WO2017126542A1 - 酸化還元酵素の活性測定方法 - Google Patents
酸化還元酵素の活性測定方法 Download PDFInfo
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- WO2017126542A1 WO2017126542A1 PCT/JP2017/001522 JP2017001522W WO2017126542A1 WO 2017126542 A1 WO2017126542 A1 WO 2017126542A1 JP 2017001522 W JP2017001522 W JP 2017001522W WO 2017126542 A1 WO2017126542 A1 WO 2017126542A1
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- Prior art keywords
- hao
- measurement
- hydroxylamine
- microorganisms
- activity
- Prior art date
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/24—Dialysis ; Membrane extraction
- B01D61/243—Dialysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
- C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
- C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12Y107/03004—Hydroxylamine oxidase (1.7.3.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/24—Earth materials
Definitions
- the present invention relates to a method for measuring HAO activity, which comprises contacting hydroxylamine oxidoreductase (HAO) with hydroxylamine in the presence of a tetrazolium salt.
- HAO hydroxylamine oxidoreductase
- the present invention also relates to a method for producing HAO.
- the present invention also relates to a method for screening an HAO inhibitor comprising contacting HAO with hydroxylamine and a candidate compound in the presence of a tetrazolium salt.
- the present invention includes culturing microorganisms in a sample storage container containing microorganisms installed separately in a liquid medium storage container, and the sample storage container including microorganisms has pores. Also provide.
- Nitrogen fertilizers that are particularly important as chemical fertilizers are over-administered to farmland as ammonia nitrogen (NH 4 + ). However, most of it is nitrified, leading to the outflow of nitrogen fertilizer from farmland and the generation of greenhouse gases (N 2 O).
- Existing nitrification inhibitors have problems such that they cannot be used at 20 ° C. or higher due to volatility, have weak effects, have carcinogenicity and residual toxicity, and new nitrification inhibitors are expected to solve these problems.
- Existing nitrification inhibitors are thought to target ammonia monooxygenase (AMO).
- HAO hydroxylamine oxidoreductase
- the present inventor has found a method for measuring the activity of hydroxylamine oxidoreductase, which is measured in a reaction system in which a large amount of hydroxylamine is present relative to a tetrazolium salt, particularly a resazurin salt of a tetrazolium salt, and has completed the present invention.
- a sample containing non-denaturing HAO after gel electrophoresis, and by performing an enzyme reaction in the gel and visualizing the reaction product by fluorescence measurement, the presence or absence of the enzyme and the strength of the activity. has been found to be effectively analyzed, and the present invention has been completed.
- the gist of the present invention is as follows.
- a method for measuring the activity of HAO comprising contacting hydroxylamine oxidoreductase (HAO) with hydroxylamine in the presence of a tetrazolium salt.
- HAO hydroxylamine oxidoreductase
- tetrazolium salt is a resazurin salt.
- HAO is purified from a sample containing HAO, and the HAO activity is determined by the measurement method according to any one of [1] to [8] in order to discriminate a fraction containing HAO in the purification.
- a method for manufacturing HAO to be measured. [10] The production method according to [9], comprising drying the purified HAO.
- a method for screening an HAO inhibitor comprising contacting HAO with hydroxylamine and a candidate compound in the presence of a tetrazolium salt.
- a method for culturing microorganisms comprising culturing microorganisms in a sample storage container containing microorganisms installed separately in a liquid medium storage container, wherein the sample storage container including microorganisms has pores.
- the culture method according to [12] wherein the volume ratio of the sample containing the liquid medium and the microorganism is 10: 1 to 1000: 1.
- a method for measuring HAO activity which has not been known so far, and is capable of performing a homogeneous assay with a very low sensitivity and a low price.
- the HAO activity measurement method according to the present invention is suitable for high-throughput screening, and can be detected directly from live bacteria, directly from samples such as soil, or directly from non-denaturing polyacrylamide gels. It is.
- the HAO activity measurement according to the present invention is possible even in the presence of normal oxygen, real-time measurement is possible, and higher sensitivity can be obtained by fluorescence measurement.
- the HAO activity measurement method according to the present invention does not require an electron carrier such as PMS.
- difficult-to-cultivate microorganisms particularly microorganisms in soil, compost or activated sludge can also be effectively cultured.
- the HAO production method according to the present invention does not perform ammonium sulfate fractionation, the purification time is greatly shortened and the scale-up is facilitated.
- the HAO obtained by the present invention can be stored for a long time.
- the activity of HAO in the sample can be quantified.
- a candidate compound as a HAO inhibitor is treated in advance for these samples, the HAO inhibitory effect of the candidate compound can be easily analyzed.
- FIG. 1 shows one embodiment of a method for producing HAO according to the present invention.
- 1 shows one embodiment of soil direct HAO activity measurement according to the present invention.
- FIG. 2 shows a gel image of HAO fluorescently stained with resazurin after non-denaturing polyacrylamide gel electrophoresis of purified HAO of N. europaea and disrupted supernatant of bacterial cells according to the present invention.
- the band indicated by an arrow in the figure is HAO.
- the present invention provides a method for measuring HAO activity, which comprises contacting hydroxylamine oxidoreductase (HAO) with hydroxylamine in the presence of a tetrazolium salt.
- HAO hydroxylamine oxidoreductase
- the HAO activity measurement method of the present invention detects a change in absorbance of a tetrazolium salt, preferably a resazurin salt of a tetrazolium salt, or a change to a fluorescent substance.
- the HAO activity measuring method of the present invention it is preferable to measure at pH 4.0 to 8.0.
- the pH may be measured from 4.6 to 7.6.
- the buffer can be selected from citrate-phosphate buffer, potassium phosphate buffer, HEPES-NaOH buffer, Tris-HCl buffer, and the like. Since the HAO activity (initial speed) changes when the buffer and pH are changed, these conditions can be selected depending on the experimental purpose. For example, conditions with the highest HAO activity that reaches the maximum fluorescence intensity in about 15 minutes are suitable for rapid measurement and measurement when the HAO concentration is low.
