JPH0630571B2 - Thermostable mannitol dehydrogenase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to mannitol dehydrogenase having excellent thermostability. Especially Actinobacillus
cillus) to a thermostable mannitol dehydrogenase obtained from a microorganism belonging to the genus Cillus. The oxygen of the present invention is serum,
It is used for measuring the enzymatic activity of α-amylase and the like in urine.

(Prior Art) Escherichia coli (Escherichia coli) has been used as a microorganism that conventionally produces mannitol dehydrogenase.
Aerobacter aerogene
s), Aspergillus oryza
e) Aspergillus niger, Aspergillus candidu
s) is known.

However, the mannitol dehydrogenase produced by these microorganisms was not sufficiently thermostable.

Journal of Bacteriology Vol. 89, No. 2, No. 32
On page 6 (1965), Aspergillus Orize
It is described that NAD-linked mannitol dehydrogenase derived from gillus oryzae) has a residual activity of 80% when treated at pH 7.6, 37 ° C. for 3 minutes.

(Problems to be Solved by the Invention) The inventors of the present invention have made various studies for the purpose of finding out mannitol dehydrogenase which is superior in thermostability to conventional mannitol dehydrogenase produced by microorganisms.

(Means for Solving Problems) The present inventors have found the mannitol dehydrogenase excellent in thermostability from microorganisms belonging to the genus Actinobacillus and arrived at the present invention. That is, the present invention is a thermostable mannitol dehydrogenase characterized by having the following properties.

Reaction formula: Substrate specificity: Acts specifically on mannitol.

Optimum pH: 9.0 to 11.0 Optimum temperature: 50 ° C. Km value: about 9.7 to 10 ⁻⁴ M Molecular weight: about 57,000 (gel filtration method) Isoelectric point: 3.9 ± 0. 1 (Focus electrophoresis with carrier ampholite) Thermal stability: pH 7.6, stable up to 45 ° C. for 3 minutes treatment.

Further, the present invention is characterized in that a thermostable mannitol dehydrogenase-producing bacterium having the above-mentioned properties belonging to the genus Actinobacillus is cultured in a nutrient medium, and a thermostable mannitol dehydrogenase having the following properties is collected from the culture. Is a method for producing thermostable mannitol dehydrogenase.

The mannitol dehydrogenase of the present invention is produced by a microorganism belonging to the genus Actinobacillus, for example, Actinobacillus sp. CRH-1271 (Microtechnology Research Institute No. 7721).

The mannitol dehydrogenase of the present invention is obtained by culturing the above strain in a nutrient medium, separating and purifying from the culture.

As the carbon source of the nutrient medium, creatine, creatinine, glucose, sucrose, fructose, starch,
Molasses, alcohols, organic acids can be used, peptone, meat extract, yeast extract, corn steep liquor, etc. can be used as natural nutrition sources, and creatine, creatinine, ammonia, ammonium sulfate, ammonium chloride, urea, etc. can be used as nitrogen sources. And potassium phosphate, potassium chloride, sodium chloride, magnesium sulfate and the like can be used as the inorganic salts.

Each of these nutrient sources can also be used alone,
Also, they can be used in combination. Culture of the strain can be carried out by shaking culture or aeration-agitation culture. Generally, culture temperature is 25-35 ℃, medium pH is 6.5-
The mannitol dehydrogenase is produced and accumulated in the cells when the culture is carried out for 1 to 2 days. Culture conditions depend on the strain used, medium composition, etc.
It is natural to set the maximum production amount of mannitol dehydrogenase.

When collecting the mannitol dehydrogenase produced and accumulated by the method, the culture solution is subjected to an operation such as centrifugal separation and filtration to collect the cells, and the obtained cells are crushed with beads or sonicated. Mannitol dehydrogenase is taken out from the bacterial cells by the operations described above. In isolating mannitol dehydrogenase from the thus obtained crude enzyme solution, a method generally used for enzyme purification can be used. For example, salting out, organic solvent, dialysis,
Methods such as isoelectric point precipitation ion exchange method and gel filtration can be used in combination. In order to obtain the purified mannitol dehydrogenase, for example, the crude enzyme solution is centrifuged to obtain the supernatant. Furthermore, the ammonium sulfate salting out fraction (0.35-0.55 saturation) is obtained from the supernatant. After one-component dialysis, DEAE-Sepharose CL4B ion exchanger is adsorbed and eluted. Highly purified mannitol dehydrogenase can be isolated by concentrating the active fraction and then performing gel filtration on Sephaacrylic S200.

The activity of the obtained enzyme (crude enzyme solution or purified enzyme) is measured by the following method. One unit is the enzyme activity that produces 1 μmol NADH per minute under the following conditions.

Reagent for activity measurement (A) 6.0 mM NAD solution (0.429 g of NAD was dissolved in distilled water to 100 ml)
(B) 0.5 M mannitol (0.911 g of mannitol is dissolved in distilled water to make 10.0 ml) (C) Enzyme solution (enzyme preparation in ice-cooled 60 mM phosphate buffer pH 7.6 0.07 ~ Dilute to 0.30 U / ml) Procedure (1) Add 1.0 ml of 60 mM phosphate buffer pH 7.6 to 0.2 ml of the above reagent (A) and 0.2 ml of the above reagent (A) into a test tube, mix and mix at 37 ° C. Preheat with.

