JPH0212558B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for efficiently producing aldose-1-epimerase. Conventionally, aldose-1-epimerase [EC5.1.3.3] (hereinafter abbreviated as mutarotase) is
Penicillium notatum
notatum) and various animal tissues (especially nephrocytes,
It is known to exist in the intestines, liver, crystalline lens, etc.) and plant tissues, and its action is based on α-D-glucose and β-D-glucose.
An enzyme that catalyzes the conversion of glucose. Mutarotase is known to more rapidly quantify glucose in serum by combining with glucose oxidase (EC1.1.3.4: β-D glucose is the only substrate).
It is strongly desired to provide a method for industrially producing metallotase with excellent stability at low cost. In order to meet such demands, the present inventors have conducted intensive research on a method for industrially producing mutarotase at low cost. As a result, when microorganisms belonging to the genus Aspergillus are cultured, highly stable mutarotase is found in the bacterial cells. It was discovered that this can be produced in significant quantities, and the present invention was completed. That is, the present invention is a method for producing mutarotase, which comprises culturing a microorganism belonging to the genus Aspergillus and capable of producing mutarotase in a nutrient medium, and collecting mutarotase from the culture. Bacterial strains that can be used in the present invention include, for example, Aspergillus niger.
All bacteria that belong to the genus Aspergillus and produce mutarotase can be used in the method of the present invention, such as ATCC6274 (Feikoken Bibori No. 10948) and Aspergillus niger IAM2020. According to the present invention, by culturing filamentous fungi belonging to the genus Aspergillus in a nutrient medium, mutarotase is produced and accumulated outside or inside the fungi, and therefore, known methods and improved methods that will be initiated in the future Enzyme powder can be obtained by purification and drying. More specifically, the mutarotase-producing bacteria of the genus Aspergillus are cultured in a suitable nutrient medium, for example, a medium containing suitable carbohydrates, nitrogen sources, and inorganic salts, and mutarotase is accumulated in the bacterial cells. Examples of carbohydrates that can be used include refined sugars such as glucose, sucrose, maltose, starch hydrolyzate, and starch, as well as sugar alcohols such as glycerol and sorbitol, and organic acids such as citric acid and succinic acid. Nitrogen sources include yeast extract, peptone, meat extract, corn stew liquor, casein hydrolyzate, defatted soybean,
Ammonium salts, nitrates, etc. are used. As the inorganic salts, metal salts such as sodium, potassium, magnesium, and calcium, and salts such as sulfuric acid, phosphoric acid, hydrochloric acid, and nitric acid can be used. When mutarotase is produced outside the bacterial body, it is purified by the usual method after bacterial cell isolation, but when it is produced inside the bacterial body, the resulting bacterial body containing mutarotase is filtered or centrifuged. The enzymes are extracted by separating the enzymes by suspending them in an appropriate buffer, and then crushing them by known methods such as grinding, ultrasonication, mechanical compression, or autogenous digestion. After separating the insoluble matter from the extract by filtration or centrifugation, enzyme preparation is performed from the obtained filtrate or supernatant by a known method such as salting out with ammonium sulfate, Glauber's salt, etc., or solvent precipitation using acetone, alcohol, etc. get goods. To obtain a more highly purified enzyme preparation,
It is preferable to perform an adsorption/elution method applying ion exchange, a gel filtration method, and a heat treatment within a stable pH range to heat-inactivate impure proteins and other enzymes that are unstable to heat. In particular, this heat treatment is an effective method for significantly increasing the specific activity of the enzyme by using it at various stages of purification. To measure the activity of mutarotase, a freshly prepared glucose solution (special grade glucose reagent, manufactured by Wako) is used. Add 2 ml of glucose measurement reagent (Diacolor GC, manufactured by Toyobo Co., Ltd.) and 0.5 ml of enzyme solution to 0.5 ml of glucose solution (0.2 mg/ml), and analyze with a double beam spectrophotometer UV-210A manufactured by Shimadzu Corporation for 30 minutes.
The rate of glucose oxidation is measured from the rate of increase in pore density at 558 nm at 558 nm. On the other hand, as a control, the same operation was performed with the above composition without the addition of enzymes, the natural conversion rate of α-D-glucose to β-D-glucose was measured, and this amount was subtracted to yield 1 μmol per minute. The amount of enzyme converted to β-D-glucose is defined as 1 unit. Figure 1 shows the effect of mutarotase on glucose oxidation. In FIG. 1, -Δ- indicates α-glucose produced by mutarotase, -〇- indicates glucose in an equilibrium state caused by mutarotase, -●- indicates glucose in an equilibrium state, and -▲- indicates α-glucose. Figure 2 shows the influence of mutarotase concentration on the glucose oxidation rate. The reaction rate is 2×10 ^-2
Since linearity deviates in a range exceeding (μmole/ml, min), the enzyme concentration is diluted and measured so that it falls within this measurement range. In addition, the optimum pH and substrate specificity are measured by recording with a polarimeter. The measurement method is 1% α-D as a substrate.
- Assuming that the change in metarotation light of the glucose solution over time is a first-order reaction, calculate the reaction rate constant with and without the addition of mutarotase, and subtract the reaction rate constant with no addition from the reaction rate constant with the addition of mutarotase. It was defined as enzyme activity. The physicochemical properties of mutarotase obtained by the present invention were investigated in Aspergillus niger.
The following describes the enzyme produced by ATCC6274 (Feikoken Bibori No. 10948). (1) Action This enzyme converts α-D-glucose into β-D-glucose, or converts β-D-glucose into α-D-glucose.
- Catalyze the reaction that converts to glucose. (2) Substrate specificity In addition to α-D-glucose, this enzyme
D-galactose, β-D-fructose, β
-L-arabinose and α-D-xylose,
Acts on β-maltose, β-lactose, and β-D-cellobiose (see Table 1).

