Method for producing cytochrome c peroxidase

Abstract

To provide a novel method for producing heme iron-containing cytochrome c peroxidase.SOLUTION: A method for producing cytochrome c peroxidase includes: a PHB accumulation step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in the medium for 10 hours or more to cause the halophilic bacterium to accumulate poly-3-hydroxybutyrate; a 3HB secretion step of microaerobically culturing the halophilic bacterium in the medium while adjusting the pH to cause the poly-3-hydroxybutyrate accumulated within the halophilic bacterium to be secreted into the medium as 3-hydroxybutyrate; and a recovery step of recovering the cytochrome c peroxidase from the medium. The pH during microaerobic culture is adjusted to 9.0 or more and 12.0 or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing cytochrome c peroxidase. [Background technology]

[0002] Heme iron is a complex consisting of iron and porphyrin, which is more easily digested and absorbed in the body than non-heme iron and is less susceptible to absorption inhibition by other substances. For this reason, heme iron is used as a useful iron supplement in dietary supplements.

[0003] However, most heme irons are poorly soluble in water. Therefore, when heme iron is used in foods, problems such as a poor texture and uneven distribution within the food may occur. This has significantly limited the use of heme iron in foods. Therefore, attempts have been made to prevent the above problems from occurring.

[0004] Patent Document 1 discloses a method for producing cytochrome c, which includes a PHB accumulation step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium to cause the bacterium to accumulate poly-3-hydroxybutyrate, and a recovery step of recovering cytochrome c from the medium. The heme iron produced by this method is water-soluble and therefore less susceptible to the above-mentioned problems. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-152571 Summary of the Invention [Problem to be solved by the invention]

[0006] Although there is a method for efficiently producing cytochrome c as a heme-containing protein, as disclosed in Patent Document 1, it is not possible to produce other heme-containing proteins.

[0007] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel method for producing cytochrome c peroxidase containing heme iron. [Means for solving the problem]

[0008] The characteristic features of the method for producing cytochrome c peroxidase according to the present invention to achieve the above object are as follows: a PHB accumulation step in which a halophilic bacterium belonging to the genus Halomonas is aerobically cultured in a medium for 10 hours or more to cause the halophilic bacterium to accumulate poly-3-hydroxybutyrate; a 3HB secretion step in which the halophilic bacterium is microaerobically cultured in the medium while adjusting the pH, and the poly-3-hydroxybutyric acid accumulated in the cells of the halophilic bacterium is secreted into the medium as 3-hydroxybutyric acid; and a recovery step of recovering the cytochrome c peroxidase from the medium, The pH of the medium during the microaerobic culture is adjusted to 9.0 or more and 12.0 or less.

[0009] As a result of extensive research into methods for producing heme iron, the present inventors discovered that when halophilic bacteria belonging to the genus Halomonas are cultured microaerobically, adjusting the pH of the medium to between 9.0 and 12.0 releases cytochrome c peroxidase containing heme iron into the medium, leading to the completion of the present invention.

[0010] That is, according to the above-described characteristic configuration, cytochrome c peroxidase containing heme iron can be produced. Because the cytochrome c peroxidase produced by this production method is a fermented natural product, it can be ingested as heme iron as is. Furthermore, by decomposing cytochrome c peroxidase using known techniques, heme iron with a higher purity can be obtained. Furthermore, because cytochrome c peroxidase has the enzymatic activity of decomposing hydrogen peroxide into water, it can be expected to function not only as heme iron but also as a physiological function of cytochrome c peroxidase. Because cytochrome c peroxidase is a water-soluble heme-containing enzyme, it can be used in applications where water solubility is required. Furthermore, in the above-mentioned characteristic configuration, halophilic bacteria belonging to the genus Halomonas can also produce cytochrome c, which is expected to have physiological functions as an electron carrier for cytochrome c and heme iron.

[0011] Furthermore, a further characteristic feature of the method for producing cytochrome c peroxidase according to the present invention is The amount of iron sulfate heptahydrate in the medium is set to 2 mg / 100 mL or more and 10 mg / 100 mL or less.

