JP2011063571A - Method for forming urease inhibiting active substance and urease inhibiting active substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce a urease inhibiting active substance from a konjac fly powder being industrial waste produced from konjac roots. <P>SOLUTION: A konjac fly powder produced in milling konjac roots and usually disposed of as a waste product is immersed in a methanol solution, the resultant methanol solution is subjected to vacuum filtration to obtain a methanol extract. Next, the methanol extract is concentrated in vacuo, and water is added to the concentrate. The resultant methanol solution is extracted with ethyl acetate to obtain an ethyl acetate extract, and the ethyl acetate extract is fractionated by column chromatography to obtain a urease inhibiting active substance. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing a urease inhibitory active substance and a urease inhibitory active substance produced by the method.

Urease is an enzyme that breaks down urea into ammonia and carbon dioxide, and is widely distributed in various living organisms such as bacteria, fungi, and plants. This enzyme was first crystallized from jujube by Sumner in 1926, and in 1975, it was also revealed that it had a structure containing nickel in the active site. However, despite the fact that this enzyme has a long history and many studies have been made, there are many unclear aspects of urease catalytic activity, and it is an enzyme for which further research is expected.
It is presumed that plants use urea to obtain ammonia, which is a nitrogen source necessary for growth, from the growth environment using urea, and are also involved in the conversion of nitrogen form in the plant body.

On the other hand, problems may be caused by ammonia generated by urease. Helicobacter pylori and Proteus mirabilis urease from the family Enterobacteriaceae are also involved in human diseases.
The former H. pylori is closely related to chronic gastritis and peptic ulcer and is characterized by being able to grow in a strongly acidic environment (pH 1-2) in the stomach where normal bacteria cannot grow. This is because urea in the cell surface can convert urea in the stomach into ammonia, neutralize gastric acid in the vicinity of the cell, and make the pH environment suitable for growth.
The latter Proteus mirabilis is a bacterium found especially in patients with urinary tract infections. The urease possessed by this bacterium is involved in the formation of deposits that cover the urinary catheter used in the treatment. The formation process is caused by a pH change caused by ammonia produced by urease, similar to H. pylori. In the body of urinary tract infection patients, Proteus mirabilis adheres to a catheter placed for treatment and forms a biofilm. The action of urease derived from fungi generates ammonia from urea in the body, and a rapid pH increase of urine and biofilm causes precipitation of calcium and magnesium phosphate crystals in the biofilm. Crystal deposition in the catheter can lead to blockage of the tube and can cause additional illnesses such as pyelonephritis.

In agriculture, urease that converts nitrogen fertilizer to ammonia, which is a nitrogen source that can be used by plants, is important, but if hydrolysis occurs too quickly and excess ammonia is generated, soil alkalinization can occur. There are problems such as causing it and causing damage to plants by ammonia.
Those already known as urease inhibitors include compounds such as fluorofamide, hydroxyurea, hydroxamic acid, and acetohydroxamic acid. However, some of them exhibit biotoxicity and instability. For example, acetohydroxamic acid has not been put to practical use because it exhibits teratogenicity to rats.

From the above, urease inhibitory active substances are not only important for deepening knowledge of enzymes, but can be used for a wide range of applications such as pharmaceutical use or agricultural use.
The present inventor has been searching for urease inhibitory active substances by enzyme inhibition tests using urease derived from peanuts for spices, fungi and methanol extracts of edible plants. Compounds that have been isolated so far include sulfoquinovosyl diacylglycerol, 5-geranyloxy-7-methoxycoumarin, 5-geranyloxypsolaren and the like.

  On the other hand, in Japanese Patent Application Laid-Open No. 2005-206493 (Patent Document 1), “Anti-Helicobacter pylori agent, Helicobacter pylori bacterium comprising an extract extracted from moss with an organic solvent and / or supercritical carbon dioxide as an active ingredient. Prophylactic and therapeutic agents for gastric or duodenal ulcers caused by the above, and preventive foods and beverages have been proposed.

JP 2005-206493 A

  In the above-mentioned Patent Document 1, cocoon is mentioned as an example of preferable moss, and ethyl acetate is described as an organic solvent used as an extraction solvent. Specific examples of isolation and purification of an active substance in an extract are described. Such a method is not disclosed.

  An object of the present invention is to provide a purification method and the like for isolating and purifying a urease inhibitory active substance from sputum.