- the measurement conditions are suitable for a process that requires quick measurement.
- potassium phosphate buffer and citrate-phosphate buffer at a pH of about 5.6, or HEPES-NaOH buffer and Tris-HCl buffer at a pH of about 6.8 can be used.
- the measurement conditions are suitable for a process that requires performing a large amount of assays in parallel, such as drug screening, and suppressing blur between assays.
- a potassium phosphate buffer having a pH of around 7.0 can be used.
- the sample may be a solid or liquid containing bacterial communities, preferably soil, compost or activated sludge, and the activity of HAO contained in them may be directly measured.
- the activated sludge is sewage during purification treatment including floating sludge in which microorganisms are abundant, and means both sludge and sewage.
- the activity measurement method of the present invention enables HAO-specific fluorescent staining from a large number of protein bands separated by non-denaturing polyacrylamide gel electrophoresis. In non-denaturing polyacrylamide gel electrophoresis, HAO is separated while retaining activity in the gel.
- the tetrazolium salt By contacting HAO in the gel with hydroxylamine in the presence of a tetrazolium salt, the tetrazolium salt is converted into a fluorescent substance. Furthermore, by using a gel imager light source and a fluorescence observation filter, a fluorescent substance in the gel can be detected with high sensitivity. Accordingly, blue light (440-500 nm) can be used as an excitation light source, more preferably cyan light (wavelength 480-530 nm), and even more preferably green light (490-580 nm). An orange filter can be used as a filter for fluorescence observation.
- native-PAGE, blue native-PAGE, etc. which are non-denaturing polyacrylamide gel electrophoresis methods
- the sample to be subjected to electrophoresis may be purified HAO, crushed supernatant of gonococcal cells, soil sample, activated sludge, and the like, and a sample to which an HAO inhibitor is added. If purified HAO whose concentration or activity is known in advance is used as a marker, the HAO activity can be simply quantified.
- the HAO activity measurement method of the present invention can be applied to high-throughput screening, and can screen for inhibitors of HAO, which is a central enzyme of nitrification reaction.
- the present invention also includes culturing microorganisms in a sample storage container containing microorganisms installed separately in a liquid medium storage container, and the sample storage container including microorganisms has pores, culture of microorganisms Provide a method.
- the volume ratio of the liquid medium to the sample containing the microorganism is 10: 1 to 1000: 1, preferably 100: 1 to 800: 1, more preferably 200: 1 to 600: 1. Most preferably, it may be 400: 1.
- the liquid medium storage container may be a bottle of 1 to 50 L, preferably 3 to 30 L, more preferably 20 L, but is not limited thereto, and the liquid medium storage container is scaled up. In addition, it is easy to scale up the sample storage container containing microorganisms.
- the sample storage container containing microorganisms may have any shape, but is, for example, a cylinder, a tube, a dish, or the like, and preferably a tube.
- a sample storage container containing microorganisms has pores, and the pores have a molecular weight cut-off of 50 KDa or more to make it difficult to suppress growth due to insufficient replacement of medium components, and 1000 KDa or less to prevent passage of microorganisms ( MWCO).
- the sample storage container containing the microorganism is preferably a dialysis tube having such a molecular weight cut off.
- the sealing port of the sample storage container containing microorganisms is preferably sterilized.
- difficult-to-cultivate microorganisms as described in Journal of Environmental Biotechnology Vol.7, No.2, 69-73, 2007, and more particularly very low It is possible to cultivate difficult-to-culture microorganisms that reach the stationary phase at a cell number level of turbidity (OD 600 is 0.2 or less).
- Examples of such microorganisms are ammonia-oxidizing bacteria, ammonia-oxidizing archaea or nitrite-oxidizing bacteria, iron-reducing bacteria, sulfur-reducing bacteria, and anammox bacteria.
- ammonia-oxidizing bacteria include the genus Nitrosomonas , Nitrosococcus , and Nitrosospira. For example, but not limited to.
- microorganisms can be effectively cultured even if they are difficult-to-cultivate microorganisms, and the labor of centrifuging the medium can be greatly simplified when collecting the cells.
- hydroxylamine oxidoreductase HEO
- HEO hydroxylamine oxidoreductase
- the method for producing HAO of the present invention may include purifying HAO from a sample containing HAO, and may include a step of measuring the activity of HAO to discriminate a fraction containing HAO in the purification.
- the step of measuring the activity of HAO is preferably, but not limited to, the method for measuring the activity of HAO of the present invention may be applied.
- microorganisms are cultured by the method for culturing microorganisms of the present invention, the cells of microorganisms containing HAO are crushed, the supernatant fraction is applied to a column, and the activity of HAO is measured.
- the method includes detecting a fraction containing HAO and applying it to a column under low oxygen concentration conditions as necessary.
- the column used in the method for producing HAO of the present invention include, but are not limited to, an anion exchange column, a gel filtration column, a hydroxyapatite column, a strong anion exchange column, and a strong cation exchange column.
- the production of HAO is preferably carried out under low oxygen concentration conditions, more preferably under 0.5% oxygen concentration conditions.
- the method for producing HAO of the present invention may include a step of drying HAO purified for storage.
- HAO Hydroxylamine oxidoreductase was produced. HAO was purified from ammonia oxidizing bacteria and stored in solution or under low oxygen concentration conditions after drying.
- Strain 1 Nitrosomonas europaea NBRC14298 (purchased from NBRC * 1 )
- Strain 2 Nitrosococcus oceani ATCC19707 (purchased from US ATCC * 2 )
- Strain 3 Nitrosomonas sp. NBRC108559 (purchased from NBRC)
- Strain 4 Nitrosomonas cryotolerans ATCC49181 (purchased from ATCC)
- Strain 5 Nitrosospira multiformis ATCC25196 (purchased from ATCC) * 1 NBRC: Biotechnology Center, National Institute for Product Evaluation and Research * 2 ATCC: American type culture collection
- the mixture was adjusted to pH 7.8 with an aqueous sodium hydroxide solution and then autoclaved.
