JPH01148184A - Heat-resistant mannitol dehydrogenase and production thereof - Google Patents

Abstract

PURPOSE:To provide a mannitol dehydrogenase obtained from a microbial strain belonging to genus Actinobacillus and having excellent thermal stability compared with the enzyme produced by conventional microbial strain. CONSTITUTION:The objective heat-resistant mannitol dehydrogenase is an enzyme catalyzing the reaction of formula I and is stable up to 45 deg.C by the treatment at pH7.6 for 3min. It can be prepared by culturing a microbial strain capable of producing the heat-resistant mannitol dehydrogenase and belonging to genus Actinobacillus in a nutrient medium and separating the heat-resistant mannitol dehydrogenase from the cultured product. The strain capable of producing the heat-resistant mannitol dehydrogenase is e.g., Actinobacillus sp. CRH-1271 (FERM P-7721).

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to mannitol dehydrogenase with excellent heat resistance. In particular, Actinobacillus (Acti
The enzyme of the present invention relating to heat-stable mannitol dehydrogenase obtained from a microorganism belonging to the genus P. nobacillus is used for measuring the activity of enzymes such as α-amylase in serum and urine.

(Prior art) As a microorganism that produces mannitol dehydrogenase, E. coli (IEsche-ricia coli)
hia coli) + Aerobacter aerogenes, Aspergillus origiae
yzae) Aspergillus niger (eight spergil)
lus nigerL Aspergillus didas (Asp
ergillus candidus).

However, the mannitol dehydrogenase produced by these microorganisms did not have sufficient thermostability.

Journal of Bacteriology Vol. 89 No. 2 No. 326 (1965) describes N.
AD-linked mannitol dehydrogenase is p
H7, 6.37℃. It is described that it has 80% residual activity after treatment for 3 minutes.

(Problems to be Solved by the Invention) The present inventors have made extensive studies with the aim of finding a mannitol dehydrogenase that has better thermal stability than mannitol dehydrogenase produced by conventional microorganisms.

(Means for Solving the Problem) The present inventors have discovered that Actinobacillus
The present invention has been accomplished by discovering a mannitol dehydrogenase with excellent thermostability from a microorganism belonging to the genus P. llus. That is, the present invention catalyzes the following reaction and has a pH of 7.6.
．． It is a heat-stable mannitol dehydrogenase characterized by being stable up to 45°C when treated for 3 minutes.

Reaction formula: The present invention also relates to actinobacillus
The present invention is a method for producing heat-stable mannitol dehydrogenase, which comprises culturing a heat-stable mannitol dehydrogenase-producing bacterium belonging to the genus S. llus in a nutrient medium, and collecting heat-stable mannitol dehydrogenase from the culture.

The mannitol dehydrogenase of the present invention is a microorganism belonging to the genus Actinobacillus, for example, the genus Actinobacillus sp. CRH-1271
(Feikoken Bokuyori No. 7721) etc. are produced.

The mannitol dehydrogenase of the present invention can be obtained by culturing the above-mentioned strain in a nutrient medium, and separating and purifying the culture.

Creatine, creatinine, glucose, sucrose, fructose, starch, blackstrap molasses, alcohols, and organic acids can be used as carbon sources for nutrient media, while peptone, meat extract, yeast extract, and cornstarch liquor can be used as natural nutrient sources. Creatine, creatinine, ammonia, ammonium sulfate, ammonium chloride, urea, etc. can be used as nitrogen sources, and potassium phosphate, potassium chloride, sodium chloride, magnesium sulfate, etc. can be used as inorganic salts.

Each of these nutritional sources can also be used alone;
They can also be used in combination. When culturing a bacterial strain, it can usually be carried out by shaking culture or aerated agitation culture. In general, it is preferable that the culture temperature is 25 to 35°C and the medium pH is 6.5 to 7.5. Usually, mannitol dehydrogenase is produced and accumulated in the bacterial cells when the culture is carried out for 1 to 2 days. It goes without saying that culture conditions should be set so as to maximize the production of mannitol dehydrogenase, depending on the strain used, medium composition, etc.

To collect the mannitol dehydrogenase produced and accumulated by this method, the culture solution is subjected to operations such as centrifugal filtration to collect bacterial cells from the culture solution, and the obtained bacterial cells are crushed with beads or ultrasonic. Mannitol dehydrogenase is extracted from the bacterial cells by the following operations. In isolating mannitol dehydrogenase from the crude enzyme solution thus obtained, a method commonly used for enzyme purification can be used. For example, methods such as salting out, organic solvent, dialysis, isoelectric precipitation, ion exchange, and gel filtration can be used in combination. To obtain purified mannitol dehydrogenase, for example, the crude enzyme solution is centrifuged to obtain a supernatant. Furthermore, the ammonium sulfate salting out fraction of the supernatant (0.35 to 0.55 saturation)
get. - After liquid dialysis, [IEAE-Sepharose CL4
Adsorb and elute on B ion exchanger. After concentrating the active fraction,
Highly purified mannitol dehydrogenase can be isolated by gel filtration using Sephacryl S200.

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

Reagent for activity measurement (^) 6. OmMNAD solution (0,429g NAD
(B) 0.5M mannitol (Dissolve 0,911g of mannitol in distilled water to make 100m) (C) Enzyme solution (60mM of enzyme preparation pre-cooled on ice) Dilute to 0.07-0.30U/+d with phosphate buffer pH 7.6)
Step (1) Add the above reagent (＾)1.0d (B)0 to a test tube.
．． 2mg of 60-M phosphate buffer 9H1,6 for 1.0d
, add 0.8 m of distilled water, mix and preheat at 37°C.