[Table] (3) Optimal pH and stable pH range The optimal pH of this enzyme is between 4.0 and 8.0 under the above activity measurement conditions (see Figure 3).
In FIG. 3, -0- indicates relative activity, -△- indicates catalytic rate constant, and -▲- indicates natural rate constant. The stable pH range of this enzyme is determined by treatment at 20℃ for 24 hours.
The pH is in the range of 5 to 10 (see Figure 4). Furthermore, the most stable pH is around 5.5-9. (4) Range of optimal temperature for action The optimal temperature for action of this enzyme is around 60°C under the activity measurement conditions described above. (5) Inactivation conditions due to pH, temperature, etc. This enzyme was treated at pH 6.0 and at 50℃ for 30 minutes.
At 60°C and 70°C, 95% and 75% of the activity remains, respectively, but when treated at 75°C, most of the activity is lost (see Figure 5). The time course of inactivation of this enzyme at 75℃ is a single molecule reaction, and the heat inactivation rate constant is 0.053 mm ^-1 , that is, the activity decreases in 6 minutes.
50% become inactive (see Figure 6). (6) Molecular Weight The molecular weight of this enzyme was calculated to be 130,000 by SDS polyacrylamide gel electrophoresis (see Figure 7). In Figure 7, 〇 marks start from the top left.
RNA・P _T −β′, RNA・P−β′, RNA・P−
β, RNA・_PT −β, RNA・_PT ⁻ I, serum albumin, RNA・_PT −α, RNA・P−α,
RNA P _T -I, tribusin inhibitor. △ indicates mutarotase. The molecular weight was determined to be 260,000 by gel filtration using Cephadex G-200 (see Figure 8). Therefore, this enzyme has a molecular weight of 130,000
It was determined to be a dimer consisting of two subunits. In FIG. 8, the circles indicate ferritin, catalase, aldolase, α-amylase, serum albumin, and egg albumin in order from the top left.
△ indicates mutarotase. Next, the novel method for producing mutarotase according to the present invention will be explained using an example using Asbergillus niger ATCC6274 strain (Feikoken Bibori No. 10948). Example 3% seuclose, 5% potato starch, 0.2% yeast extract (manufactured by Oriental Yeast), NaNO ₃ 2
%, MgSO ₄ .7H ₂ O 0.5% was placed in a 500 ml Meyer flask and steam sterilized at 120°C for 10 minutes. As an inoculum, use 1 slant of Aspergillus niger ATCC6274 (Feikoken Bacteria No. 10948) and 15 ml of sterile water.
ml and blended to prepare a spore suspension, inoculate 5 ml and incubate at 180 nm for 2 days at 30°C.
Culture it under cm conditions and use it as an inoculum. 5 ml of the above-mentioned culture was inoculated into the same medium as above (but without starch) and cultured at 30°C for 1 day. For the main culture, 5% of the seed culture was inoculated into a 100 fermenter (manufactured by Marubishi, liquid volume 70, medium composition is the same as the seed culture), and aeration (35/min) was carried out at 30℃ for 24 hours.
The pH was adjusted to 5.0 to 5.3 with 10% NaOH while culturing with stirring (240 rpm). After the cultivation was completed, the culture solution was filtered through gauze while being cooled in ice to obtain 4.2 kg of wet bacterial cells. The 3.6 kg of wet bacterial cells were disrupted with a homogenizer together with 0.02M Tris/0.05M acetate buffer (pH 6.0), and the 8.2 bacterial cell suspension was ground with Dyno Mill (manufactured by Shinmaru Enterprises).
The mixture was separated using a refrigerated centrifuge (Tomy Seiko RS-18) at 10,000 rpm for 20 minutes to obtain a crude enzyme solution of 7.78. Add 40% ammonium sulfate to the obtained crude enzyme solution.
Add to saturation and leave overnight, then 10 at 8000 rpm.
The precipitate was removed by cooling centrifugation for 1 minute, and ammonium sulfate was further added to the supernatant to reach 80% saturation. After standing overnight, the mixture was cooled and centrifuged at 8000 rpm for 10 minutes to recover the novel mutarotase as a precipitate. Add 500ml of 0.02M Tris, 0.05M acetate buffer (pH 6.0) to this precipitate, dialyze overnight while changing the same buffer several times, and remove the resulting precipitate by centrifugation to obtain 530ml of enzyme solution. Ta. Enzyme solution after dialysis
DEAE− equilibrated with 530 ml buffer (pH 6.0)
Cephadex A-50 (Pharmacia Afine)
Column (6.0φ x 40cm) packed with (manufactured by Chemical Co., Ltd.)
After adsorbing the novel mutarotase and washing with the same buffer, the salt concentration in the buffer was increased linearly from 0.05M to 0.5M, and the activity at a salt concentration of around 0.35M in eluate 2.4 was increased. Classification (fraction)
No. 122-134) 320 ml was collected (see Figure 9). Ammonium sulfate was added to 120 ml of the 320 ml to achieve 90% saturation, and the resulting salting out product was dissolved in the same buffer and concentrated to 20.8 ml. 5 ml of this solution was mixed with 0.02M Tris and 0.05M in cellophane tubes. Dialysis was performed overnight in acetate buffer (pH 6.0). 70% of this dialysate
After heat treatment at ℃ for 10 minutes, the mixture was centrifuged at 12,000 rpm for 10 minutes to remove the precipitate and obtain 5 ml of supernatant. This liquid 5
ml of Cephacryl S-300 equilibrated in advance with 0.02M Tris/0.05M acetate buffer (pH 6.0).
The active fraction was collected through a column (2φ x 72cm) packed with (manufactured by Pharmacia, Sweden). Fraction No. 25 is considered to be a protein denatured due to heat treatment. Here, Fraction No. 35
It can be seen that the protein peak and the peak of mutarotase activity coincide (see Figure 10). Next, use an ultrafiltration device with a molecular weight of 20,000 (UHP-76 manufactured by Toyo Roshi) to obtain fraction No.
5 ml of 35 was concentrated to 1 ml. The enzyme activities in the above purification steps are shown in Table 2. Protein concentration was determined by measuring the absorbance at 280 and 320 nm.
OD ₈₀ −OD ₃₂₀ was calculated, and ΔOD=1 was expressed as 0.5 mg of protein per ml of enzyme solution.