[0012] The present inventors have found that when adding ferrous sulfate heptahydrate to a medium at a concentration of 2 mg / 100 mL or more and 10 mg / 100 mL or less and adjusting the pH of the medium to 9.0 or more and 11.0 or less during microaerophilic cultivation of halophilic bacteria belonging to the genus Halomonas, the amount of cytochrome c peroxidase released into the medium increases.

[0013] Furthermore, a further characteristic feature of the method for producing cytochrome c peroxidase according to the present invention is The halophilic bacterium is Halomonas sp. KM-1 strain (FERM BP-10995).

[0014] The inventors of the present application have confirmed through experiments that when Halomonas sp. KM-1 strain is used as a halophilic bacterium and poly-3-hydroxybutyrate is allowed to accumulate within the cells of the Halomonas sp. KM-1 strain, cytochrome c peroxidase can be produced. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a photograph showing the change in color of the medium when the pH of the medium is adjusted to 8.6 to 12. [Figure 2] 1 is a photograph showing the results of SDS-PAGE when the pH of the medium was 9.0 and 11.0. [Figure 3] 1 is a photograph showing the results of SDS-PAGE when the amount of iron sulfate heptahydrate blended was 10 mg / 100 mL. DETAILED DESCRIPTION OF THE INVENTION

[0016] The method for producing cytochrome c peroxidase according to the present invention will be described below. Preferred examples are provided below to illustrate the present invention more specifically. Various modifications are possible within the scope of the present invention, and the present invention is not limited to the following descriptions.

[0017] [Outline of the method for producing cytochrome c peroxidase] The method for producing cytochrome c peroxidase of the present invention comprises culturing halophilic bacteria belonging to the genus Halomonas to release cytochrome c peroxidase into a medium, and recovering the cytochrome c peroxidase from the medium.

[0018] Specifically, the method for producing cytochrome c peroxidase of the present invention involves the following steps. (1) a step of aerobically culturing halophilic bacteria belonging to the genus Halomonas in a medium to allow the halophilic bacteria to accumulate poly-3-hydroxybutyrate (PHB accumulation step); (2) a step of microaerobically culturing the halophilic bacteria in the medium while adjusting the pH after the PHB accumulation step to allow the poly-3-hydroxybutyrate accumulated in the halophilic bacteria to be secreted into the medium as 3-hydroxybutyrate (3HB secretion step); and (3) a recovery step of recovering cytochrome c and cytochrome c peroxidase from the medium.

[0019] [PHB accumulation process] The PHB accumulation step in the production method of the present invention is a step of aerobically culturing halophilic bacteria belonging to the genus Halomonas (hereinafter also simply referred to as halobacteria) in a medium to allow the halophilic bacteria to accumulate poly-3-hydroxybutyrate.

[0020] The halophilic bacteria used in the PHB accumulation process grow aerobically in a medium containing inorganic salts and one or more organic carbon sources, and have the property of accumulating PHB within their own cells while simultaneously releasing cytochrome c and cytochrome c peroxidase into the medium. Furthermore, when the accumulated PHB is secreted as 3HB, they also have the property of releasing more cytochrome c and cytochrome c peroxidase into the medium than when PHB was accumulated.

[0021] These halophilic bacteria have an optimum growth temperature of 0.1 to 1.0 M, and can sometimes grow in a salt-free medium. The above-mentioned halophilic bacteria belonging to the genus Halomonas usually grow in a medium with a pH of about 5 to 12.

[0022] An example of such a halophilic bacterium is the Halomonas sp. KM-1 strain. The Halomonas sp. KM-1 strain was deposited on July 10, 2007, at the National Institute of Advanced Industrial Science and Technology (National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, 305-8566, Japan) under accession number FERM P-21316. This strain has now been transferred to international depository status under accession number FERM BP-10995. The 16S rRNA gene of the Halomonas sp. KM-1 strain has been registered with DDBJ under accession number AB477015. The cytochrome released by the Halomonas sp. KM-1 strain during PHB accumulation and 3HB secretion is cytochrome c.