- the mixture was adjusted to pH 7.8 with an aqueous sodium hydroxide solution and then autoclaved.
- NBRC medium 1201 Strain 3: Nitrosomonas sp. NBRC108559 Medium used for culture
- the mixture was adjusted to pH 7.8 with an aqueous sodium hydroxide solution and then autoclaved.
- Trace element mixing solution (trace element solution) The following components were dissolved in 1 L of pure water.
- the microorganism was cultured in two ways : a normal culture method and a culture method using a dialysis tube according to the present invention.
- the dialysis tube method the cells were suspended in 100 mL of medium, 50 mL was taken from the suspension, sealed in a 50 mL dialysis tube, and the tube was submerged in 20 L of medium and cultured. According to this method, the culturing time becomes long, but it is not necessary to collect bacterial cells by centrifuging a large amount of medium.
- the collected cells were placed in a 50 mL plastic test tube after completely removing the culture medium, filled with nitrogen gas to obtain a low oxygen concentration condition, and then stored in a ⁇ 80 ° C. freezer until enzyme purification was performed.
- the amount of each bacterial cell was about 1 to 2 g per 20 L medium.
- the cells in about 30 mL of the pre-cultured medium were collected by centrifugation at 7,000 g and resuspended in 50 mL of new medium.
- the resuspended cells were placed in an autoclave-sterilized dialysis tube (Spectra / Pore (R) 6 Dialysis Membrane MWCO: 50 kD: SPECTRUM) and sealed with an autoclave-sterilized dialysis tube clip (SPECTRUM).
- the enclosure was sterilized with sterilizing ethanol (Wako Pure Chemical Industries, Ltd.) and isodine scrub (Meiji Seika Pharmacia). .
- the dialysis tube in which the bacterial cells were sealed was placed in a 20 L plastic bottle (Nalgene® Clearboy: Thermo Fisher Scientific) containing 20 L medium, and culture was performed. Incubation was performed in a room at a room temperature of 26 ° C. by feeding 2.5 L / min of air through an autoclaved vent filter (Millex-FG: Millipore) with an air pump. Three weeks later, since the pH indicator in the medium became yellow (pH 6.0), the culture was terminated, and the culture solution containing the cells was collected from the dialysis tube. The culture solution was transferred to a 50 mL plastic centrifuge tube and collected by centrifugation at 7,000 g for 15 minutes.
- Nitrogen gas was sealed in a centrifuge tube containing the bacterial cell pellets, the conditions were low, and the sample was stored frozen in a freezer at -80 ° C. until enzyme purification was performed.
- the amount of each bacterial cell was about 1 to 2 g per 20 L medium.
- a device for purifying under low oxygen concentration conditions without inactivating oxygen-sensitive HAO HAO derived from bacteria other than Nitrosomonas europaea and Nitrosococcus oceani is very sensitive to oxygen and takes 2 to 3 days. Most of it was inactivated, so it was necessary to purify under low oxygen concentration conditions. Since Nitrosomonas europaea and Nitrosococcus oceani can be expected to retain their activity by purifying under low oxygen concentration conditions, they were purified in the same manner.
- All buffers were degassed by aeration of nitrogen gas for 1 hour or more in advance to obtain low oxygen concentration conditions.
- Nitrogen gas used for aeration showed a detection limit of 0.00% on the low concentration oxygen monitor JKO-O2 Ver.3 (Zico Corporation) (hereinafter referred to as oxygen concentration of 0.01% or less).
- the degassed buffers were confirmed to have a detection limit of 0.01 mg / ml or less with a dissolved oxygen meter (MultiLine 3410 model, optical DO electrode FDO925 model, WWT company).
- a plastic bag was placed on the fraction collector of the chromatography system, and nitrogen gas was always allowed to flow there to perform purification while maintaining low oxygen concentration conditions.
- the oxygen concentration in the plastic bag was 0.01% or less.
- nitrogen gas was constantly fed into a glass bottle containing a degassed buffer solution to prevent oxygen from dissolving and to maintain a low oxygen concentration condition.
- a buffer solution for measurement (100 mM potassium phosphate buffer, 300 mM sodium chloride) was prepared. The work was carried out in a glove box under low oxygen concentration conditions (oxygen concentration of 0.01% or less). The prepared solution was stored at 4 ° C.
- a buffer solution at pH 7.0 was prepared for screening for HAO inhibitors, and a buffer solution at pH 5.6 was prepared for confirmation of fractions containing HAO during HAO purification and for measurement of soil direct HAO activity.
- the screening compound library was subjected to HAO fluorescence activity measurement by the resazurin method to verify the inhibitory effect of these compounds.
- Forty compounds were dissolved in DMSO for molecular biology (Wako Pure Chemical Industries) or ultrapure water at a concentration of 100 mM, and further a 16-fold dilution series (100 mM, 50 mM, 25 mM, 12.5 mM, 6.25 mM, 3.13 mM, 1.56 mM, 0.78 mM, 0.39 mM, 0.19 mM, 97 ⁇ M, 48.8 ⁇ M, 24.4 ⁇ M, 12.2 ⁇ M, 6.1 ⁇ M, 3.1 ⁇ M) The density is 1/200 of these).
- a similar dilution series was prepared for phenylhydrazine, a known HAO inhibitor.
- Compound of 0.5 ⁇ L at each concentration is supplied to a 384-well microplate (MICROPLATE, 384 Well, NON-BINDING, F-BOTTOM, BLACK, Greiner Bio-One) using a 16-channel electric pipette (VIAFLO II 12.5 ⁇ L, INTEGRA) To empty wells.
