JPS6348514B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to superoxide dismutase as a novel enzyme. (Prior art) Generally, superoxide dismutase (hereinafter simply referred to as SOD) causes dismutation of the substrate O 2 - by the following reaction 2O ₂ ^- +2H ⁺ -→H ₂ O ₂ ⁺ _{O 2} . It is known as an enzyme that can Conventionally, such SOD is, for example, SOD (Mark) extracted from cow red blood cells.
owitz, J. Biol. Chem vol. 234, p. 40, 1959), and it is also known that this SOD contains Cu and Zu as metal atoms related to activity. (Problems to be Solved by the Present Invention) Incidentally, not only the above-mentioned SOD, but enzymes in general are stable under heat, and therefore, the industrial application of enzymes has been limited. In order to solve these problems, the main purpose of the present invention is to develop superoxide dismutase as a new enzyme, and also to provide a heat-stable enzyme. (Means for Solving the Problems) The present invention is a novel superoxide dismutase developed to achieve the above object, and is a dimer with a molecular weight of 48,000±3,000 and a subunit molecular weight of approximately 24,000. It has a structure and contains Mn as a metal related to activity, and has a diameter of 250 to 800 nm.
Visible and ultraviolet spectrum in the range of around 280nm and
It is characterized by having a maximum absorption value around 470 nm and being obtained from a bacterial species belonging to the genus Gluconobacter. (Example) Examples of the present invention will be described below. Example 1 This example describes an exemplary method for purifying superoxide dismutase. That is, in this example, first, bacteria of Gluconobacter selinus (IF03268) are tested and cultured on a medium containing the following components in the following grams. Yeast extract powder 5g Glycerol 10g K ₂ HPO ₄ 1g This medium was sterilized at 110°C for 20 minutes. Bacteria that had been precultured in the same medium were added to this and cultured aerobically for 24 hours. After that, the bacteria were collected using a centrifuge and further washed twice with 0.85% (W/W) saline solution.
SOD was extracted from the bacteria. Next, the bacteria washed as described above were suspended in 0.1M phosphate buffer (PH7.8), and the bacteria were disrupted using a Dynomill. The disruption of the bacteria and subsequent operations were all carried out at 4°C. Ammonium sulfate was then added to this solution until it reached 45% saturation. The resulting precipitate was removed using a centrifuge, and ammonium sulfate was added to the resulting solution until it reached 100% saturation. Most of the SOD was present in the resulting precipitate. Next, this precipitate was dissolved in a small amount of 0.1M phosphate buffer (PH7.8) and dialyzed against about 10 times the amount of 0.1M phosphate buffer (PH7.8) overnight. During this period, the dialysis fluid was exchanged three times. Ammonium sulfate was added to the dialyzed solution until it was 60% saturated, the precipitate was removed by centrifugation, and the supernatant was equilibrated with 0.1M phosphate buffer containing 60% saturated ammonium sulfate.
DEAE-Toy opeal
It was applied to a column and SOD was adsorbed onto the column. Next, the column with this SOD adsorbed was washed with 0.1M phosphate buffer containing 60% saturated ammonium sulfate, SOD was eluted with 0.1M phosphate buffer containing 50% saturated ammonium sulfate, and fractionated using a fraction collector. I took it. Then, the fractionated components containing SOD are collected, and ammonium sulfate is added to this solution until it reaches approximately 100% saturation to precipitate SOD. This solution was allowed to stand overnight, and the precipitate was collected using a centrifuge. SOD was present in the majority in this precipitate. Next, a small amount of 0.01M phosphate buffer was added to this precipitate to dissolve the precipitate, and this solution was dialyzed against 0.01M phosphate buffer overnight. During this time, add 3
Exchanged twice. This solution was applied to a DEAE-Toyo Pearl column equilibrated with 0.01M potassium phosphate buffer.
Fractionated components of SOD that were not adsorbed were collected on this column. Ammonium sulfate was added to the fractionated components thus collected until 100% saturation was achieved to precipitate SOD. This precipitate was collected by centrifugation,
Dissolve this in a small amount of 0.02M phosphate buffer,
Dialysis was performed overnight against 0.02M phosphate buffer (PH7.2). Next, add this solution to 0.02M phosphate buffer (PH7.2)
The mixture was applied to a Sephadex G-100 column equilibrated with This eluate was fractionated using a fraction collector to obtain SOD active fraction components. The SOD obtained by the above procedure showed a single band in polyacrylamide gel electrophoresis, and homogeneity was confirmed by ultracentrifugation analysis. The molecular weight of this SOD was measured by high performance liquid chromatography using Finepak SIL AF-102 (manufactured by JASCO Corporation) as a known column. In this measurement, the following markers with known molecular weights were used. Molecular weight glutamate dehydrogenase 290000 lactate dehydrogenase 142000 enolase 67000 adenyl kinase 32000 cytochrome c 12400 As a result of measurement, the molecular weight of the above SOD was approximately 50000. Next, in order to determine the subunits of the protein structure of SOD, polyacrylamide gel electrophoresis with sodium dodecyl sulfate was performed, and as a result, a single type of subunit with a molecular weight of about 24,000 was observed.