In order to solve the above-mentioned problems, as a first invention, the soot flying powder generated during the milling of the straw and used as a waste is immersed in a methanol solution, filtered under reduced pressure to obtain a methanol extract,
The methanol extract is concentrated under reduced pressure and then hydrated, and the methanol solution is extracted with ethyl acetate to obtain an ethyl acetate extract.
There is provided a method for producing a urease inhibitory active substance, wherein the ethyl acetate extract is fractionated by column chromatography to obtain a urease inhibitory active substance.

The kite flying powder is a by-product when the kite is produced from the kite. Although the kite fly powder accounts for about 40% of the koji, it has a peculiar taste and smell and is not suitable for food use and is currently treated as industrial waste.
In the present invention, as a result of screening, the flying powder that has been found to be active is effectively used to isolate and purify a urease inhibitory active substance from the methanol extract of flying powder and to determine the structure by NMR.

  In the step of obtaining the ethyl acetate extract, the methanol solution is preferably extracted multiple times with an equal amount of ethyl acetate using a separatory funnel.

In the fractionation step in the column chromatography, half of the ethyl acetate extract was adsorbed on Wakogel C-200, then suspended in n-hexane and loaded on the top of the column. Elution is performed by a stepwise method using a mixed solvent of hexane-ethyl acetate-methanol, and is obtained as a fraction eluted with 20-30% ethyl acetate.
Next, the fractionation step in column chromatography was carried out by adsorbing half of the ethyl acetate extract to Wakogel C-200 and then suspending it in n-hexane and packing it at the top of the column. It is eluted by a stepwise method using an n-hexane-acetone mixed solvent, and the eluted 6-15% acetone elution fraction is obtained as the urease inhibitory active substance.

  Further, as the second invention, the carbon chain length of the urease inhibitory active substance obtained by the production method of the first invention is measured, and the urease inhibitory activity that increases as the carbon chain length increases is measured. A method for measuring urease inhibitory activity is provided.

  Furthermore, as a third invention, a urease inhibitory active substance obtained by the production method of the first invention is provided.

   The urease inhibitory active substance contains a saturated or unsaturated fatty acid having 18 to 22 carbon atoms. The unsaturated fatty acid is preferably docosenoic acid.

  Furthermore, this invention provides the fertilizer and soil improvement agent containing the said urease inhibitory active substance. For example, the soil conditioner contains 5 to 15% by weight of urease inhibitory active substance and 0.5 to 2% nitrogen.

Furthermore, the present invention provides a urinary catheter containing the urease inhibitory active substance.
For example, a urease inhibitory active substance is added to a hexane solution of a fatty acid, and the solution is applied to the surface of a catheter, or kneaded into a balloon catheter forming material and a resin.

  In the present invention, it is possible to isolate and purify a urease inhibitory active substance by utilizing the fly powder that is an industrial waste generated when processing a koji product by milling koji, and the urease inhibitory activity Substances can be widely used as fertilizers, land improvers, urinary catheters and the like.

It is process drawing which shows the production | generation method of the urease inhibitory active substance of this invention. (A) (B) is a graph which shows the NMR spectrum of a urease inhibitory active substance. (A)-(D) are graphs showing gas chromatographic analysis of urease inhibitory active substances. It is a graph which shows the inhibition rate of a linoleic acid. (A)-(F) are graphs which show inhibitory activity from the relationship between the density | concentration of fatty acids and the inhibition rate. It is a graph of a line Weber-Burk plot.

[Isolation and purification of urease inhibitory active substance]
In the step shown in FIG. 1, a urease inhibitory active substance is purified from the fly powder of konjac.
Specifically, 10 kg of flying powder is soaked in methanol, concentrated and then separated with ethyl acetate, and the resulting ethyl acetate extract is purified by Wakogel C-200 column chromatography in various mixed solvent systems. Obtained 180 mg of urease inhibitory active substance.
Below, each process is explained in full detail.

(First step # 1 methanol extraction step)
Spear flying powder (10 kg) generated during the milling of the koji and regarded as waste was immersed in a methanol solution (15 L) for 7 days and filtered under reduced pressure to obtain a methanol extract (10 L).

