JPH0556947B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel pyruvate dehydrogenase and a method for producing the same. More specifically, the present invention relates to a novel pyruvate dehydrogenase with excellent thermostability produced by a microorganism of the genus Lactopacillus and a method for producing the same. The novel pyruvate dehydrogenase of the present invention is an enzyme that generates acetyl phosphate, carbon dioxide, and a reduced electron acceptor from pyruvate, phosphate, and an electron acceptor. It is used to measure enzyme activities such as pyruvate transaminase, glutamate-oxaloacetate transaminase, lactate dehydrogenase, and pyruvate kinase. (Prior art) The so-called Pyruvate Dehydrogenase is a conventionally known pyruvate dehydrogenase.
Pyruvate forming somplex: Lipoamide
oxidoreductase (EC1, 2, 4, 1),
Pyruvate: Ferricytochrom b ₁ oxidoreductase
(EC1, 2, 2, 2), Pyruvate: Ferredoxin
Oxidoreductase (EC1, 2, 7, 1) is known (Enzyme Handbook, supervised by Bunji Maruo and Nobuo Tamiya, publisher: Asakura Shoten). These enzymes and the novel pyruvate dehydrogenase of the present invention have different reaction modes. In addition, pyruvate oxidase (EC1, 2, 3,
3) (Enzyme Handbook, supervised by Bunji Maruo and Nobuo Tamiya, publisher: Asakura Shoten), but it is known that pyruvate oxidase acts best on oxygen as an electron transport system. [Fed.Proc.Volume 13 734
~738 pages (1954)]. (Problems to be Solved by the Invention) Conventionally, colorimetric determination of pyruvate in serum or urine has been carried out using pyruvate oxidase, but pyruvate oxidase does not have sufficient heat resistance and is sensitive to dissolved oxygen. Due to these problems, it was unsuitable for dry chemistry. (Means for Solving the Problems) Based on the above background, the present inventors have developed a pyruvate dehydrogenase produced by microorganisms that has superior thermostability than conventional pyruvate oxidase and is not affected by dissolved oxygen. As a result of intensive research to find out, a novel pyruvate dehydrogenase with excellent thermostability was discovered from a microorganism belonging to the genus Lactobacillus. That is, the present invention is a novel pyruvate dehydrogenase characterized by having the following properties. Catalyze the following reaction. Pyruvate + phosphoric acid + electron acceptor --- → acetyl phosphate + CO ₂ + reduced electron acceptor Acts specifically on pyruvic acid. As electron carriers, 1-methoxyphenazine methosulfate (PMS), methylene blue, ferricyanide and 2,6-dichlorophenol indophenol (DCPIP) rather than oxygen
easy to act on. Stable pH is around 5-8. The optimum temperature is approximately 55°C. Stable up to 50℃ when treated at PH6.3 for 15 minutes. The present invention also provides a novel pyruvate dehydrogenase characterized by culturing a strain belonging to the genus Lactobacillus that produces a novel pyruvate dehydrogenase having the above-mentioned properties, and collecting the novel pyruvate dehydrogenase from the culture. This is the manufacturing method. As the microorganism used in the present invention, any strain of microorganism can be used as long as it has a novel ability to produce pyruvate dehydrogenase with excellent thermal stability. Suitable examples of microorganisms used in the present invention include Lactobacillus delbrittii subsp. lactes JCM 1063, JCM 1248, Lactobacillus delbrittii IFO 3202, IFO
3534, Lactobacillus salivarium subsp. salivarium JCM1231. In producing the enzyme of the present invention, a novel pyruvate dehydrogenase-producing bacterium with excellent thermostability is cultured by a conventional method. As for the medium composition, either a synthetic medium or a natural medium can be used as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients that can be assimilated by the bacterial strain used. For example, glucose, sucrose, dextrin, pyruvic acid, molasses, etc. are used as carbon sources. As nitrogen sources, inorganic or organic nitrogen compounds are used, such as nitrogen-containing natural products such as peptone, meat extract, yeast extract, and casein hydrolyzate, and amino acids such as ammonium chloride, ammonium sulfate, ammonium citrate, and glutamic acid. Ru. Culture is usually carried out by shaking culture or aerated agitation culture. The culture temperature ranges from 20℃ to 50℃, preferably
Around 37℃, culture pH in the range of 5 to 8, preferably PH
It is best to control it to 6-7. It can be carried out under conditions other than these if the strain used grows. The culture period is usually 1 to 4 days, and new pyruvate dehydrogenase is produced and accumulated within the bacterial cells. A commonly used purification method may be used to purify the enzyme of the present invention. For example, any method such as ultrasonic crushing, mechanical crushing using glass beads, French press, surfactant, etc. may be used for the extraction method. Furthermore, for the extract, known salting methods such as ammonium sulfate and Glauber's salt, metal coagulation methods such as magnesium chloride and calcium chloride, coagulation methods such as protamine and polyethyleneimine, and DEAE (diethylaminoethyl) cephalose and CM (carboxymethyl) cephalose are used. It can be purified by ion exchange chromatography such as Next, Clatobacillus delbrichii, which is an example of the novel pyruvate dehydrogenase of the present invention,
We describe the physicochemical properties of pyruvate dehydrogenase produced by IFO3202. (1) Action Pyruvate + phosphoric acid + electron acceptor ---→ acetyl phosphate + CO ₂ + reduced receptor Substrate specificity Oxaloacetate, DL-lactic acid, acetic acid or α
- Does not react with ketoglutaric acid and is specific for pyruvate. The enzyme activity was shown when only oxygen was used in the electron transport chain and when various electron acceptors were added. Relative activity is shown with the activity in the presence of oxygen alone as 100. Also, a known pyruvate oxidase that shows a similar reaction formula [Fed.
Proc. 13, pp. 734-738 (1954)].

