured in a nutrient medium containing 1% by weight or more of yeast extract. Less than,
The present invention will be described in detail. First, the bacterial strain Leuconostoc lactis SHO47 of the present invention (hereinafter, referred to as “SHO47”)
And it may be referred to as SHO47 strain), again leuco
Nostoc lactis SHO54 share (, SHO5
4 strains) are shown in Tables 1 to 4.

[0008]

[Table 1]

[0009]

[Table 2]

[0010]

[Table 3]

[0011]

[Table 4]

From the mycological properties shown in Table 1, Bargey's Manual of Systematic Bacteriology
And Methods in Microbiology (METHODS
IN MICROBIOLOGY) Vol. 16, as a result of the search based on pp. 147-178, is SHO47 shares, Leuconostoc
It was found to be a bacterium belonging to Leuconostoc lactis , but it was different from existing strains and could be judged to be a new strain, so Leuconostoc lactis SHO47
FERM P-13 on November 17, 1993
A deposit number of 970 was obtained.

As a result of a similar search, the SHO54 strain
Leuconostoc lactis (Leuconostoc lacti
s ), a new strain belonging to Leuconostoc
Lactis SHO54 named, 1993 11 May 17
Deposited at the depositary institution as described above, and FERM P-1397
An accession number of 1 was obtained. This SHO47 strain and SHO
As a nitrogen source to be added to the nutrient medium when each of the 54 strains is cultured, inorganic or organic substances such as ammonium sulfate, ammonium chloride, peptone, meat extract, and yeast extract can be used.

As the carbon source, glucose, sucrose, lactose, galactose and the like can be used. Further, as the inorganic salts, potassium, sodium, zinc, iron, magnesium, manganese and other salts,
Vitamins and the like may be used. However, when these bacterial strains are efficiently and massively grown in a short time, 1% by weight of yeast extract is contained in the medium as shown in the test examples described later.
Add above.

The culture is performed under anaerobic or microaerobic conditions. Culture temperature is from 20 ° C to 42 ° C, preferably 30 ° C
~ 42 ° C, optimally between 35 ° C and 42 ° C. Medium pH
Is 5.8 to 7.2, preferably 6.0 to 7.0, and most preferably 6.4 to 6.7. Under these conditions, 3 to 20
By culturing for 6 hours, preferably for 6 to 12 hours.
Cells having PGDH-producing ability can be obtained.

In order to actually isolate and purify 6PGDH,
For example, the SHO47 strain or the SHO54 strain may be cultured as described above, and after the culture, the cells may be recovered from the culture solution by an operation such as centrifugation or filtration, and a crude enzyme solution may be extracted from the cells and purified. For the extraction method, any of ultrasonic crushing, French press, surfactant treatment, lysozyme treatment and the like may be used. After such treatment, cell debris is removed by centrifugation to obtain a crude enzyme solution. For the crude enzyme solution, the desired 6PGDH can be isolated and purified by combining chromatography such as ion exchange chromatography, affinity chromatography, hydrophobic chromatography, and gel filtration chromatography. Q-Sepharose FF (manufactured by Pharmacia), DEAE-Sepharose column (manufactured by Pharmacia) and the like are used for ion exchange chromatography, and Procion Blue H-ERD (I) is used for affinity chromatography.
CI), Blue Sepharose CL-6B column (Pharmacia), hydrophobic chromatography, octyl-Sepharose CL-4B column (Pharmacia), gel filtration chromatography, Sephadex column, etc. Can be In addition to these column chromatography, nucleic acid removal by treatment with streptomycin sulfate or protamine sulfate or salting out of protein by treatment with ammonium sulfate may be performed.

Next, the SHO47 strain and SHO of the present invention
The physicochemical properties of 6PGDH obtained from 54 strains are shown below. (1) Action: catalyzes the reaction of the following reaction formula.

[0018]

Embedded image

(2) Substrate specificity: Assuming that the activity value is 100 when NAD is used as a coenzyme, it is 1 or less when NADP is used. (3) Optimum pH: 6.5 to 8.5 (temperature 30 ° C.) (4) Stable pH: 5 to 9 (5) Range of suitable temperature for action: range from 20 ° C. to 50 ° C.
(MES buffer pH 7.2) (6) Stability: 50 mM MES buffer (pH 7.2)
Medium 2mM ethylene

It has a residual activity of 90% or more after standing at room temperature for 30 days in the presence of diaminetetraacetic acid (hereinafter abbreviated as EDTA). (7) Molecular weight: about 130,000 to 260,000 (Sephacryl S-3
00 gel chromatography method, p
About 130,000 when performed at H6.0. (about 260,000 when performed at pH 8.0) (8) Activity measuring method: 1.7 mM 6PG, 2.0 mM
NAD and 8.0 mM magnesium chloride (MgC
glycyl-glycine buffer solution (pH 7.5) containing l ₂ )
, And the mixture was gently mixed, and the change in absorbance at 340 nm was measured with a spectrophotometer. The measurement was performed at 30 ° C. 1 micromolar NAD per minute
The amount of the enzyme for producing H was defined as 1 unit (U).

Next, the effects of the yeast extract in the mass propagation method of the present invention will be described in detail with reference to test examples. Test Example SHO47 strain and SHO54 strain were inoculated respectively into 400 ml (in a 500 ml Erlenmeyer flask) of MRS medium manufactured by DIFCO, and cultured at 40 ° C. for 12 hours.