(Distribution extraction process of 2nd process # 2)
About 10 L of the obtained methanol extract was concentrated under reduced pressure to about 100 ml, 900 ml of water was added, and this was extracted three times with an equal amount of ethyl acetate using a separatory funnel.
The inhibition rate of the ethyl acetate phase was 32% at 1000 ppm (final).

[Separation step of inhibitory active substance contained in ethyl acetate extract]
(Concentration step of the third step # 3)
The extracted ethyl acetate was concentrated under reduced pressure to obtain an ethyl acetate extract (15.6 g).

(Wakogel C-200 column chromatography step in the fourth step # 4)
After 7.8 g of 15.6 g of the ethyl acetate extract obtained in the third step was adsorbed to Wakogel C-200 (15.6 g), it was suspended and filled in advance with n-hexane. Place on top of column (78 g), n-hexane-ethyl acetate-methanol mixed solvent system (100% n-hexane, 20%, 30%, 40%, 0%, 100% ethyl acetate, 90%, 80%, 50% methanol).
A urease inhibitory activity test was performed, and the fraction containing the active substance was confirmed. As a result, activity was observed in the fraction eluted with 20% to 30% ethyl acetate.

(Wakogel C-200 column chromatography step in the fifth step # 5)
4 g of the 20% -30% ethyl acetate elution fraction obtained in the fourth step was adsorbed onto Wakogel C-200 (6 g) in the same manner as in the previous step, and then suspended and filled in advance with n-hexane. The column was placed on the top of a column (40 g) and eluted by a 3% stepwise method using an n-hexane-acetone mixed solvent system. When the urease inhibitory activity test was conducted and the fraction containing the active substance was confirmed, strong inhibition was observed in the 6-15% acetone-eluted fraction.

(Wakogel C-200 column chromatography in the sixth step # 6)
The 6% acetone-eluted fraction with the largest amount among the fractions obtained in the fifth step was adsorbed on Wakogel C-200 (4.5 g) as in the previous step, and then suspended with n-hexane in advance. It was placed on the top of a turbid and packed column (30 g) and eluted by a 1% stepwise method using an n-hexane-acetone mixed solvent system. A urease inhibitory activity test was performed, and the fraction containing the active substance was confirmed. As a result, 180 mg of active substance was obtained in the 7% acetone-eluted fraction.

  As described above, when the methanol extract obtained from 10 kg of flying powder was subjected to solvent fractionation, activity was observed in the ethyl acetate phase. The ethyl acetate phase was concentrated under reduced pressure and then purified by Wakogel C-200 column chromatography. As a result, 180 mg of urease inhibitory active substance was obtained. For purification, enzyme inhibition test using juvenile bean-derived urease, ultraviolet absorption in TLC, and coloration with sulfuric acid reagent were used as indicators.

[Structural analysis and identification of compounds for urease inhibitory active substances]
The structure analysis and structure determination of the urease inhibitory active substance obtained from the flying powder in the above step will be described below.

  The following NMR spectrum analysis and gas chromatography analysis method were employed as experimental methods for structural analysis and structure determination.

(NMR spectrum analysis)
The urease inhibitory active substance was dried under reduced pressure with a vacuum pump to remove the solvent, and dissolved in CDCl ₃ as a solvent for NMR measurement. A ¹ H-NMR spectrum and a ¹³ C-NMR spectrum were measured using JNM-AL400 manufactured by JEOL. The chemical shift value is indicated by δ with the signal of the NMR measurement solvent as an internal standard.

(Gas chromatography analysis)
The NMR spectrum strongly suggested that the active substance was linoleic acid.
Therefore, gas chromatographic analysis was performed, and the active substance was identified by comparing with a sample of fatty acid.
About 10 mg of the active substance was dissolved in 2 ml of MeOH, and 0.5 ml of a methylating agent Trimethyl silyl diazomethane 2.0 M Solution in hexane was added.
After stirring for 5 minutes while stirring with a stir bar, it was confirmed by ¹ H-NMR that the reaction had progressed.
6 mg of fatty acid methyl ester of the active substance was obtained. The fatty acid methyl ester thus obtained was subjected to gas chromatography [column: TC-FFAP (φ0.25 × 60 mm), carrier gas N ₂ flow rate: 1.18 ml / min, column temperature: temperature increase 120 → 240 ° C. (10 ° C. / Min), detector: FID].