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

(3) 340mm with a spectrophotometer controlled at 37℃
The change in absorbance is recorded for 4 to 5 minutes, and the change in absorbance per minute is determined from the initial linear portion.

(4) For blind testing, use 60ml of enzyme solution (C) instead of 0.1ml.
+wM'J acid buffer pl! 7.6 and perform the same operation as above to determine the change in absorbance per minute.

Calculation formula % Formula % 6.22 = millimolar molecular extinction coefficient of NADll (c
m”/5icr.

mole) 1.0 = optical path length (1) Mannitol dehydrogenase produced by Actinobacillus sp, CRH-1271 (Feikoken Bacteria No. 7721) has the following properties.

■ Substrate specificity: Acts specifically on mannitol.

■ Optimum pH: 9.0 to 11.0 (see Figure 1) ■
Optimal temperature: 50°C (see Figure 2) ■ K-value: Approximately 9.7X10-'M ■ Molecular weight: Approximately 57°000 (gel filtration method) [6] Isoelectric point: 3.9±0.1 (Focused electrophoresis method using carrier ampholite) ■ pH stability: pl+9 or less (see Figure 3) ■ Temperature stability: 845°C or less (pH 7, 6, 3 minute treatment) (Table 4)
(Refer to the figure) (Example) The present invention will be specifically explained using examples.

Example 1 0.2% creatine, 0.5 polypeptone, 0.5% yeast extract, 1.4% KJPOn, 0.3% KHIPO4
,0,01%M,SQ.

・7HzO-PI17. 50jd of medium consisting of O
The mixture was placed in a 00 m Sakaro flask and sterilized in an autoclave at 121°C for 10 minutes. Actinobacillus (Actin
genus obacillus sp.

One loopful of CRH-1271 (Feikoken Bibori No. 7721) was inoculated into the above medium, and cultured with shaking at 30°C for 20 hours.
It was used as a seed culture solution. Separately, the above seed culture solution 50d was inoculated into a 101 volume jar fermenter containing a medium 62 sterilized under the same conditions. Culture was performed at 300 rpm, air flow rate 31/min, and 30°C for 16 hours. The enzyme activity of the obtained culture solution was 0.2
It was 90/d.

The culture solution 62 was centrifuged, the cells were collected and suspended in 50mM'J acid buffer, and crushed using a bead crusher (Dynomil i [OL>) as 11.The cell suspension was centrifuged, and the supernatant was Ammonium sulfate was added to the supernatant to a saturation of 0.35, followed by centrifugation to obtain a supernatant.
．． Ammonium sulfate was added to saturate the mixture with 55%, and the mixture was centrifuged to obtain a precipitate. 50aMIJ acid buffer pi+7.5.25(l
d was redissolved. Re-dissolve solution in 50mM phosphate buffer PH
Sephadex G-25 column (2
Desalted in step 1). Transfer the desalting solution to DEAR-Sepharose CL
It was adsorbed onto a 4B column 50d and eluted with 0.4M NaCl. The physicochemical properties of the enzyme were measured using the mannitol fraction of DEAR-Sephalol as a sample, and the results were as described above. The eluate was concentrated by ultrafiltration and subjected to molecular sieving using a Sephacryl S-200 column. The specific activity of the active fraction was 0.81 U/mg protein.

(Effects of the Invention) According to the present invention, a thermostable mannitol dehydrogenase that is stable up to 45° C. can be obtained by treatment at pH 7, 6, and for 3 minutes.

[Brief explanation of the drawing]

FIG. 1 shows the relationship between pi and activity of the enzyme obtained according to the present invention. Figure 2 shows the relationship between temperature and activity. Figure 3 shows the relationship between pH and activity when treated at 25°C for 16 hours with each po. FIG. 4 shows the relationship between temperature and activity when treated at p I+ 7 and 6 for 3 minutes at each temperature. FIG. 5 shows the temperature stability of a conventional enzyme (derived from Aspergillus oryzae). Patent applicant Toyobo Co., Ltd. Akuta IIi! I し遁p)((pH-n-early 2X!I;ah i("C) early 3 Figure H $4 Figure early 5rg Procedural amendment (method) 1. Indication of the case 1988 Patent Application No. 306809 2. Name of the invention: Heat-resistant mannitol dehydrogenase and its manufacturing method 3; Relationship with the amended person's case Patent applicant: 2-2-8 Dojimahama, Kita-ku, Osaka February 3, 1988 (Shipping mortar: 2, 1988) 5. Subject of amendment 6. Contents of amendment The name of the invention is corrected to "Heat-stable mannitol dehydrogenase and its manufacturing method."

Claims (3)

[Claims] (1) A heat-resistant mannitol dehydrogenase that catalyzes the following reaction and is stable up to 45°C after treatment at pH 7.6 for 3 minutes. Reaction formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (2) The thermostable mannitol dehydrogenase according to claim 1, which has the following properties. [1] Substrate specificity: Acts specifically on mannitol. [2] Optimum pH: 9.0-11.0 [3] Optimum temperature: 50°C [4] Km value: Approximately 9.7 x 10^-^4M [5] Molecular weight: Approximately 57,000 (gel Filtration method) [6] Isoelectric point: 3.9±0.1 (Focal electrophoresis method using carrier ampholite) (3) Actinobacillus
s) A method for producing heat-stable mannitol dehydrogenase, which comprises culturing heat-stable mannitol dehydrogenase-producing bacteria belonging to the genus in a nutrient medium, and collecting heat-stable mannitol dehydrogenase from the culture.