- VIAFLO II 12.5 ⁇ L, INTEGRA 16-channel electric pipette
- the composition is about 50 ⁇ L for the buffer for 2-fold dilution, 25 ⁇ L for 400 ⁇ M resazurin for 4-fold dilution, and 0.5 ⁇ L for 5 nM HAO).
- the plate is inserted into a fluorescent plate reader with reagent injector (infinite M1000 PRO, Tecan Co., Ltd.), 4 ⁇ l of 4 mM hydroxylamine stock solution for dilution is added at 25 ⁇ L per well, and 10 times the amount of hydroxylamine for resazurin in the reaction system. (Molar ratio).
- IC 50 50% inhibitory concentration
- GraphPad Prism 6 GraphPad
- Storage conditions Both are stored in a light-proof bottle and stored at 4 ° C.
- a 0.5 g soil sample was placed in a 12-well plate (Corning). 250 ⁇ L of pH 5.6 potassium phosphate buffer for 2-fold dilution and 125 ⁇ L of resazurin stock solution for 4-fold dilution were added. Rotating and shaking at 300 rpm for 5 minutes. 125 ⁇ L of a 4-fold dilution hydroxylamine stock solution was added, and the amount of hydroxylamine with respect to resazurin was made 10 times (molar ratio) in the reaction system.
- the netwell protrudes about 5 mm on the plate, it could not be inserted into the plate reader (infinite M1000 PRO, Tecan Co.). Therefore, the upper part of the netwell was cut with a heating wire cutter for styrene foam, and then inserted into a plate and inserted into a plate reader.
- Measurement was performed using a plate reader (infinite M1000 PRO, Tecan Co., Ltd.) and fluorescence measurement (excitation wavelength: 562 nm: wavelength width: 20 nm, measurement wavelength: 592 nm: wavelength width: 20 nm) in the kinetic mode every minute. While measurement was not being performed, the plate was shaken using a shake function of a plate reader and incubated at room temperature (26 ° C.).
- the value derived from HAO present in the soil sample was determined from the difference in relative fluorescence intensity between the untreated well and the inhibitor-added well at the incubation time of 60 minutes when the fluorescence intensity was highest.
- the position of this band was present at a position equivalent to the purified HAO band derived from N. europaea used as a marker, and was confirmed to be a HAO band. From the fluorescence intensity of the band, it was possible to easily quantify the HAO activity in the sample.
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Abstract
Description
[1] テトラゾリウム塩の存在下でヒドロキシルアミン酸化還元酵素(HAO)をヒドロキシルアミンと接触させることを含む、HAOの活性測定方法。
[2] テトラゾリウム塩が、レサズリン塩である、[1]記載の測定方法。
[3] テトラゾリウム塩に対するヒドロキシルアミンが、少なくとも3倍量のモル比で接触させる、[1]または[2]記載の測定方法。
[4] テトラゾリウム塩に対するヒドロキシルアミンが、少なくとも5倍量のモル比で接触させる、[1]~[3]のいずれか一項記載の測定方法。
[5] テトラゾリウム塩に対するヒドロキシルアミンが、少なくとも10倍量のモル比で接触させる、[1]~[4]のいずれか一項記載の測定方法。
[6] pH4.0から8.0で測定する、[1]~[5]のいずれか一項記載の測定方法。
[7] pH4.6から7.6で測定する、[1]~[6]のいずれか一項記載の測定方法。
[8] 測定対象が、土壌、堆肥、活性汚泥又はそれらを非変性状態で電気泳動したポリアクリルアミドゲルである、[1]~[7]のいずれか一項記載の測定方法。
[9] HAOを含む試料からHAOを精製することを含み、精製においてHAOが含まれる画分を判別するために[1]~[8]のいずれか一項記載の測定方法によりHAOの活性を測定する、HAOの製造方法。
[10] 精製されたHAOを乾燥することを含む、[9]記載の製造方法。
[11] テトラゾリウム塩の存在下でHAOをヒドロキシルアミン及び候補化合物と接触させることを含む、HAOインヒビターのスクリーニング方法。