Therefore, the molecular weight of the SOD obtained is estimated to be about 24,000 x 2, or about 48,000. Therefore, considering both the observation of this subunit and the results of the high performance liquid chromatography described above, the molecular weight of SOD in the present invention can be estimated to be in the range of about 48,000±3,000. Furthermore, from this, it is estimated that the above SOD has a dimeric structure in which the molecular weight of the subunits is approximately 24,000. Next, when the visible and ultraviolet spectra of the SOD solution in this example were measured, the results shown in FIG. 1 were obtained. That is, the maximum absorption value was observed in the vicinity of about 280 nm in the ultraviolet region and in the vicinity of about 470 nm in the visible region. In addition, the results of metal analysis by atomic absorption spectrometry,
Mn and Cu were detected, but the above SOD was 5mM.
Since it is not inactivated by KCN, this SOD is a Mn-SOD containing Mn as a metal related to activity.
It can be estimated that In addition, the activity measurement of SOD is carried out by Arg.Biol.Chem., 38
(2) 471-473 (1974), by a method similar to that described by K. Asada et al. That is, 50mM potassium phosphate (PH7.8), 0.1mM MEDTA, 0.1mM xanthine, 10μM cytochrome c (derived from horse heart) were added to the optical cell (for 1ml).
Add SOD to make the total volume 0.99ml. Add 0.01 ml of xanthine oxidase to this to start the reaction, determine cytochrome c reduction from the initial rate of increase in absorbance at 550 nm, and let this value be v.
If the cytochrome c reduction rate when SOD is not added is V, the amount of SOD enzyme is proportional to V/v.
Under these conditions, the amount of SOD enzyme that inhibits cytochrome c reduction by 50% (ie, V/v = 2) was defined as one enzyme unit. The SOD of this example uses superoxide ion (O ₂ ^- ) as a substrate and has the effect of causing disproportionation of this O ₂ ^- . Therefore, by blending the SOD of this example into products containing oxidizing components such as oils and fats, which generally produce peroxides due to light, heat, and other causes, and which can significantly alter or deteriorate quality. , O ₂ ^- can be caused to disproportionate, thereby preventing product deterioration and deterioration due to peroxides. In addition, when the stable PH of the above SOD was tested, it was as follows. First, 1/100M Britton was obtained by adjusting the pH of 0.98g/phosphoric acid, 0.6g/acetic acid, and 0.62g/boric acid with 0.2N sodium hydroxide.
−While adjusting the pH with Robinson buffer,
SOD was left at 37°C for 30 minutes at various pH ranges, and the relative activity of SOD was measured. As a result, as shown in Figure 2, the above SOD is PH6.0 ~
It was found to be stable within the range of 11.0. Next, in order to determine the optimal pH of the above SOD,
Xanthine - xanthine, oxidase - at 25℃
Cytochrome c system with 1/20M of the above composition
The relative activity of SOD was measured at various pH ranges in Britton-Robinson's buffer. As a result, the second
As shown in the figure, in the basic region of PH 8.5~
It was observed that the relative activity was high in the range of 10.5.
As a result, the optimal PH range for the above SOD is 8.5 ~
10.5. Example 2 This example is an example for testing the peroxide production suppressing effect of SOD. First, the following model was set up as a peroxide production system, and the antioxidant effect was investigated. 0.55M phosphate buffer (PH6.9) 25ml polyoxyethylene nonyl phenyl ether
1.25g Linoleic acid 0.28g When a mixture with the above composition is left in a thermostat at 50°C, peroxide is generated. The SOD of Example 1 was added to the above system at a concentration of 0.1 mg/ml, and the system was left in a thermostat at 50°C, and the lipid peroxide suppressing effect was examined after 3 days. The amount of lipid peroxide was measured using the Rodan iron method of Mitsuda et al. (Nutrition and Shokuryo Vol. 19, No. 3, p. 210, 1966). That is, 75%-ethanol to 0.1 ml of sample solution.
4.7ml, 35% - ammonium rhodanide aqueous solution 0.1
ml, 0.1 ml of a 1N hydrochloric acid solution of 0.02M ferrous chloride was added, and the absorbance at 500 nm was measured after exactly 3 minutes. As a reference example, the same amount of bovine serum albumin was added instead of the above SOD, and the same amount of bovine serum albumin was added instead of SOD, and a similar test was conducted for comparison, and the following table is shown. 1
The following results were obtained. All numerical values in the table are expressed in absorbance.