[0023] In addition, in view of the growth characteristics of the above-mentioned halophilic bacteria, the halophilic bacteria used in the PHB accumulation step of the present invention are not limited to Halomonas sp. KM-1 strain, as long as they are halophilic bacteria belonging to the genus Halomonas that accumulate poly-3-hydroxybutyrate. In addition to Halomonas sp. KM-1 strain, other halophilic bacteria used in the PHB accumulation step include Halomonas pantelleriensis (ATCC 700273) and Halomonas campisalis (ATCC 7000597).

[0024] Furthermore, analysis of 16S ribosomal RNA sequences has revealed that, in addition to the above-mentioned halophilic bacteria, Halomonas nitritophilus, Halomonas alimentaria, and the like may also be used as halophilic bacteria belonging to the genus Halomonas in the PHB accumulation step.

[0025] A gene may be introduced into the halophilic bacterium belonging to the genus Halomonas. The gene to be introduced is not particularly limited as long as it expresses a function that improves the production efficiency of cytochrome c peroxidase in the production method of the present invention. Examples include a gene that increases the expression level of cytochrome c peroxidase, a gene that expresses a function that increases the accumulation of PHB in the bacterium, and a gene that degrades PHB. Various general methods can be used to introduce recombinant DNA into the bacterium and to transform it using the recombinant DNA.

[0026] The medium used in the PHB accumulation step contains inorganic salts and an organic carbon source. The pH of the medium is not particularly limited as long as it satisfies the growth conditions of the halophilic bacteria, but specifically, it should be about pH 5 to 12, and more preferably pH 8.8 to 12. It is preferable to use an alkaline medium, as this effectively prevents contamination with other bacteria.

[0027] The medium may be a liquid medium or a solid medium.

[0028] The inorganic salts to be added to the culture medium used in the PHB accumulation step are not particularly limited, and examples include phosphates, nitrates, carbonates, sulfates, and metal salts such as sodium, magnesium, potassium, manganese, iron, zinc, copper, and cobalt.

[0029] For example, when sodium is used as an inorganic salt, NaCl, NaNO3, NaHCO3, Na2CO3, etc. may be used.

[0030] It is preferable to use compounds that serve as nitrogen and phosphorus sources for the halophilic bacteria as these inorganic salts.

[0031] The nitrogen source may be, but is not limited to, nitrates, nitrites, urea, ammonium salts, etc., and may be compounds such as NaNO3, NaNO2, NH4Cl, etc.

[0032] The amount of nitrogen source used may be appropriately set within a range that does not affect the growth of the bacterial cells and achieves the purpose of producing cytochrome c peroxidase. Specifically, the amount of nitrate used is usually about 500 mg or more, more preferably about 1000 mg or more, and even more preferably about 1250 mg or more per 100 ml of medium at the initial stage of culture.

[0033] The phosphorus source may be a phosphate, monohydrogen phosphate, dihydrogen phosphate, or the like, and is not particularly limited, but may be, for example, a compound such as K2HPO4 or KH2PO4.

[0034] The amount of phosphorus source used may be determined appropriately from the same viewpoint as the amount of nitrogen source used. Specifically, the amount of dihydrogen phosphate used is usually about 50 to 400 mg per 100 ml, and more preferably about 100 to 200 mg per 100 ml.

[0035] These inorganic salts may be used alone or in combination of two or more.

[0036] Inorganic salts including other compounds may be used at a total concentration of usually about 0.1 to 2.5M, preferably about 0.2 to 1.0M, and more preferably about 0.2 to 0.5M.

[0037] The organic carbon source to be added to the medium used in the PHB accumulation step is not particularly limited and includes, for example, tryptone, yeast extract, soluble starch, ethanol, n-propanol, acetic acid, sodium acetate, propionic acid, waste glycerol, waste molasses, wood saccharification solution, hexoses such as psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, gulose, idose, galactose, and talose, pentoses such as ribulose, xylulose, ribose, arabinose, xylose, lyxose, and deoxyribose, disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose, and sugar alcohols such as erythritol, glycerin, mannitol, sorbitol, and xylitol.

[0038] The concentration of the organic carbon source may be appropriately set within a range in which accumulation of PHB progresses and the objective of producing cytochrome c peroxidase is achieved.

[0039] In the production method of the present invention, halophilic bacteria belonging to the genus Halomonas are cultured in a medium with a relatively high salt concentration, so there is almost no risk of contamination or proliferation of other bacteria, etc. Therefore, the medium may or may not be sterilized, and the culture can be carried out using simple equipment.