[Table] Abbreviations indicate the following. 1-MethoxyPMS: 1-methoxyphenazine methosulfate DCPIP: 2,6-dichlorophenol indophenol These results show that the enzyme of the present invention is more likely to act on electron acceptors other than oxygen as an electron transport system. (3) Optimal PH New reaction PH of pyruvate dehydrogenase
We sought the influence of The buffer solution used was 50mM K-phosphate buffer. The results of measuring the novel pyruvate dehydrogenase activity at each pH are shown in Figure 1, and the optimum pH was around 6.2 to 6.3. (4) Stable PH buffer includes 50mM acetate buffer (PH3-6),
50mM K-phosphate buffer (PH5.5-8), 50mM
Using Tris-HCl buffer (PH8-9),
The mixture was incubated at ℃ for 20 hours and its residual activity was measured. As a result, as shown in Figure 2, the stable pH is 5~
It was around 8. (5) Optimal temperature Enzyme activity was measured at each temperature. As shown in Figure 3, the optimum temperature was around 55°C. (6) Thermostability Novel pyruvate dehydrogenase at 50mM
After heating at 40-60°C for 15 minutes in K-phosphate buffer, pH 6.3, residual activity was measured. As a result, as shown in Figure 4, it was stable up to 50°C. (7) Material effects The following table shows the enzyme activity as relative activity when the following substances are removed from the reaction system shown in the enzyme activity measurement method. In addition, in the case of phosphoric acid removal, 50mM acetate buffer PH6.3 was used as a buffer solution.

[Table] Abbreviations indicate the following. TPP: Thiamine pyrophosphate FAD: Flavin adenine dinucleotide From the above, it was found that this enzyme requires TPP, FAD, and Mg ²⁺ as cofactors, and phosphate as a substrate. Enzyme activity was measured using the following various metals instead of magnesium sulfate in the reaction system shown in the enzyme activity measurement method. The concentration of each metal in the reaction solution was 10 mM, and the relative activity was expressed with the activity of magnesium sulfate as 100.