[12] 液体培地格納容器内に隔離して設置された微生物を含む試料格納容器内で、微生物を培養することを含み、微生物を含む試料格納容器は、細孔を有する、微生物の培養方法。
[13] 液体培地と微生物を含む試料の体積比が、10:1~1000:1である、[12]記載の培養方法。
[14] 細孔の分画分子量が、50K~1000KDaである、[12]または[13]記載の培養方法。
[15] 微生物を含む試料格納容器が、透析チューブである、[12]~[14]のいずれか一項記載の培養方法。
[16] 微生物を含む試料格納容器の封入口が、殺菌されている、[12]~[15]のいずれか一項記載の培養方法。
[17] 微生物が、難培養性微生物である、[12]~[16]のいずれか一項記載の培養方法。
[18] 微生物が、OD600が0.2以下の濁度で静止期に達する微生物である、[1]~[17]のいずれか一項記載の培養方法。
ヒドロキシルアミン酸化還元酵素を製造した。HAOはアンモニア酸化細菌から精製し、溶液状態または乾燥後に低酸素濃度条件下で保存した。
1.1.1 菌株
以下5種のアンモニア酸化細菌を利用した。
菌株1:Nitrosomonas europaea NBRC14298 (NBRC*1より購入)
菌株2:Nitrosococcus oceani ATCC19707 (米国ATCC*2より購入)
菌株3:Nitrosomonas sp. NBRC108559 (NBRCより購入)
菌株4:Nitrosomonas cryotolerans ATCC49181 (ATCCより購入)
菌株5:Nitrosospira multiformis ATCC25196 (ATCCより購入)
*1NBRC:独立行政法人製品評価基盤研究機構バイオテクノロジーセンター
*2ATCC: American type culture collection
使用した培地の組成は、以下の通りであった。
1.1.2.1 培地1
菌株1:Nitrosomonas europaea NBRC14298
菌株5:Nitrosospira multiformis ATCC25196
の培養に使用する培地
菌株2:Nitrosococcus oceani ATCC19707
菌株4:Nitrosomonas cryotolerans ATCC49181
の培養に使用する培地
菌株3:Nitrosomonas sp. NBRC108559
の培養に使用する培地
以下成分を純水1Lに溶解させた。
微生物は、通常の培養方法と、本発明による透析チューブを利用した培養方法の2通りで培養した。透析チューブ法では、100mLの培地に菌体を懸濁し、そこから50mLをとって、50mL透析チューブに封入し、チューブを20Lの培地に沈めて培養した。この方法によれば、培養時間は長くなるが、大量の培地の遠心分離による菌体回収が不要になった。
各菌株を、菌株に適した30mLの培地を入れた、換気フィルター付50mLプラスチック試験管(バイオリアクターチューブ、TPP社)にて培養した。アンモニア酸化細菌は亜硝酸を分泌するため、増殖に伴いpHが低下し、pH6程度になると定常状態となり増殖が停止する。そのため、培地中に含まれるpH指示薬のフェノールレッドが黄色(pH6.0付近)になるまで1週間程度培養した。
透析チューブを用いない通常の培養では、前培養された30mLの培養液を20L培地入りの20Lプラスチックボトル(Nalgene クリアボーイ:サーモフィッシャーサイエンティフィック社)に入れ、培養を行った。培養は、オートクレーブ済みベントフィルター(Millex-FG:ミリポア社)を通した空気をエアーポンプで毎分2.5L送り込み、室温26℃の部屋にて行った。1週間後、培地中のpH指示薬が黄色(pH6.0)になったため、培養を終了した。培養液は500mLのプラスチック遠沈管に移し、7, 000gの遠心分離を複数回行ない集菌した。集菌した菌体は、培地を完全に除去後に50mLプラスチック試験管に入れ、窒素ガスを封入し低酸素濃度条件にした後、酵素精製作業を行うまで-80℃のフリーザーで保存した。各菌の菌体量は、20Lの培地あたり1~2g程度であった。
前培養された少量の菌体を透析チューブに封入した後、大量の培地が入った大型ボトルにて継続的に培養した。具体的には、以下の通りに行った。
1.2.1 破砕と上清画分の回収
凍結保存されていた約1gの菌体を流水で溶解後、50mLのバッファーA(pH7.5、20mMトリス塩酸緩衝液)に懸濁した。懸濁液を、超音波破砕器(UD-201, トミー社)にてOUTPUT 5.5、DUTY 50にて 15分間破砕した。破砕液を、40,000gで30分間の遠心分離を行った。HAOが含まれる上清画分を35mL回収した。
バッファーAで平衡化された5mLのHiTrap Q HP (GE healthcare社)を直列に3本つなげたカラムに上清画分を添加した。次に、バッファーAとバッファーB(pH7.5、20mMトリス塩酸緩衝液、1M塩化ナトリウム)を用いて、0~500mMの塩化ナトリウム濃度のリニアグラジェントにより溶出を行った。溶出液の分画は3mLずつ行った。HAOを含む画分を検出し、約10mLの溶液を回収した。HAOを含む画分の検出は、後述の1.2.7に記載の方法で行った。
回収した陰イオンカラム溶出画分を、バッファーB (pH7.0、20mMトリス塩酸緩衝液、150mM塩化ナトリウム)で平衡化されたゲルろ過カラムHiLoad 26/600 Superdex 200 prep grade (GE healthcare社)に添加した。次にバッファーBを1.5mL/分で送液し、溶出させた。溶出液の分画は3mLずつ行った。HAOを含む溶出画分を12mL回収した。
回収したゲルろ過カラム溶出画分を、5mLのCHT Ceramic Hydroxyapatite担体(Type I、粒子径20μm、BioRad社)が充填され、バッファーC(pH7.5、20mMリン酸カリウム緩衝液)で平衡化された内径1cmのカラムに添加した。次に、バッファーCとバッファーD (pH7.5、500mMリン酸カリウム緩衝液)を用いて、20~500mMのリン酸カリウム緩衝液のリニアグラジェントにより溶出を行った。溶出液の分画は2mLずつ行った。HAOを含む画分を12mL回収した。
ハイドロキシアパタイトカラムの溶出画分を、バッファーAで平衡化された強陰イオン交換カラムのMonoQ 10/10 GL(GE healthcare社)に添加した。溶出は、バッファーAとバッファーBを用い、0~500mMの塩化ナトリウム濃度のリニアグラジェントにて行った。溶出液の分画は2mLずつ行った。最終的に、HAOを含む画分を12mL回収した。なお、Nitrosospira multiformis由来HAOのみMonoQには吸着されなかったため、強陽イオン交換カラムのMonoS 10/100 GL (GE healthcare社)を利用した。
Nitrosomonas europaea及びNitrosococcus oceani以外の菌由来のHAOは、酸素に非常に弱く、2~3日かかる精製中にその大部分が失活してしまうため、低酸素濃度条件で精製する必要があった。Nitrosomonas europaea及びNitrosococcus oceaniも低酸素濃度条件で精製することで活性の保持が期待できるため、これらも同様に精製を行った。