[0040] The halophilic bacteria in the PHB accumulation step are cultured under aerobic conditions. The conditions for the aerobic culture are not particularly limited as long as they allow the bacteria to grow and a significant amount of PHB to accumulate within the bacteria.

[0041] Specifically, the halophilic bacterium is inoculated into approximately 5 ml of medium and pre-cultured overnight with shaking at a predetermined stirring speed and temperature. The bacterial cells obtained by pre-culture are then diluted approximately 100-fold in a medium placed in an Erlenmeyer flask, fermenter, jar fermenter, or the like, and main culture (corresponding to aerobic culture in the PHB accumulation step in this application) is carried out.

[0042] The culture temperature for the main culture can usually be set within a range of about 20 to 45°C, but is preferably set within a range of about 30 to 37°C. Furthermore, when an Erlenmeyer flask is used, the stirring speed can usually be set within a range of about 120 to 250 rpm, but is preferably set within a range of about 120 to 180 rpm. When a fermenter or jar fermenter is used, it is preferable to supply oxygen at an oxygen supply rate comparable to the above. Furthermore, the culture time for the main culture is not particularly limited as long as it is a time period during which PHB accumulation occurs, but is preferably a time period during which the amount of PHB accumulated within the halophilic bacterium becomes approximately constant, for example, 10 to 60 hours.

[0043] In the PHB accumulation step, halophilic bacteria belonging to the genus Halomonas may be aerobically cultured under these culture conditions. Specifically, the dissolved oxygen concentration in the medium during aerobic culture is not particularly limited, but is usually 2 mg / L in the absence of bacterial cells, and preferably 5 mg / L or higher.

[0044] The culture method in the PHB accumulation step includes, but is not limited to, batch culture, semi-batch culture, continuous culture, and the like. However, considering that the halophilic bacteria used in the method of the present invention are highly unlikely to be contaminated with other bacteria, long-term continuous culture for efficient cytochrome production is also possible. The culture environment may be one in which the medium is exposed to air, and may be adjusted by actively blowing an oxygen-containing gas onto the surface of the medium or by blowing such a gas into the medium. The culture environment may be either a non-sterile or sterile environment.

[0045] In the method for producing cytochrome c peroxidase according to the present invention, the PHB accumulation step is carried out so that halophilic bacteria accumulate PHB, and cytochrome c peroxidase is released from the halophilic bacteria into the medium, although the mechanism behind this is not clear.

[0046] [3HB secretion process] The 3HB secretion step in the production method of the present invention is a step that follows the PHB accumulation step, in which halophilic bacteria are microaerobically cultured in a medium while adjusting the pH, and poly-3-hydroxybutyrate accumulated within the halophilic bacteria is secreted into the medium as 3-hydroxybutyric acid.

[0047] Specifically, in the 3HB secretion step, after the PHB accumulation step, aeration is stopped, a pH adjuster is added to adjust the pH to within a predetermined range, and the halophilic bacteria are cultured under microaerophilic conditions.

[0048] The conditions for microaerobic culture are not particularly limited as long as they allow PHB accumulated within the bacterial cells to be secreted into the medium as 3HB.

[0049] When microaerobic cultivation is continued, the pH of the medium tends to decrease due to the production of organic acids. The pH of such a medium can be appropriately confirmed using a known pH measuring device or a jar fermenter equipped with such a device.

[0050] In the 3HB secretion step, the pH is adjusted and / or maintained within a predetermined range. "Adjusting and / or maintaining" means maintaining the pH within a predetermined range by adding a pH adjuster while checking the pH, or simply adjusting the pH at the start of culture by adding a pH adjuster, without subsequently adjusting the pH.

[0051] The pH adjusted and maintained during the 3HB secretion step is preferably 9.0 or higher and 12.0 or lower, more preferably 9.5 or higher and 11.0 or lower.