From the above NMR spectrum analysis, as shown in FIG. 2 (A), there are two double bonds from the signal of δ _c 180.4 in the ¹³ C-NMR spectrum and from the signal of δ _c 128.7 to 130.2. I found out that
Also, as shown in FIG. 2 (B), from the signal of δ _H 0.87 of the ¹ H-NMR spectrum, the methyl proton at the end of the alkyl chain is 3H, from the overlapped signal near δ _H 1.30, the methylene proton is 14H, From the signal of δ _H 1.60, the methylene proton at the β position of the carboxyl group is 2H, from the signal of δ _H 2.03 the methylene proton at the allylic position is 4 _H , and from the signal of δ _H 2.33, the α position of the carboxyl group From the signal of methylene proton at 2H, δ _H 2.75, the methylene proton sandwiched between double bonds was from 2H, and from the signal of δ _H 5.34, it was found that an olefinic proton was present.
From the above spectrum, it was suggested that the active substance was linoleic acid. This possibility was strongly suggested by NMR comparison of the sample linoleic acid.

After methylating the isolated urease inhibitory active substance, as shown in FIGS. 3 (A) to (D), it was subjected to gas chromatography analysis, and the retention times of the fatty acid preparations were compared.
Since the retention time 12.87 min of the linoleic acid standard coincided with the retention time 12.90 min of the main component of the active substance, this active substance was identified as linoleic acid.
For the other mixtures, the peak with a retention time of 12.35 min was identified as oleic acid and the peak with a retention time of 13.56 min was identified as linolenic acid. A substance showing a peak with a retention time of 10.27 min could not be identified. However, since the activities of linoleic acid and the active substance were similar, it was confirmed that the center of activity was linoleic acid.

The NMR spectrum of the isolated urease inhibitory active substance is shown below.
¹ H-NMR (400 MHz, CDCl ₃ ) δ 0.87 (3H, t), 1.30 (14H, m), 1.61 (2H, quint), 2.03 (4H, q), 2.33 ( 2H, t), 2.75 (2H, m), 5.34 (4H, m).
¹³ C-NMR (100 MHz, CDCl ₃ ) δ 14.0, 22.6, 24.6, 25.6, 27.2, 27.2, 29.0, 29.0, 29.1, 29.3, 29.6, 31.5, 34.0, 127.8, 128.0, 130.0, 130.2, 180.0.
The NMR spectrum of the sample linoleic acid is shown below.
¹ H-NMR (400 MHz, CDCl ₃ ) δ 0.87 (3H, t), 1.30 (14H, m), 1.60 (2H, quint), 2.03 (4H, q), 2.33 ( 2H, t), 2.75 (2H, t), 5.34 (4H, m).
¹³ C-NMR (100 MHz, CDCl ₃ ) δ 14.1, 22.6, 24.6, 25.6, 27.2, 27.2, 29.0, 29.1, 29.1, 29.3, 29.6, 31.5, 34.1, 127.9, 128.1, 130.0, 130.2, 180.4.

[Method for measuring urease inhibitory activity of active substance]
Next, a method for measuring urease inhibitory activity of an active substance isolated and purified from the flying powder will be described. Specifically, the intensity of activity of the isolated linoleic acid is measured.

Prepare the following reagents:
(Preparation of buffer)
5.331 g of MES was dissolved in 250 ml of milli-Q water, and adjusted to pH 6.0 by adding 0.1 M NaOH.
(Enzyme preparation)
Jack bean derived urease (manufactured by Toyobo) was used. To a solution of 0.136 g of 20 mM KH ₂ PO _{4 in} 50 ml of milli-Q water, 0.02 M NaOH was added to adjust the pH to 7.0.
The following operation was performed while cooling with ice. 1 ml of 0.5 M EDTA was added to it, and it was made up to 100 ml with Milli-Q water (A).
Add 960 μl of (A) to 40 μl of 5% BSA (B).
(A) 49.5 ml of (B) added with 500 μl of urease 7.5 mg dissolved in eppen tubes in 700 μl aliquots, stored in a refrigerator, and diluted 10 times immediately before use This was used as an enzyme solution.
(Preparation of coloring reagent)
Color solution A was prepared by dissolving 5 g of phenol and 25 mg of sodium nitroprusside in Milli-Q water to make 500 ml. A color developing solution B was prepared by dissolving 5 g of NaOH in 500 ml of milli-Q water, and adding 0.3 ml of a sodium hypochlorite solution to 19.7 ml of this solution immediately before use.
(Adjustment of urea)
12.0 mg of urea was dissolved in 100 ml of milli-Q water, and then diluted 10 times to obtain a 0.2 mM solution.