HAOの精製には、クロマトグラフィーシステムのAKTA Explore (GE healthcare社)を用いた。HAOを含む画分の判別は、AKTAによる3波長の吸光測定{280nm (タンパク質)、409nm(ヘム)、463 nm (HAO特有のヘムP460)}、レサズリン法によるHAO蛍光活性測定(pH5.6のリン酸クエン酸緩衝液を使用)、SDS-PAGEによるバンド確認を併用して行った。
1.3.1 溶液による保存(短期保存)
酸素濃度0.01%以下のグローブボックス中で、O-リング付きの1.5mLスクリューキャップチューブ(深江化成社)に保存した。
5nMのHAO溶液(バッファーB)を384穴マイクロプレート(Greiner bio-one社)の各ウェルに0.1μLずつスポットし、室温で自然乾燥させた。1日程度常温で放置した後、後述の蛍光法で活性を測定したところ、乾燥前にくらべ80%程度の活性が残っていた。乾燥後は、プレートにアルミシートを張り、低温で保存すれば長期保存が可能であった。すべての操作は、酸素濃度が0.01%以下の低酸素濃度条件下のグローブボックス内で行った。
2.1 試薬調製法
測定のために長期保存可能な3種のストック溶液を調製した。
超純水を用い4mM 塩酸ヒドロキシルアミン溶液(東京化成工業社)を調製し、10mLずつ15mLサイズのプラスチックチューブに分注した。このとき、超純水は事前に十分脱気し、作業は低酸素濃度条件下のグローブボックス内(酸素濃度0.01%以下)で行った。調製済みの溶液は、-80℃で冷凍保存した。
前述のヒドロキシルアミンと同様に、400μMレサズリンナトリウム溶液(和光純薬工業社)を調製した。作業は低酸素濃度条件下のグローブボックス内(酸素濃度0.01%以下)で行った。-80℃で冷凍保存した。
測定用緩衝液(100mMリン酸カリウム緩衝液、300mM塩化ナトリウム)を調製した。作業は低酸素濃度条件下のグローブボックス内(酸素濃度0.01%以下)で行った。調製済みの溶液は、4℃で保存した。HAOインヒビターのスクリーニング用にはpH7.0の緩衝液を、HAO精製時のHAOを含む画分の確認用および土壌ダイレクトHAO活性測定用にはpH5.6の緩衝液を用意した。
384穴マイクロプレート(MICROPLATE, 384 WELL, NON-BINDING, F-BOTTOM, BLACK, Greiner Bio-One社)と、分注装置搭載型モノクロメーター式マイクロプレートリーダー(infinite M1000 PRO: テカン社)を利用して測定した。
液量は、1アッセイ100μLで384穴全面を使用した。プレート1枚分で、38.4mL分のアッセイ溶液が必要であった。まず、4倍希釈用のテトラゾリウム塩のストック溶液10mL、2倍希釈用の緩衝液のストック溶液20mL、5μMのHAO溶液400μLをゆっくり混和した。この時、HAO溶液だけではなく、プレート上で乾燥保存されているHAOも利用可能である。混合液は、16チャンネル電動ピペット(VIAFLO II 125 μL, INTEGRA社)を使用して、75μLずつ384穴マイクロプレートの各ウェルに分注した。
基質以外の試薬が分注された384穴マイクロプレートを、プレートリーダーに挿入した。まず、プレートリーダーに備え付けられたインジェクターによって、基質である4倍希釈用のヒドロキシルアミンストック溶液を全ウェルに25μLずつ添加し、反応系において、レサズリンに対するヒドロキルアミンを10倍量(モル比)とした。その後、ただちに蛍光測定を開始した(励起波長462nm:波長幅20nm、測定波長482nm:波長幅 20nm)。測定はカイネティックスモードで行い、全ウェルを1分毎に1回測定し、それを5時間続けた。測定は常温で行った。測定値は、2時間程度で最大蛍光強度に達した。活性の比較には、酵素活性の初速、または2時間未満の時点での活性値が利用可能であった。
3.1 HAOインヒビタースクリーニングへの適用
3.1.1 試薬条件の検討
レサズリン法によるHAO蛍光活性測定をHAOインヒビターのスクリーニングに用いることを検討した。具体的には、化合物の溶媒として多用されるジメチルスルホキシド(DMSO)濃度、化合物の非特異的阻害の原因となるケミカルアグリゲーション防止のための界面活性剤濃度を検討した。前述の活性測定法に沿い、ストック溶液の混合時に同時にこれらの添加物類も混合することにより検討した。その結果、一般的に使われる0.03%Triton-X 100(界面活性剤)では、特に蛍光測定の妨害効果は見られなかった。しかし、DMSOは、その濃度と比例して顕著な蛍光の低下が見られたが、0.5%程度のDMSO濃度であれば、十分な測定(DMSOなしの場合に比べて半分程度の活性値)が可能である事がわかった。
レサズリン法を用いたスクリーニング実験に供するHAOインヒビター候補化合物を絞り込むため、まずin silicoスクリーニングを行った。
スクリーニング用化合物ライブラリに対してレサズリン法によるHAO蛍光活性測定を行い、それら化合物の阻害効果を検証した。40種の化合物は、分子生物学用DMSO(和光純薬工業社)または、超純水に 100mMの濃度で溶解させ、さらに2倍希釈系列を16系列(100mM、50mM、25mM、12.5mM、6.25mM、3.13mM、1.56mM、0.78mM、0.39mM、0.19mM、97μM、48.8μM、24.4μM、12.2μM、6.1μM、3.1μM)作成した(終濃度はこれらの1/200の濃度となる)。ポジティブコントロールとして、既知のHAOインヒビターであるフェニルヒドラジンも同様な希釈系列を作成した。各濃度の化合物0.5μLを16チャンネル電動ピペット(VIAFLO II 12.5 μL, INTEGRA社)にて、384穴マイクロプレート(MICROPLATE, 384 WELL, NON-BINDING, F-BOTTOM, BLACK, Greiner Bio-One社)の空ウェルに添加した。緩衝液・レサズリン溶液・HAO溶液の各ストック溶液の混合液を400ウェル分準備し、384ウェルに対し1ウェルあたり75μLを16チャンネル電動ピペット(VIAFLO II 125 μL, INTEGRA社)で添加した(1ウェルあたりの組成は、2倍希釈用緩衝液を50μL、4倍希釈用400μMレサズリンを25μL、5nM HAOを0.5μLとなる)。プレートは、試薬インジェクター付蛍光プレートリーダー(infinite M1000 PRO, テカン社)に挿入し、4倍希釈用4mMヒドロキシルアミンストック溶液を1ウェルあたり25μL添加し、反応系において、レサズリンに対するヒドロキシルアミンを10倍量(モル比)とした。2時間のインキュベート後に蛍光測定(励起波長562nm:波長幅20nm、測定波長592nm:波長幅 20nm)を行った。蛍光強度が化合物無添加の場合の10%以下に低下した候補化合物Aをヒット化合物として見出した。