[0052] Halophilic bacteria belonging to the genus Halomonas can usually be cultured under moderately high salt concentrations and alkaline conditions, resulting in little contamination by contaminating bacteria. However, some lactic acid bacteria can grow in environments with moderately high salt concentrations and a pH of 8.4 or less. If such bacteria contaminate the culture system of the present invention, they may consume 3-hydroxybutyric acid or a salt thereof secreted by the halophilic bacteria belonging to the genus Halomonas as a substrate for lactic acid fermentation, and further decrease the pH of the medium.

[0053] Therefore, in the present invention, when halophilic bacteria belonging to the genus Halomonas are cultured in a non-sterilized medium and / or in a non-sterile environment to secrete 3HB into the medium, it is preferable to adjust and maintain the pH of the medium during the 3HB secretion step at a pH of 9.0 or higher and 12.0 or lower. Because a pH higher than 12.0 reduces the amount of cytochrome c peroxidase produced, it is preferable to keep the pH of the medium below 12.0.

[0054] The pH adjuster used for adjusting the pH is not particularly limited as long as it is an alkaline substance, but sodium hydroxide or potassium hydroxide can be used. The timing of adjusting the pH is not particularly limited as long as it is after the PHB accumulation step, but it is preferably after the amount of PHB accumulated in the halophilic bacterium has become approximately constant.

[0055] In the method for producing cytochrome c peroxidase according to the present invention, a 3HB secretion step is carried out, and PHB accumulated in the halophilic bacterium is secreted as 3HB. This increases the amount of cytochrome c peroxidase released into the medium from the halophilic bacterium, although the mechanism behind this is not clear.

[0056] [Recovery process] The recovery step in the production method of the present invention is a step of recovering cytochrome c peroxidase from the medium after the PHB accumulation step or the 3HB secretion step.

[0057] In the recovery step, cytochrome c peroxidase may be recovered using a known method. Recovery refers to stopping the culture in the PHB accumulation step or 3HB secretion step when cytochrome c peroxidase is present in the medium, and separating the medium containing cytochrome c peroxidase from the halophilic bacterial cells. For example, when the PHB accumulation step or 3HB secretion step is performed using a liquid medium, the culture in these steps is stopped, and the culture solution obtained in these steps is separated from the halophilic bacterial cells using a separation means, as necessary, to obtain the culture solution.

[0058] Specific separation techniques that can be used include known solid-liquid separation procedures such as centrifugation and filtration. The method for terminating the culture is also not particularly limited. For example, after the PHB accumulation step or the 3HB secretion step, the halophilic bacteria may be sterilized by heating, acid treatment, or the like, or a solid-liquid separation procedure may be performed to separate the halophilic bacteria from the medium.

[0059] The method for confirming the presence of cytochrome c peroxidase in the medium may vary depending on the bacterial species, medium components, culture conditions, etc., and should be appropriately determined taking these factors into consideration. For example, the presence of cytochrome c peroxidase can be confirmed by sampling the medium over time and subjecting it to an analytical method such as capillary electrophoresis.

[0060] Methods for separating cytochrome c peroxidase from the culture medium include column chromatography and membrane separation. For example, the culture medium after the 3HB secretion step contains cytochrome c and 3HB in addition to cytochrome c peroxidase. Because the molecular sizes of cytochrome c and cytochrome c peroxidase are significantly different from those of 3HB, they can be separated using an ultrafiltration membrane. In the method for producing cytochrome c peroxidase of the present invention, the amount of cytochrome c peroxidase produced increases with an increase in the pH of the medium during microaerobic culture, but the amount of cytochrome c produced does not. [Example]

[0061] The present invention will be described in more detail below with reference to examples, although it goes without saying that the present invention is not limited to these examples.

[0062] In the following examples, a medium based on SOT Modified 5 (Spirulina platensis Medium Modified 5) shown in Table 1 was used. This medium is Spirulina platensis Medium (National Institute for Environmental Studies website), and the amounts of NaHCO3 and Na2CO3 were adjusted, the nitrogen source NaNO3 was increased five-fold, the phosphorus source K2HPO4 was increased four-fold, and FeSO4·7H2O was increased two-fold. The pH of the medium after adjustment was 9.4 ± 0.1, and it was used as is without sterilization procedures such as autoclaving.

[0063] [Table 1]

[0064] During the culture, the above medium was supplemented with a 26% aqueous sucrose solution.