(2) Add 0.1 ml of the above enzyme solution (C) to start the reaction.

(3) Record the change in absorbance at 340 nm for 4 to 5 minutes with a spectrophotometer controlled at 37 ° C, and determine the change in absorbance per minute from the initial linear portion.

(4) Instead of 0.1 ml of enzyme solution (C) in the blind test, 60 mM phosphate buffer p
H7.6 is added, and the same operation as above is performed to obtain the change in absorbance per minute.

a formula 6.22 = NADH millimolar extinction coefficient (cm ² / micromole)
1.0 = optical path length (cm) Actinobacillus sp. CRH-1271
No.) produced mannitol dehydrogenase having the following properties.

Substrate specificity: Acts specifically on mannitol.

Optimum pH: 9.0 to 11.0 (see Fig. 1) Optimum temperature: 50 ° C (see Fig. 2) Km value: about 9.7 x 10 ^-4 M Molecular weight: about 57,000 (gel filtration) Method) Isoelectric point: 3.9 ± 0.1 (focus electrophoresis using carrier amphrite) pH stability: pH 9 or less (see Fig. 3) Temperature stability: 45 ° C or less (pH 7.6, treatment for 3 minutes) ) (4th
(See the drawings) (Examples) The present invention will be specifically described with reference to Examples.

Example I 0.2% creatine, 0.5 polypeptone, 0.5% yeast _{extract, 1.4% K 2 HPO 4,} 0.3% KH 2 PO 4, 0.01% MgSO 4 · 7H 2 O,
Put 50 ml of medium consisting of pH 7.0 into a 500 ml Sakaguchi flask,
It was sterilized by autoclaving at 121 ° C for 10 minutes. Actinobacillus sp. CRH-1271 (Microtechnology Research Institute
No. 721), 1 platinum loop of the above medium was inoculated and shake-cultured at 30 ° C. for 20 hours to obtain a seed culture solution. Separately, 50 ml of the above seed culture was inoculated into a 10-volume jar fermenter containing medium 6 sterilized under the same conditions. 300 rpm, aeration rate 3 / min, 16 at 30 ℃
Incubated for hours. The enzyme activity of the obtained culture solution was 0.29 U / ml.

The culture solution 6 was centrifuged, the bacterial cells were collected and suspended in 50 mM phosphate buffer, and the beads were crushed as 1 (Dynomill KDL).
It was crushed by. The disrupted cell suspension was centrifuged to obtain a supernatant. Ammonium sulfate was added to the supernatant so that the concentration was 0.35, and the mixture was centrifuged to obtain a supernatant. Ammonium sulfate was further added to the supernatant so that the concentration became 0.55, and the mixture was centrifuged to obtain a precipitate. Redissolved in 250 ml of 50 mM phosphate buffer pH 7.5. After redissolving, it was desalted on a Sephadex G-25 column (2) equilibrated with 50 mM phosphate buffer pH 7.5. Deaerate the solution with DEAE-Sepharose CL4B
It was adsorbed on a column of 50 ml and eluted with 0.4 M NaCl. DEAE-
The physicochemical properties of the enzyme were measured using the mannitol fraction of cephalol as a sample, and the results were as described above. The eluate is concentrated by ultrafiltration and subjected to molecular sieves on a Sephaacrylic S-200 column. The specific activity of the active fraction is 0.81 U / mg
It was protein.

(Effect of the Invention) According to the present invention, a thermostable mannitol dehydrogenase having a pH of 7.6 and stable for up to 45 ° C. for 3 minutes is obtained.

[Brief description of drawings]

FIG. 1 shows the relationship between pH and activity of the enzyme obtained by the present invention. FIG. 2 shows the relationship between temperature and activity. Figure 3 shows the pH when treated at 25 ℃ for 16 hours at each pH.
And the relationship between activity. FIG. 4 shows the relationship between temperature and activity when treated at pH 7.6 for 3 minutes at each temperature. FIG. 5 shows the temperature stability of a conventional enzyme (derived from Aspergillus oryzae).

Claims (2)

[Claims] 1. A thermostable mannitol dehydrogenase characterized by having the following properties. Reaction formula: Substrate specificity: Acts specifically on mannitol. Optimum pH: 9.0 to 11.0 Optimum temperature: 50 ° C. Km value: Approximately 9.7 × 10 ⁻⁴ M Molecular weight: Approximately 57,000 (gel filtration method) Isoelectric point: 3.9 ± 0. 1 (Focus electrophoresis with carrier ampholite) Thermal stability: pH 7.6, stable up to 45 ° C. for 3 minutes treatment. 2. A thermostable mannitol dehydrogenase-producing bacterium belonging to the genus Actinobacillus having the following properties-cultivated in a nutrient medium, and a thermostable mannitol dehydrogenase having the following properties-collected from the culture. And a method for producing thermostable mannitol dehydrogenase. Reaction formula: Substrate specificity: Acts specifically on mannitol. Optimum pH: 9.0 to 11.0 Optimum temperature: 50 ° C. Km value: Approximately 9.7 × 10 ⁻⁴ M Molecular weight: Approximately 57,000 (gel filtration method) Isoelectric point: 3.9 ± 0. 1 (Focus electrophoresis with carrier ampholite) Thermal stability: pH 7.6, stable up to 45 ° C. for 3 minutes treatment.