On the other hand, 600 ml of each of mediums 1 to 3 having the same composition as in Example 1 except that the yeast extract content was changed to 0.5%, 1% or 2%, was prepared. Each 30 ml of the culture solution of the SHO54 strain was inoculated and cultured at 40 ° C. for 10 hours while stirring at 200 rpm under aeration-free conditions while adjusting the pH to 6.4 with 4N NaOH. After cultivation, 200 ml of each culture
Was centrifuged in the same manner as in Example 1, and the cell yield was calculated.
Further, a crude extract was prepared from the collected cells, and 6PGDH activity was measured. Table 5 shows the results.

[0023]

[Table 5]

As is evident from the results in Table 5, all cultured cells had the same 6PGDH activity per kg of wet cells, but when the yeast extract was used in a medium containing 1% or more, About 4 when the yeast extract is 0.5% medium
The cell yield was ８8 times. From this, 6PGDH
When a large amount of E. coli was obtained, that is, when the bacterial strain was grown in large quantities, it was confirmed that the yeast extract content in the culture medium of the SHO47 strain or the SHO54 strain was 1% or more.

[0025]

Next, the bacterial strain of the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following Examples. Example 1 Glucose 3.0% (representing wt%, the same applies hereinafter), yeast extract 1.0%, peptone 0.5%, trisodium citrate dihydrate 0.5%, sodium acetate 0.2 %, Magnesium sulfate heptahydrate 0.02%, manganese sulfate
25 liters of a medium consisting of 0.005% tetra-pentahydrate, 0.1% (vol%) of Twin 80, pH 6.4 was charged into a 30-liter jar fermenter, and sterilized by autoclaving at 121 ° C. for 15 minutes. , Leuconostoc
Lactis was inoculated (Leuconostoc lactis) SHO47 strain (FERM P-13970), 7 hours at 40 ° C., stirred at 100 rpm, and incubated while adjusting conditions unvented, the pH in 4N NaOH to 6.4. The cells were collected by centrifugation to obtain about 160 g of the cells. The obtained cells were stored in a frozen state.

Next, about 100 g of the frozen cells was added to 20 mM containing 2 mM each of EDTA and 2-mercaptoethanol.
The suspension was suspended in 1 liter of phosphate buffer (pH 7.0), and Triton X-100 was 0.01%, lysozyme was 0.2 mg / ml, and DNase I was 0.2 mg / m2.
After stirring at room temperature for 2 hours, cell debris was removed by centrifugation to obtain a crude enzyme solution containing 6PGDH, and the content of 6PGDH in the cells was measured to be 180,000 U
/ Kg wet cells.

Acetic acid was added to the crude enzyme solution to adjust the pH to 5.8, and a Procion Blue H-BRD column (Procion Blue H-BRD was preliminarily equilibrated with 20 mM phosphate buffer (pH 5.75) was purchased from ICI. Purchase), 1
Elution was performed with a 00 mM phosphate buffer (pH 6.8). After concentration, a 20 mM Tris-HCl buffer (pH 8.
After passing through a Q-Sepharose FF column (Pharmacia) equilibrated in step 0) and eluting with a solution in which KCl was added to the equilibration solution, the active fraction eluted near a KCl concentration of 0.40 to 0.45 M did. After concentrating the eluted fraction, ammonium sulfate was added to a concentration of 0.5 M, and a 20 mM phosphate buffer (pH: 1 M) containing 1 M ammonium sulfate was previously added.
Octyl Sepharose column C equilibrated in 5.5)
The solution was passed through L-4B (Pharmacia) and eluted with the same buffer. The active fractions were collected, concentrated and then desalted. 6PGDH thus obtained was detected as a single band in polyacrylamide gel electrophoresis, and a purified enzyme preparation was obtained. The activity yield is about 55%.
Showed a specific activity of about 110 units per 1 mg of enzyme, and the purification degree was about 33 times assuming that the crude enzyme solution was 1. Example 2 Leuconostoc was prepared using the same medium and conditions as in Example 1.
・ Lactis SHO54 (FERM P-13971)
Was cultured to obtain about 180 g of bacterial cells. The obtained cells were similarly stored in a frozen state.

Next, a crude extract was obtained from these cells in the same manner as in Example 1, and the content of 6PGDH in the cells was measured. As a result, a value of 150,000 U / kg wet cells was shown. further,
A purified enzyme preparation was obtained from the crude extract in the same manner as in Example 1. The activity yield is about 55%, about 130 mg / mg enzyme.
It showed a specific activity of unit, and its purification degree was about 25 times when the crude enzyme solution was 1.

[0029]

As described above in detail, the novel bacterial strain of the present invention and the method for mass-producing the same make it possible to efficiently obtain 6PGDH which acts specifically on NAD and has excellent stability. Become. 6PGDH thus obtained can be widely used for various measurement reagents due to its excellent stability.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/04 C12R 1:46) (72) Inventor Hitomi Ohara Hitoshi Nishi-no-Kyowabaracho, Nakagyo-ku, Kyoto-shi, Kyoto Shimazu Works Sanjo Plant ( 72) Inventor Masato Yahata 1 Nishinokyo Kuwabaracho, Nakagyo-ku, Kyoto-shi, Kyoto Shimazu Manufacturing Co., Ltd. Sanjo Plant (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 1/20 C12N 1/38 WPI / DIALOG BIOSIS / DIALOG

Claims (3)

(57) [Claims] 1. Leuconostoc lactis SHO47 producing 6-phosphogluconate dehydrogenase that specifically acts on nicotinamide adenine dinucleotide
(FERM P-13970). 2. Leuconostoc lactis SHO54 producing 6-phosphogluconate dehydrogenase which specifically acts on nicotinamide adenine dinucleotide.
(FERM P-13971). 3. The leuconostoc lacti according to claim 1.
Scan SHO47 (FERM P-13970) or according to claim 2, Leuconostoc lactis SHO54 (FE
RM P-13971) in a nutrient medium containing 1% by weight or more of yeast extract.