[Table] Specific activity at the stage from ammonium sulfate fraction to purified enzyme solution
4.91U/mg protein, approximately 270% compared to crude enzyme solution
doubled. The purified enzyme was subjected to disk polyacrylamide gel electrophoresis and SDS polyacrylamide gel electrophoresis. As shown in FIG. 11, it can be seen that the protein was purified to almost a single band. In FIG. 11, A shows disk polyacrylamide gel electrophoresis, and B shows SDS polyacrylamide gel electrophoresis. In addition, disk polyacrylamide gel electrophoresis method uses 2.5% gel for concentration and 2.5% gel for separation at pH 9.
7.5% was used. The amount of sample is based on 200μg,
The electrophoresis current was 4 mA per gel, bromophenol blue was used as the indicator dye, and after staining with amide black for 1 hour, the gel was dipped in 7% acetic acid.
Destaining was performed by occasionally adding acetic acid. In addition, 7.5% acrylamide gel (pH 7.2) was used for SDS polyacrylamide gel electrophoresis. The amount of sample (diluted with 0.1% SDS solution) was based on 2 μg per gel, and the electrophoresis current was 8 mA per gel.
Bromophenol blue was used as the indicator dye, and after staining with Comassie blue for 1 hour, 7%
After overnight immersion in acetic acid (containing 25% methanol), destaining was performed through a current of 10 mA per gel.

[Brief explanation of drawings]

FIG. 1 shows the effect of mutarotase on glucose oxidation. Figure 2 shows the effect of mutarotase concentration on glucose oxidation rate. Figure 3 shows the optimum mutarotase obtained by the method of the present invention.
Indicates pH. Figure 4 shows the stable pH of the enzyme.
FIG. 5 shows the temperature-dependent inactivation conditions of the enzyme at pH 6.0. Figure 6 shows the rate of heat inactivation of the above enzyme at 75°C. Figure 7 shows the determination of molecular weight by SDS polyacrylamide gel electrophoresis. FIG. 8 shows the measurement of the molecular weight of Cephadex G-200 by the gel filtration method. Figure 9 shows the purification of mutarotase by DEAE-Sephadex A-50. Figure 10 shows the purification of mutarotase by Sephacryl-S-300. FIG. 11 shows discret polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis B of mutarotase.

Claims (1)

[Claims] 1 Belongs to the genus Aspergillus, aldose-1-
Aldose-1, which comprises culturing a microorganism capable of producing epimerase in a nutrient medium, and collecting aldose-1-epimerase from the culture.
- Method for producing epimerase.