[Table] An increase in absorbance after 3 days from day 0 represents an increase in the amount of lipid peroxide. Moreover, the amount of lipid peroxide and the absorbance are proportional. From this and the above test results, the SOD of this example was found to have an extremely excellent peroxide production suppressing effect. Example 3 This example is an example for testing the heat resistance of the above SOD. The heat resistance of SOD in the above example was compared with SOD from Serratia marsetuscens, which grows near room temperature like Gluconobacter. Each SOD was prepared in 0.01M phosphate buffer (PH
7.2) and heated at 60℃ for 5 minutes each.
The activity was measured after cooling in ice. The activity was also measured before heating to 60℃, and
Inactivation due to heating was observed. The results are shown in Table 2 below.

[Table] As described above, the SOD in this example was found to have significantly better heat resistance than the SOD derived from bacteria that grows near room temperature. In the above example, SOD extracted and purified by Gluconobacter selinus was used, but the bacterial species is not limited to this.In short, the SOD of the present invention can be obtained from a bacterial species belonging to the genus Gluconobacter. It is fine as long as it is. Furthermore, the method for purifying SOD is not limited to the above example. (Effects of the Invention) (a) As described above, the present invention has led to the production of a completely new superoxide dismutase from a species of the genus Gluconobacter. (b) In addition, superoxide dismutase obtained from bacterial species that grow at room temperature generally has weak heat resistance, but the superoxide dismutase of the present invention is obtained from a bacterial species of the genus Gluconobacter that grows at room temperature. However, compared to other superoxide dismutases derived from bacterial species that grow at room temperature, such as those obtained from Serratia marsetuscens,
It had very good heat resistance. Therefore, there is a practical benefit of expanding the range of industrial applications of enzymes. (c) Furthermore, the bacterial species of the genus Gluconobacter has been used in the food industry for a long time, for example in the production of raw materials for vinegar and vitamin C, and their safety has been confirmed. The superoxide dismutase of the present invention is also recognized to be highly safe.

[Brief explanation of the drawing]

FIG. 1 is a chart of visible and ultraviolet spectra of superoxide dismutase as an example. Figure 2 is a diagram showing the correlation between oxygen activity and PH.

Claims (1)

[Claims] 1. A substance that uses O ₂ ^- as a substrate, has an action of causing disproportionation of O ₂ ^- , and has a stable PH range of 6.0 to 11.0 and an optimal PH range of 8.5 to 10.5. In some superoxide dismutases, the molecular weight is
It has a dimer structure with a subunit molecular weight of 48,000±3,000 and approximately 24,000, and metals related to activity are
A novel superoxide dismutase containing Mn, having maximum absorption values around 280 nm and around 470 nm in the visible and ultraviolet spectrum in the range of 250 to 800 nm, and being obtained from a bacterial species belonging to the genus Gluconobacter. 2. The novel superoxide dismutase according to claim 1, wherein the bacterial species belonging to the genus Gluconobacter is Gluconobacter selinus.