[0065] Example 1 50 L of the medium supplemented with the sucrose solution was placed in a 90 L jar fermenter, and Halomonas sp. KM-1 was cultured aerobically for 40 hours (PHB accumulation process). After that, aeration was stopped, and the culture was cultured under microaerophilic conditions for 5 hours while adjusting the pH to 8.6, 9.0, 10.0, 11.0, or 12.0 using sodium hydroxide. The PHB accumulated within the cells was secreted as 3HB (3HB secretion process). Subsequently, a red component containing heme iron was separated from the concentrate by membrane separation of the appropriate molecular weight fraction (recovery process).

[0066] Figure 1 shows the color change of the concentrate when the pH of the medium during microaerobic culture was adjusted to 8.6, 9.0, 10.0, 11.0, or 12.0. As is clear from Figure 1, the red color deepened as the pH of the medium increased, with the red color of the medium being the most intense at pH 11.0. This confirmed that increasing the pH of the medium increased the amount of cytochrome c peroxidase produced. Furthermore, when the medium was adjusted to pH 12.0, the red color of the medium became lighter than when adjusted to pH 11.0, and the intensity of the red color was equivalent to that of the medium adjusted to pH 10.0. This suggests that when the pH of the medium during microaerobic culture exceeds 12.0, the production of cytochrome c peroxidase may be suppressed.

[0067] Figure 2 shows the results of SDS-PAGE protein separation of cytochrome c peroxidase produced when the medium pH was adjusted to 9.0 and 11.0 during microaerobic culture. The band detected around 6.5 kDa is cytochrome c, and the band detected around 37 kDa is cytochrome c peroxidase. Figure 2 shows that there is no difference in the size of the cytochrome c band between pH 9.0 and 11.0, suggesting that the amount of cytochrome c produced is not affected by pH. On the other hand, the size of the cytochrome c peroxidase band was larger at pH 11.0 than at pH 9.0, indicating that the amount of cytochrome c peroxidase produced was greater at pH 11.0 than at pH 9.0. These results confirm that the darker red color of the medium with increasing pH is due to the increased production of cytochrome c peroxidase.

[0068] Example 2 The test was carried out in the same manner as in Example 1, except that the amount of FeSO4·7H2O added was 10 mg / 100 mL and the pH of the medium in the 3HB secretion step was adjusted to 9.5.

[0069] The concentrated solution was subjected to protein separation by SDS-PAGE, and the results are shown in Figure 3. As shown in Figure 3, a cytochrome c peroxidase band was detected around 37 kDa. The band detected around 6.5 kDa was identified as cytochrome c5 (Accession Number WP_010626206.1). The band around 37 kDa was excised and sequenced, and was identified as cytochrome c peroxidase (Accession Number WP_010628388.1).

[0070] From the above, it was confirmed that in the production of cytochrome c peroxidase using halophilic bacteria belonging to the genus Halomonas, cytochrome c peroxidase can be produced by adjusting the pH of the culture medium in the 3HB secretion step to between 9.0 and 12.0, and by adding ferrous sulfate heptahydrate to the medium at a concentration of 2 mg / 100 mL or more. [Industrial Applicability]

[0071] The present invention can be applied to a method for producing cytochrome c peroxidase.

Claims

1. a PHB accumulation step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium for 10 hours or more to cause the halophilic bacterium to accumulate poly-3-hydroxybutyric acid; a 3HB secretion step of microaerophilically culturing the halophilic bacterium in the medium while adjusting the pH, and secreting the poly-3-hydroxybutyrate accumulated in the cells of the halophilic bacterium as 3-hydroxybutyric acid into the medium, a recovery step of recovering cytochrome c peroxidase from the medium; The method for producing cytochrome c peroxidase comprises adjusting the pH during the microaerobic culture to 9.0 or higher and 12.0 or lower.

2. 2. The method for producing cytochrome c peroxidase according to claim 1, wherein the amount of iron sulfate heptahydrate in the medium is 2 mg / 100 mL or more and 10 mg / 100 mL or less.

3. 3. The method for producing cytochrome c peroxidase according to claim 1, wherein the halophilic bacterium is Halomonas sp. KM-1 strain (FERM BP-10995).

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