スクリーニングによって得られた候補化合物AのHAOに対する50%阻害濃度の決定をレサズリン法によるHAO蛍光測定によって行った。ポジティブコントロール用のインヒビターとして、Nitrosomonas europaea由来HAOの非可逆的インヒビターとして公知のフェニルヒドラジン(東京化成工業社)を用いた。
候補化合物Aの硝化抑制効果(アンモニア酸化細菌の硝化活性の阻害作用)の検証をアンモニア酸化細菌(Nitrosomonas europaea)の生菌を利用して行った。アンモニア酸化細菌として、各菌は大量培養後(pH6.0付近に達したら集菌)に同容量のフレッシュな培地に交換した。菌体を384穴Deep well plate (BIO-BIK 社)に100μLずつ分注した。各インヒビターの希釈系列溶液(DMSOに溶解)を0.5μL添加した(n=4または3)。2000rpmで1分間撹拌した。空気透過型プレートシール(細胞用培養プレートシール:AeraSeal社)を張ったのち、常温で12時間インキュベートした。インキュベート後、2000rpmで1分間撹拌した。各ウェル1μLの培地を、50μLのGreiss Reagent A液入りの384穴マイクロプレートに移した。各ウェルに50μLのGreiss Reagent B液を追加した。15分発色させた後、プレートリーダーで吸光(545nm)を測定。GraphPad Prism6ソフトウェアでマニュアルに従って50%阻害濃度(IC50)を計算した。候補化合物AのIC50は1.8μMであった。この実験は、Greiss Reagentの文献(Giustarini, D., Rossi, R., Milzani, A., & Dalle-Donne, I. (2008). Nitrite and nitrate measurement by Griess reagent in human plasma: evaluation of interferences and standardization. Methods in enzymology, 440, 361-80. doi:10.1016/S0076-6879(07)00823-3)を参照した。
A: 2 × Sulfanilamide ストック溶液
2% (w/w) sulfanilamide (和光純薬工業社)
12%(v/v) phosphoric acid (リン酸) 生化学用(和光純薬工業社)
B: 2 × NED ストック溶液
0.2% (w/w) N-(1-naphthyl)ethylenediamine(NED)(和光純薬工業社)
保存条件:どちらも遮光ビンに入れ4℃保存
土壌サンプルなどの微生物群集からダイレクトにHAO活性を測定する方法を開発した。この方法により、土壌のアンモニア酸化細菌の菌体量や硝化活性を推定することが可能となった。
レサズリンを含むHAO蛍光活性測定法の溶液を、非変性ポリアクリルアミドゲル電気泳動であるネイティブ-PAGEを行ったHAOを含む試料に適用することにより、ゲル内のHAO活性を可視化した。
ネイティブ-PAGEのサンプルには、上記項目1.2.1にて調製したN. europaeaの破砕上清を使用した。また、マーカーとして上記項目1.3.1にて保存したN. europaea由来の精製HAOを使用した。それぞれの溶液は、サンプルバッファー(0.4%ブロモフェノールブルー、50%グリセロール、pH7.5、5mMトリス塩酸塩)と1:1で混合して、ネイティブ-PAGEに供した。
ネイティブ-PAGE電気泳動にはe-PAGEL15%ポリアクリアルアミド既成ゲル(ATTO社)を用い、取り扱い説明書に従って行った。泳動層にゲルをセットした後、各ウェルに10μLのサンプルをそれぞれ2レーンずつアプライした。ゲル電気泳動は、ネイティブ-PAGE用泳動バッファー(25mMトリス塩酸、192mMグリシン)を用い、300V、20mAで80分間、室温(約25℃)で通電した。
電気泳動の後、ゲルをレサズリン含有緩衝液 30mL(上記項目2.1.2に記載の4倍希釈用レサズリン溶液10mLと上記項目2.1.3に記載の2倍希釈用測定用緩衝液(pH5.6) 20mLを混合した溶液)中で5分以上振盪反応させ、ゲル中にレサズリン含有緩衝液を浸透させた。
ゲルをレサズリン含有緩衝液からアクリルトレイ上に移し、余分な溶液をキムワイプで除去した。ゲルに上記項目2.1.1で調製した基質のヒドロキシルアミン溶液1mLをまんべんなくかけ、数秒後に余分な溶液をキムワイプで除去した。30秒ほど反応させた後、シアン色(波長480-530nm)のゲルイメージング光源Cyanoview(ATTO社)、およびオレンジフィルター(ATTO社)により、バンドの蛍光染色を確認し、デジタルカメラにて撮影した(図5)。その結果、N. europaeaの破砕上清のレーンで蛍光を発しているバンドが1つあることが確認された。このバンドの位置は、マーカーとして使用したN. europaea由来の精製HAOのバンドと同等な位置に存在しており、HAOのバンドであることが確認された。バンドの蛍光強度から、サンプル中のHAO活性を簡易的に定量することが可能であった。
Claims (18)
- テトラゾリウム塩の存在下でヒドロキシルアミン酸化還元酵素(HAO)をヒドロキシルアミンと接触させることを含む、HAOの活性測定方法。
- テトラゾリウム塩が、レサズリン塩である、請求項1記載の測定方法。
- テトラゾリウム塩に対するヒドロキシルアミンが、少なくとも3倍量のモル比で接触させる、請求項1または2記載の測定方法。
- テトラゾリウム塩に対するヒドロキシルアミンが、少なくとも5倍量のモル比で接触させる、請求項1~3のいずれか一項記載の測定方法。
- テトラゾリウム塩に対するヒドロキシルアミンが、少なくとも10倍量のモル比で接触させる、請求項1~4のいずれか一項記載の測定方法。
- pH4.0から8.0で測定する、請求項1~5のいずれか一項記載の測定方法。
- pH4.6から7.6で測定する、請求項1~6のいずれか一項記載の測定方法。
- 測定対象が、土壌、堆肥、活性汚泥又はそれらを非変性状態で電気泳動したポリアクリルアミドゲルである、請求項1~7のいずれか一項記載の測定方法。
- HAOを含む試料からHAOを精製することを含み、精製においてHAOが含まれる画分を判別するために請求項1~8のいずれか一項記載の測定方法によりHAOの活性を測定する、HAOの製造方法。
- 精製されたHAOを乾燥することを含む、請求項9記載の製造方法。
- テトラゾリウム塩の存在下でHAOをヒドロキシルアミン及び候補化合物と接触させることを含む、HAOインヒビターのスクリーニング方法。
- 液体培地格納容器内に隔離して設置された微生物を含む試料格納容器内で、微生物を培養することを含み、微生物を含む試料格納容器は、細孔を有する、微生物の培養方法。
- 液体培地と微生物を含む試料の体積比が、10:1~1000:1である、請求項12記載の培養方法。
- 細孔の分画分子量が、50K~1000KDaである、請求項12または13記載の培養方法。
