JPH1084946A - Production of lactobacillus fermentum uku-2 strain and d-lactate dehydrogenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain Lactobacillus fermentum UKU-2 strain (FERM P-15779) having an ability to produce D-lactate dehydrogenase so that D-lactate dehydrogenase can be mass-produced which is widely used for a medical field, analysis of foods, etc. SOLUTION: Lactobacillus fermentum UKU-2 strain (FERM P-15779) has an ability to produce D-lactate dehydrogenase. The strain is cultured under anaerobic conditions to harvest D-lactate dehydrogenase to produce D-lactate dehydrogenase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a strain capable of producing D-lactate dehydrogenase (hereinafter abbreviated as D-LDH) and a method for producing D-LDH using the same. Lactobacillus fermentum with high D-LDH producing ability
The present invention relates to a UKU-2 strain and a method for producing D-LDH using the same.

[0002]

2. Description of the Related Art In recent years, enzymes have advantages such as that the reaction takes place under mild conditions, that the binding to a substrate is specific, and that the reaction is easy to optically quantify. It is attracting attention and is widely used in the medical field, food component analysis, and the like. Among them, D-LDH is used in the field of clinical tests and the like for the determination of pyruvate in serum and urine, and for the measurement of transaminase and pyruvate kinase activities. D-LDH is also used for industrial production of D-lactic acid.

Conventionally, microorganisms producing D-LDH include genus Lactobacillus, genus Leuconostoc, and staphylococcus.
hylococcus) and other microorganisms belonging to the genus [Microbiological Reviews
views), 44, 106-139 (1980)
Year)〕. In addition, Japanese Patent Application Laid-Open No. 7-508165 discloses D
-As a microorganism having a high LDH-producing ability, a yeast transformed with a D-LDH gene of the genus Lactobacillus is disclosed, and the D-LDH content of this yeast can be increased to 10 U / mg (protein). ing.

[0004]

However, since the yield of D-LDH is still low even in this yeast, there is a problem that the efficiency of mass production of D-LDH is low. The present invention relates to a microorganism having a high ability to produce D-LDH, and a D-LD using the same.
-It is an object to provide a method for producing LDH.

[0005]

Means for Solving the Problems In order to solve such problems, the present inventors searched for a strain having a higher D-LDH-producing ability than soil, compost, chicken dung, cow dung, etc., and found that Lactobacillus (Lactobacillus ) A strain belonging to the genus having high D-LDH-producing ability was found, and the present invention was completed. That is, the first invention is characterized in that it has a D-LDH-producing ability, and is characterized by having Lactobacillus fermentum (Lactobac
illus fermentum) UKU-2 strain (FERMP-157)
79). Further, a second aspect of the present invention provides a method for producing D-LDH, which comprises culturing the above strain and collecting D-LDH from the culture.

Hereinafter, the present invention will be described in detail. The strain of the present invention is a strain isolated from compost and produces D-LDH which catalyzes a reaction represented by the following chemical formula.

[0007]

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[0008] The bacteriological properties of this strain are shown below. (A) Morphological characteristics Cell shape and size Bacillus Motility No Spore-forming ability

(B) Cultural properties Slant culture Blue colony Liquid culture No gas generation, coagulation, surface growth

(C) Physiological properties Gram staining Positive catalase activity Negative Growth range 15 ° C ungrown 45 ° C grown Oxygen attitude Facultative anaerobic Lactate DL sugars fermentability Arabinose Yes Xylose No Glucose Yes Mannose No Fructose Yes D-galactose Yes Maltose Yes Sucrose Yes Lactose Yes Trehalose No Sorbitol No Mannitol No Amygdalin No Cellobiose No Esculin No Gluconate Yes Meletitose No Melibiose Yes Raffinose Yes Rhamnose No Ribose GC content of in-cell DNA (mol%) 52

From the mycological properties shown above, Bergey's Manual of Systematic Bacteriology
Vol.2 1986 edition and microorganisms Vol.6 page3 1
As a result of a search based on the 990 edition, this strain was found to be a bacterium belonging to Lactobacillus fermentum. Since this strain has a higher D-LDH producing ability than the conventional strain, it was determined to be a new strain, and Lactobacillus fermentum was determined.
) The strain was named UKU-2 strain and deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry. The deposit number is FERM
P-15779.

In the nutrient medium used for culturing the strain of the present invention, glucose, sucrose, maltose and the like can be used as a carbon source, and nitrogen sources can be used as a carbon source.
Inorganic or organic substances such as ammonium sulfate, ammonium chloride, peptone, meat extract and yeast extract can be used. Further, as the inorganic salts, salts such as potassium, sodium, zinc, iron, magnesium, and manganese, and if necessary, trace metal salts and vitamins may be used.

[0013] Culture is usually preferably performed under anaerobic or microaerobic conditions. The culture temperature is 20 to
40 ° C is preferred, more preferably 30 to 40 ° C, and particularly preferably 35 to 40 ° C. Under these conditions, 3 to 3
By culturing for 0 hour, preferably for 6 to 10 hours,
D-LDH is produced and accumulated in the cells.