(experimental method)
The following reagents were used for the enzyme reaction.
30 mM (final) MES buffer (pH 6.0) was used as a buffer solution.
0.20 mM urea was used as the substrate. 0.2 ml of enzyme was added to 1.2 ml of buffer solution plus methanol (control) or 0.1 ml of sample, and pre-incubated at 37 ° C. for 5 minutes.
Next, 0.5 ml of the substrate was added to start the reaction, and after incubation at 37 ° C. for 20 minutes, 1 ml of each of the color developing solutions A and B was added to the reaction solution, followed by further incubation at 37 ° C. for 30 minutes.
Then, the amount of generated ammonia was measured by the absorbance at 640 nm.
That is, an inhibitor was added to this system, and the inhibitory activity was determined by comparison with an unadded control. The following formula was used for calculation of the inhibition rate.
The absorbance was measured using a Shimadzu uv mini-1240 uv-vis spectrophotometer.
Inhibition rate (%) = {(Abs. ₆₄₀ control-Abs. ₆₄₀ sample) / Abs. ₆₄₀ control} × 100

The isolated linoleic acid was tested for urease inhibitory activity.
The result is shown in FIG. The IC ₅₀ of linoleic acid was 85.0 μM. In contrast, the IC _{50 of} acetohydroxamic acid known as a urease inhibitor was 20.0 μM. Therefore, the activity of the linoleic acid isolated this time was not strong.

[Structure-activity relationship of fatty acids to urease inhibitory activity]
Since the isolated active substance was linoleic acid, the structure-activity relationship of fatty acids was examined.
Specifically, the carbon chain length of fatty acids having different structures is measured, and the urease inhibitory activity that increases as the carbon chain length increases is measured.

(experimental method)
In order to investigate the correlation due to the length of the carbon chain, IC50s of hexadecenoic acid (16: 1) oleic acid (18: 1), eicosenoic acid (20: 1), and docosenoic acid (22: 1) were determined.
In order to investigate the correlation depending on the number of double bonds, IC ₅₀ of oleic acid (18: 1), linoleic acid (18: 2), and linolenic acid (18: 3) was determined. Further, in order to investigate whether fatty acid methyl ester is also active, IC ₅₀ of linoleic acid methyl ester was determined.

The results of the experiment are shown in FIGS.
A comparison shows that the activity increases as the carbon chain lengthens. On the other hand, there was no significant difference in activity due to the difference in the number of double bonds.
In addition, linoleic acid methyl ester showed the same activity as linoleic acid. These results suggested that the urease inhibitory activity of fatty acids was influenced by the length of the carbon chain.
The longer the carbon chain, the stronger the activity of the fatty acid. In particular, docosenoic acid shown in FIG. 5 (D) has an IC ₅₀ of 14 μM and is more active than acetohydroxamic acid. It is considered that urease inhibition of fatty acids is influenced by hydrophobic interaction as a cause of the stronger activity as the carbon chain becomes longer.
From the above results, it was suggested that the urease inhibitory active substance contains a saturated or unsaturated fatty acid having 18 to 22 carbon atoms, and that the unsaturated fatty acid is preferably docosenoic acid.

[Analysis of inhibition mode of linoleic acid on urease]
(experimental method)
Reagents used in the method for measuring urease inhibitory activity of the active substance were prepared.
The following reagents were used for the enzyme reaction.
30 mM (final) MES buffer (pH 6.0) was used as a buffer solution. 0.20 mM urea was used as the substrate. 0.2 ml of enzyme was added to 1.2 ml of buffer solution and 0.1 ml of methanol (control), and preincubated at 37 ° C. for 5 minutes.
Next, 0.5 ml of the substrate is added to start the reaction. After incubation at 37 ° C. for 0, 5, 15, and 20 minutes, 1 ml each of the coloring solutions A and B is added to the reaction solution, and further incubated at 37 ° C. for 30 minutes. did.
Then, the amount of generated ammonia was measured by the absorbance at 640 nm.
For the measurement of the amount of ammonia, a calibration curve of the amount of ammonia and absorbance Abs 640 nm was prepared in advance, and the amount of ammonia generated was determined from the absorbance. The initial rate was determined from the enzyme reaction time and the amount of ammonia generated.
Similarly, initial velocities were obtained when the concentration of urea as a substrate was 0.1, 0.125, 0.143, 0.25, and 0.5 mM. Then, a line Weber-Burk plot was created by plotting the substrate concentration in the horizontal axis and the initial velocity in the vertical axis.
Next, linoleic acid having a final concentration of 70 and 140 μM was added to this system instead of methanol, and a line Weber-Burk plot was similarly prepared.