- 微生物を含む試料格納容器が、透析チューブである、請求項12~14のいずれか一項記載の培養方法。
- 微生物を含む試料格納容器の封入口が、殺菌されている、請求項12~15のいずれか一項記載の培養方法。
- 微生物が、難培養性微生物である、請求項12~16のいずれか一項記載の培養方法。
- 微生物が、OD600が0.2以下の濁度で静止期に達する微生物である、請求項1~17のいずれか一項記載の培養方法。
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Title |
---|
EWALD M. ET AL.: "Kurzzeittest fuer die Bestimmung der Dehydrogenasenaktivitaet von Belebtschlaemmen", VOM WASSER, vol. 68, 1987, pages 165 - 175, XP009512935, ISSN: 0083-6915 * |
GIUSTARINI, D.; ROSSI, R.; MILZANI, A.; DALLE-DONNE, I.: "Nitrite and nitrate measurement by Griess reagent in human plasma: evaluation of interferences and standardization", METHODS IN ENZYMOLOGY, vol. 440, 2008, pages 361 - 80 |
H. MAEDA; S. MATSU-URA; Y. YAMAUCHI; H. OHMORI: "Resazurin as an electron acceptor in glucose oxidase-catalyzed oxidation of glucose", CHEM. PHARM. BULL., vol. 49, 2001, pages 622 - 625 |
J. SCHALK; S. DE VRIES; J.G. KUENEN; M.S. JETTEN: "Involvement of a novel hydroxylamine oxidoreductase in anaerobic ammonium oxidation", BIOCHEMISTRY, vol. 39, 2000, pages 5405 - 12 |
JOURNAL OF ENVIRONMENTAL BIOTECHNOLOGY, vol. 7, no. 2, 2007, pages 69 - 73 |
NEJIDAT A. ET AL.: "Effect of Ammonia Starvation on Hydroxylamine Oxidoreductase Activity of Nitrosomonas europaea", JOURNAL OF BIOCHEMISTRY, vol. 121, 1997, pages 957 - 960, XP055508006 * |
SCHALK J. ET AL.: "Involvement of a Novel Hydroxylamine Oxidoreductase in Anaerobic Ammonium Oxidation", BIOCHEMISTRY, vol. 39, 2000, pages 5405 - 5412, XP055508001 * |
SHIMAMURA M. ET AL.: "Another Multiheme Protein, Hydroxylamine Oxidoreductase, Abundantly Produced in an Anammox Bacterium Besides the Hydrazine-Oxidizing Enzyme", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 105, no. 3, 2008, pages 243 - 248, XP022590040 * |
W.J. MAALCKE; A. DIETL; S.J. MARRITT; J.N. BUTT; M.S.M. JETTEN; J.T. KELTJENS; T.R.M. BARENDS; B. KARTAL: "Structural basis of biological NO generation by octaheme oxidoreductases", J. BIOL. CHEM., vol. 289, 2014, pages 1228 - 42 |
YUKI NISHIGAYA ET AL.: "4A07a09 Kozo Seibutsugakuteki Approach ni yoru Hydroxylamine Sanka Kangen Koso o Hyoteki to shita Shoka Yokuseizai no Kaihatsu = Structure based approach for nitrification inhibitor design targeting for hydroxylamine oxidoreductase", PROCEEDINGS OF THE ANNUAL MEETING OF JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, 2014, XP009512443, ISSN: 2186-7976 * |
YUKI NISHIGAYA ET AL.: "Kozo Kaiseki to Shinki Sogaizai o Riyo shita Ammonia Sanka Saikin Yurai no Hydroxylamine Sanka Kangen Koso ni Taisuru Shoka Yokuseizai Target to shiteno Validation", 87TH ANNUAL MEETING OF THE JAPANESE BIOCHEMICAL SOCIETY [DAI 87 KAI NIHON SEIKA GAKKAI TAIKAI PUROGURAMUGŌ], vol. 87th, 2014, pages 2P-097, XP009512873 * |
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US20190024138A1 (en) | 2019-01-24 |
CN108474020A (zh) | 2018-08-31 |
EP3406732A4 (en) | 2019-10-30 |
CN108474020B (zh) | 2022-08-02 |
JP6270237B2 (ja) | 2018-01-31 |
US10907191B2 (en) | 2021-02-02 |
EP3406732B1 (en) | 2020-11-04 |
EP3406732A1 (en) | 2018-11-28 |
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