From the cells cultured in this manner, D-LD
As a method for purifying H, it may be purified by a method generally used for purifying D-LDH. Specifically, first, the cells cultured as described above, for example,
After autolysis, sonication, crushing by French press, etc., surfactant treatment, lysozyme treatment, etc., the crude enzyme solution of D-LDH obtained by removing cell debris by centrifugation, etc. is used for chromatography. D-LD by contacting resin
H is adsorbed.

As the resin used at this time, for example, Q-Sepharose FF (manufactured by Pharmacia), DE
Ion exchange resins such as AE-Sepharose (Pharmacia), affinity chromatography resins such as Blue Sepharose CL-6B and Red Sepharose CL-6B (Pharmacia), phenyl Sepharose FF (Pharmacia), butyl toyopearl ( Gel filtration carrier or resin such as a resin for hydrophobic chromatography such as Tosoh) and Ultrogel AcA-34, Sephadex G-100 (manufactured by Pharmacia).

The contact method is not particularly limited, and may be any of a column method and a batch method.
The amount of the solution at this time is not particularly limited, but if it is higher than the adsorption capacity of the resin, it leaks without being adsorbed by the resin.
The amount is preferably 00 times, more preferably 1 to 100 times. In addition, the flow rate in the case of contact by the column method is preferably 0.001 to 1000 column volumes / hour.

Next, after washing the resin, D-LDH is eluted using an eluate. Water and buffers such as acetic acid, citric acid, phosphoric acid, imidazole, and good can be used as an eluate when D-LDH is eluted from the resin, and even if a salt such as NaCl or KCl is added to these, Good.
The salt concentration of the eluate is preferably from 0.001 to 4M, more preferably from 0.1 to 1M. The pH of the eluate is preferably from 1 to 13, and more preferably from 4 to 10. In addition, the amount of the eluate is preferably 0.1 to 1000 times, more preferably 4 to 10 times the volume of the column.

The physicochemical properties of D-LDH thus purified are shown below. (1) Action The catalyst catalyzes the reaction represented by the following reaction formula.

[0019]

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(2) Substrate specificity Km value for D-lactic acid: 18.5 mM Km value for pyruvate: 0.69 mM Km value for NAD: 0.36 mM Km value for NADH: 0.12 mM (3) Optimum pH It has an optimum pH around pH 8 (measuring temperature 30 ° C)
It is stable at 4 to 8 (measuring temperature 4 ° C). (4) Suitable temperature range up to 40 ° C. (in 100 mM phosphate buffer, pH 7.5
Measured with). (5) Molecular weight It is about 90,000 as measured by gel filtration, and SDS
It is 38,000 as measured by electrophoresis.

[0021]

Next, the present invention will be described specifically with reference to examples. The activity of D-LDH was measured by adding an enzyme solution to a Tris buffer (pH 7.7) containing 3.0 mM pyruvate and 0.25 mM NADH, gently mixing, and then using a spectrophotometer (manufactured by Hitachi, Ltd.). ) Was performed by measuring the change in absorbance at 340 nm. The measurement is 3
Perform at 0 ° C. and add 1 micromolar NADH per minute to NA
The amount of enzyme converted to D ⁺ was defined as 1 unit (U). In addition, the amount of protein was measured using a Bio-Rad protein assay kit.
It measured using II (500-0002).

Example 1, Comparative Examples 1 and 2 Glucose 3.0% (expressed in weight%; the same applies hereinafter), Yeast extract 1.0%, Peptone 0.5%, Trisodium citrate dihydrate 0 0.5%, sodium acetate
0.2%, magnesium sulfate heptahydrate 0.02%,
Manganese sulfate tetra-pentahydrate 0.005%, twin 8
0 25% of a medium (pH 6.4) containing 0.1% by volume was charged into a 30-liter jar fermenter, sterilized at 121 ° C. for 15 minutes, and then treated with Lactobacillus.
Lactobacillus fermentum UKU-
Two strains (FERM P-15779) were inoculated, stirred at 140 rpm for 6 hours at 37 ° C.,
The culture was performed while adjusting the pH to 6.4 with N NaOH.
After completion of the culture, the cells are collected by centrifugation to obtain about 250 g.
Was obtained. The obtained wet cells are crushed with a crusher (800b
ar: manufactured by Dainippon Pharmaceutical Co., Ltd.), centrifugation was performed to remove cell debris, and the D-LDH activity of the obtained solution was measured. The D-LDH activity per cell was 1,300.
10,000 U / kg (cells). In addition, D-
LDH activity was 30 U / mg (protein). These results show that the strain of the present invention contains about three times as much D-LDH as the yeast producing Lactobacillus-derived LDH obtained by conventional genetic manipulation.