[Table] As a result, enzyme activity was expressed by Mg ²⁺ , Co ²⁺ , Ca ²⁺ , Zn ²⁺ and Ni ²⁺ . From the above results, this enzyme is clearly a novel pyruvate dehydrogenase with excellent thermostability. Next, a novel method for measuring the activity of pyruvate dehydrogenase of the present invention will be described. 0.15M K-phosphate buffer, PH6.3 10ml 3mM TPP 2ml 0.15mM FAD・Na ₂ 2ml 15mM EDTA・Na ₂ 2ml 0.15M MgSO ₄ 2ml 1mM 1-methoxy PMS-10mM NTB 3ml 10% Triton X-100 1.5ml Distilled water 2.5 ml After preparing the above reaction solution, take 2.5 ml into a test tube, add 0.5 ml of 0.3M potassium pyruvate aqueous solution, and preheat at 37°C for 5 minutes. Enzyme solution 0.05ml
After adding and gently mixing, the change in absorbance per minute at 570 nm is determined using a spectrophotometer controlled at 37°C using water as a control (ΔODTest). For blind testing, 0.05 ml of enzyme dilution solution 50 mM K-phosphate buffer, PH 6.3 is added instead of the enzyme solution, and the same procedure as above is performed to determine the change in absorbance per minute (ΔOD blank). The novel pyruvate dehydrogenase activity was expressed as 1 unit (U) of the enzyme that produced 1/2 micromole of diformazan per minute under the above conditions. (Example) The present invention will be specifically illustrated below with reference to Examples. Example 1 Meat extract 0.5%, polypeptone 2%, yeast extract 1%, _{K2HPO4 ・ 3H2O1} %, _MgSO4・
Transfer 90 ml of a medium (PH6.8) containing 7H ₂ O 0.02% and MnSO ₄ 7H ₂ O 0.02% to a 500 ml Sakaguchi flask, and add 121
Autoclave was performed at ℃ for 15 minutes. After cooling, 10 ml of separately sterilized 20% sodium pyruvate aqueous solution (PH5.0) was added aseptically. A loopful of Lactobacillus delbrittii IFO3202 was inoculated into the same medium as a seed culture, and cultured with shaking at 37°C for 24 hours to obtain a seed culture solution. Next, the same medium 5.4 was transferred to a 10-year fermenter, autoclaved at 121°C for 15 minutes, and after cooling, 600 ml of a separately sterilized 20% sodium pyruvate aqueous solution was added aseptically.
Transfer 100ml of the seed culture solution to this, set the speed at 300 rpm, and the aeration rate to 2.
/min at 37°C for 24 hours. The pyruvate dehydrogenase activity at the end of the culture was 0.35 U/ml. Culture solution 6 was collected by centrifugation, and 50mM K
- Suspended in 100 ml of phosphate buffer pH7.0. The mixture was treated with an ultrasonic crusher (manufactured by Kaiyo Denki, 19KHz) for 15 minutes, and then centrifuged to obtain a supernatant. 50% supernatant
Equilibrated with mM K-phosphate buffer PH7.0
DEAE-Sepharose CL-6B (manufactured by Pharmacia)
Subjected to column chromatography, 0-0.5M
Pyruvate dehydrogenase activity was obtained in the NaCl elution fraction. After filtering the eluate using an ultrafilter,
Gel filtration was performed using Sephadex G-200 (manufactured by Pharmacia) equilibrated with 50 mM K-phosphate buffer and pH 7.0. As a result, 840U of pyruvate dehydrogenase was obtained. (Effects of the Invention) The present invention provides a novel pyruvate dehydrogenase that has excellent thermal stability and is not affected by dissolved oxygen.

[Brief explanation of the drawing]

FIG. 1 shows the optimum pH of the novel pyruvate dehydrogenase of the present invention. FIG. 2 shows the stable PH of the novel pyruvate dehydrogenase of the present invention. In the figure, ○-○ are 50mM acetate buffer, ●-● are 50mM K
- Phosphate buffer, △-△ indicates 50mM Tris-HCl buffer. FIG. 3 shows the optimum temperature of the novel pyruvate dehydronase of the present invention. FIG. 4 shows the thermostability of the novel pyruvate dehydrogenase of the present invention.

Claims (1)

[Scope of Claims] 1. A novel pyruvate dehydrogenase characterized by having the following properties. Catalyze the following reaction. Pyruvate + phosphoric acid + electron acceptor --- → acetyl phosphate + CO ₂ + reduced electron acceptor Acts specifically on pyruvic acid. As electron carriers, 1-methoxyphenazine methosulfate (PMS), methylene blue, ferricyanide and 2,6-dichlorophenol indophenol (DCPIP) rather than oxygen
easy to act on. Stable pH is around 5-8. The optimum temperature is approximately 55°C. Stable up to 50℃ when treated at PH6.3 for 15 minutes. 2. A method for producing a novel pyruvate dehydrogenase, which comprises culturing a strain belonging to the genus Lactobacillus that produces a novel pyruvate dehydrogenase having the following properties, and collecting the novel pyruvate dehydrogenase from the culture. Catalyze the following reaction. Pyruvate + phosphoric acid + electron acceptor --- → acetyl phosphate + CO ₂ + reduced electron acceptor Acts specifically on pyruvic acid. As electron carriers, 1-methoxyphenazine methosulfate (PMS), methylene blue, ferricyanide and 2,6-dichlorophenol indophenol (DCPIP) rather than oxygen
easy to act on. Stable pH is around 5-8. The optimum temperature is approximately 55°C. Stable up to 50℃ when treated at PH6.3 for 15 minutes.