FIG. 6 shows a line weaver-bark plot created based on the experimental results.
As shown in FIG. 6, it was found that Km did not change and Vmax decreased, indicating that inhibition of linoleic acid on urease was non-competitive inhibition.
Non-competitive inhibitors bind to both the enzyme and the enzyme substrate complex. This type of inhibitor is not a substrate analog and does not bind to the same site as the substrate.
Examples of such non-competitive inhibition are known among allosteric enzymes. Such non-competitive inhibitors are thought to change the conformation of the enzyme, allowing the substrate to bind, but not to catalyze the reaction.
Fatty acids such as linoleic acid that have urease inhibitory activity are not similar in structure to urea, which is a substrate. From this, it can be expected that fatty acids show non-competitive inhibition rather than competitive with urease.

  The urease inhibitory active substance produced from the above-described kite flying powder of the present invention is suitably used as a fertilizer and a soil conditioner. For example, the soil conditioner contains 5 to 15% by weight of urease inhibitory active substance and 0.5 to 2% nitrogen.

In addition, Proteus mirabilis, which causes urinary tract infections in humans, raises the pH with ammonia generated by urease, causing problems such as attachment of catheters used to treat urinary tract infections and tube embolization. Is caused. This is because when calcium is increased by Proteus mirabilis infected near the catheter, crystallization of calcium and magnesium salts present in the vicinity occurs.
Therefore, the urease inhibitory active substance obtained from the flying powder is added to the hexane solution of fatty acid, and the solution is applied to the surface of the catheter, or kneaded into the balloon catheter forming material and resin, as a component of the catheter. It can be used suitably.

Claims (10)

Soaked powder, which is generated during the milling of koji and made into waste, is immersed in a methanol solution and filtered under reduced pressure to obtain a methanol extract.
The methanol extract is concentrated under reduced pressure and then hydrated, and the methanol solution is extracted with ethyl acetate to obtain an ethyl acetate extract.
A method for producing a urease inhibitory active substance, wherein the ethyl acetate extract is fractionated by column chromatography to obtain a urease inhibitory active substance.   The method for producing a urease inhibitory active substance according to claim 1, wherein in the step of obtaining the ethyl acetate extract, the methanol solution is extracted a plurality of times with an equal amount of ethyl acetate using a separatory funnel. In the fractionation step in the column chromatography, half of the ethyl acetate extract was adsorbed on Wakogel C-200, then suspended in n-hexane and loaded on the top of the column. Elution is performed by a stepwise method using a mixed solvent of hexane-ethyl acetate-methanol, and is obtained as a fraction eluted with 20-30% ethyl acetate.
Next, the fractionation step in column chromatography was carried out by adsorbing half of the ethyl acetate extract to Wako Gel C-200, and then suspending it in n-hexane and placing it on the top of the column. The urease inhibitory active substance according to claim 1 or 2, wherein the urease inhibitory active substance is eluted as a urease inhibitory active substance by elution by a stepwise method using a mixed solvent of -hexane-acetone and eluting 6% to 15% acetone. Generation method.   A carbon chain length of a urease inhibitory active substance obtained by the production method according to any one of claims 1 to 3 is measured, and a urease inhibitory activity that increases as the carbon chain length increases is measured. For measuring urease inhibitory activity.   A urease inhibitory active substance obtained by the production method according to any one of claims 1 to 3.   The urease inhibitory active substance according to claim 5, comprising a saturated or unsaturated fatty acid having 18 to 22 carbon atoms.   The urease inhibitory active substance according to claim 6, wherein the fatty acid is docosenoic acid.   A fertilizer comprising the urease inhibitory active substance according to any one of claims 5 to 7.   A soil improver comprising the urease inhibitory active substance according to any one of claims 5 to 7.   A urinary catheter containing the urease inhibitory active substance according to any one of claims 5 to 7.

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