For comparison, a D-LDH producing strain, Leuconostoc lactis, was used.
is) SHO47 strain (FERM P-13970: Comparative Example 1)) and Leuconostoc lactis (Leuconosto
c lactis) SHO54 strain (FERM P-13971:
Comparative Example 2) was cultured in the same manner as described above. After completion of the culture, the cells were collected by centrifugation, and each of
g, 180 g of wet cells were obtained. Further, when the D-LDH content of each cell was measured in the same manner as above, 2.5 million U and 1.9 million U per kg of the cell, respectively, were obtained.
Met. Table 1 shows the above results.

[0024]

[Table 1]

From the results shown in Table 1, the strain of the present invention was
Other D-LDH producing bacteria SHO47 strain and SHO5
It turns out that it has D-LDH production ability of 8-9.5 times compared with 4 strains.

Example 2 Approximately 50 g of the wet cells obtained in Example 1 was subjected to 2 mM ED.
25 containing TA and 2 mM 2-mercaptoethanol
The suspension was suspended in mM phosphate buffer (pH 8.0) to make up to 250 ml. This was passed through a pressure crusher (800 bar: manufactured by Dainippon Pharmaceutical Co., Ltd.) to be crushed to obtain a crushed liquid. To 250 ml of the obtained crushed liquid, 38 ml of a 1% by weight aqueous solution of Uniflocker UF305 (manufactured by Unitika), which is a polymer flocculant, was slowly added for the purpose of removing nucleic acids, and the mixture was stirred for about 30 minutes. The precipitate was removed by centrifugation to obtain 300 ml of a crude enzyme solution of D-LDH.

To 300 ml of the crude enzyme solution was added dipotassium phosphate to adjust the pH to 8.0, and this was previously adjusted to a 25 mM phosphate buffer (pH 8.0) containing 2 mM EDTA and 2 mM 2-mercaptoethanol. DEAE Sepharose FF (Pharmacia) equilibrated in 0)
After passing through a column (35 ml) to adsorb D-LDH, the column was washed with the same buffer, and the same buffer was used to gradually increase the concentration of potassium chloride (0 to 0.4 M) for elution. However, when the potassium chloride concentration was around 0.3 M, D-LDH
Eluted.

Ammonium sulfate was added to the eluted fraction to a concentration of 25% by weight, and this was previously added to a 25 mM phosphate buffer (pH: 25%) containing 25% by weight of ammonium sulfate, 2 mM of EDTA and 2 mM of 2-mercaptoethanol.
8.0) and passed through a 10 ml phenyl sepharose FF (Pharmacia) column equilibrated in D-.
After the LDH is adsorbed, it is washed with the same buffer, and the ammonium sulfate concentration is gradually reduced using the same buffer (25 to 0).
Weight%).
D-LDH eluted around 0 to 20% by weight.

The eluted fractions are collected and desalted by dialysis, and then 25 mM phosphate buffer (pH 8.20) containing 2 mM EDTA and 2 mM 2-mercaptoethanol in advance.
The solution was passed through a 5 ml DEAE trisacryl (Pharmacia LKB) column equilibrated in step 0), and the solution was subjected to DL.
DH was adsorbed. The column was washed with the same buffer, and the same buffer was used to gradually increase the concentration of potassium chloride (0
〜0.3 M) When elution was carried out, D-LDH eluted at around 0.15 M potassium chloride concentration.

Ammonium sulfate was added to the eluted fraction to a concentration of 25% by weight, and a 25 mM phosphate buffer (pH 8.0) containing 25% by weight of ammonium sulfate, 2 mM of EDTA and 2 mM of 2-mercaptoethanol was previously added.
After adsorbing D-LDH by passing through 5 ml of a butyl toyopearl (manufactured by Tosoh Corporation) column equilibrated with the above, washing with the same buffer, and using the same buffer, the ammonium sulfate concentration was gradually lowered ( When the elution was performed, D-LDH eluted at an ammonium sulfate concentration of about 12 to 20% by weight. The D- thus obtained
The result of polyacrylamide gel electrophoresis of the LDH purified sample was a single band. The D-LDH thus obtained had the same physicochemical properties as described above.
Table 2 shows the results of the above purification.

[0031]

[Table 2]

[0032]

Industrial Applicability The strain of the present invention has a high D-LDH-producing ability, so that D-LDH can be mass-produced. Further, according to the production method of the present invention, D-
LDH can be efficiently mass-produced.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 225) (72) Inventor Munehiko 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Central Research Laboratory (72) Inventor Kazu Uchida No. 23 Uji Kozakura, Uji-city, Kyoto, Japan Unitika's Central Research Laboratory (72) Inventor Hitoshi Kondo 23 Uji Kozakura, Uji-city, Kyoto, Japan Unitika's Central Research Laboratory

Claims (2)

[Claims] Claims: 1. A lactobacillus fermentum (Lactob) having an ability to produce D-lactate dehydrogenase.
acillus fermentum) UKU-2 strain (FERMP-157)
79). 2. The method according to claim 1, wherein the strain according to claim 1 is cultured, and D-lactate dehydrogenase is collected from the culture.
A method for producing lactate dehydrogenase.