Urease inhibitor and fertilizer thereof

Abstract

The invention relates to application of N-methylglycine and salts thereof as urease inhibitors in nitrogen fertilizer synergism. The N-methylglycine and the salts thereof have an efficient inhibition effect on urease, and can be used as a novel nitrogen fertilizer synergist to ensure that the yield of crops is not reduced and reduce the use amount of nitrogen fertilizer at the same time.

Description

Technical Field

[0001] This invention relates to the inhibitory activity of N-methylglycine and its salts on urease and their application as nitrogen fertilizer synergists in reducing nitrogen fertilizer application in crop cultivation. Background Technology

[0002] Nitrogen (N) is one of the essential macronutrients for plant growth and development, playing a crucial role in crop yield and quality. However, the utilization rate of nitrogen fertilizer in my country is currently low (Zhou Xuan, Wu Lianghuan, Dai Feng. Effects of biochemical inhibitor combination on urea nitrogen transformation in yellow mud soil. Journal of Soil and Water Conservation, 2015, 29(5): 95-100), resulting in excessive use of nitrogen fertilizer, relatively reduced agricultural economic benefits, and various ecological and environmental problems such as soil salinization, groundwater pollution, and increased greenhouse gas emissions.

[0003] Urease in soil is a nickel-containing hydrolytic enzyme that catalyzes the decomposition of urea into ammonia and carbon dioxide. Inhibition and regulation of urease activity is one of the most effective biochemical methods to improve urea utilization. Liu Zhaohui et al. demonstrated (Liu Zhaohui, Wu Xiaobin, Tan Deshui, et al. Application and environmental effects of single-application fertilization in major grain crops in my country. Chinese Agricultural Science, 2018, 51(20): 10) that adding urease inhibitors can increase the yield of the three major grain crops of corn, wheat and rice by 5.8% to 22.8%, while significantly reducing ammonia volatilization and nitrous oxide emissions.

[0004] There are over 100 patented urease inhibitors in the global fertilizer market, mainly categorized into three types: metal salts, small organic molecules, and plant extracts. Currently, only urease inhibitors such as n-butyl thiophosphate triamine (NBPT), n-propyl thiophosphate triamine (NPPT), and hydroquinone (HQ) are in practical application. Existing urease inhibitors still suffer from numerous problems in fertilizer use, including low biological activity, low cost-effectiveness, and biosafety concerns.

[0005] N-Methylglycine, also known as sarcosine, is a white crystalline solid in its pure form, soluble in water and slightly soluble in alcohol. As a natural amino acid, N-methylglycine has the advantages of being safe, green, non-toxic, and pollution-free, and it is already being synthesized and produced on a large scale.

[0006] This study is the first to discover that N-methylglycine and its salts have significant urease inhibitory activity, and have advantages such as high cost-effectiveness and good biosafety. They can be used as a new type of nitrogen fertilizer synergist to reduce the amount of nitrogen fertilizer used while ensuring that crop yields are not reduced. Summary of the Invention

[0007] The purpose of this invention is to provide a highly active urease inhibitor, N-methylglycine, and its salts, which can be used as a nitrogen fertilizer synergist in crop cultivation for the purpose of reducing nitrogen fertilizer application.

[0008] Based on this, the present invention provides a urease inhibitor, wherein the active ingredient of the urease inhibitor is N-methylglycine.

[0009] The urease inhibitor comprises a pharmaceutically acceptable or agriculturally acceptable salt of N-methylglycine, the salt being selected from one or more of the following: hydrochloride, phosphate, citrate, malate, salicylate, fumarate, sulfonate, methanesulfonate, potassium salt, sodium salt, magnesium salt, zinc salt, or calcium salt.

[0010] The present invention also provides the application of the urease inhibitor in inhibiting urease activity.

[0011] The application involves adding the urease inhibitor to urea-containing fertilizers to inhibit the catalytic hydrolysis of urea by urease in the soil.

[0012] A nitrogen fertilizer enhancer comprising the aforementioned urease inhibitor.

[0013] The synergist is used in combination with nitrogen-containing fertilizers, which contain urea.

[0014] A fertilizer composition comprising a nitrogen-containing fertilizer and the urease inhibitor mentioned above.

[0015] The nitrogen-containing fertilizer is urea or a compound fertilizer containing urea. Based on the mass of the urea, the mass percentage of the urease inhibitor is 0.01% to 10%. In a preferred embodiment, the mass percentage of the urease inhibitor is 0.1% to 5%.

[0016] A method for improving nitrogen fertilizer utilization or reducing nitrogen fertilizer application involves simultaneously applying the urease inhibitor or the nitrogen fertilizer synergist when applying nitrogen-containing fertilizer to the soil or crops.

[0017] The nitrogen-containing fertilizer is urea or a compound fertilizer containing urea, and the application amount of the urease inhibitor is 0.01% to 10% based on the mass of the urea.

[0018] The present invention also provides a use of a urease inhibitor, wherein the urease inhibitor is used to prepare a nitrogen fertilizer synergist.

[0019] The present invention also provides a use of a urease inhibitor, wherein the urease inhibitor is used to prepare agricultural formulations that reduce ammonia volatilization and / or nitrous oxide emissions.

[0020] To implement the technical solution of this invention, the present invention provides the following technical features:

[0021] 1. Urease inhibitory activity assay The basic principle of urease inhibitor activity testing is to determine the level of urease inhibitor activity by detecting the change in the ability of urease to catalyze the decomposition of urea and release ammonia after the urease inhibitor reacts with urease. The most commonly used method is the indophenol method. First, the inhibitor reacts with urease for a period of time, then catalyzes the decomposition of urea. The resulting ammonia dissolves in a buffer solution, and indophenol is used for color development. The OD value is then measured using a spectrophotometer, and finally, the inhibition rate of the inhibitor on urease is calculated. The calculation formula is as follows:

[0022] Urease activity was tested according to the method reported by Weatherburn. The specific steps were as follows: 25 μL of urease solution (10 U / mL) and 25 μL of the test compound (concentration prepared with DMSO-phosphate buffer according to the experimental setup) were added to a 96-well plate. The plate was co-incubated at 37°C for 30 min. Then, 50 μL of phosphate buffer containing 25 mmol of urea was added, and the plate was co-incubated at 37°C for 30 min. Next, 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) were added, and the plate was co-incubated at 37°C for 30 min. The absorbance was measured at 620 nm. Each concentration was repeated 5 times. The blank sample was measured without urea and the test compound, otherwise the same as above. The control sample was measured without the test compound, otherwise the same as above. The positive control was acetoxyxamic acid. The inhibition rate and half-maximal inhibitory concentration (IC50) of the inhibitor were calculated. 50 .

[0023] 2. Field experiments on crops Three treatment groups (with the same area) were set up at the selected crop experimental sites: (1) urea (100% dosage); (2) urea (80% dosage) + urease inhibitor (N-methylglycine or its salt); (3) urea (70% dosage) + urease inhibitor (N-methylglycine or its salt). The proportion of urease inhibitor added was 0.01% to 10% of the urea quality. Each treatment was replicated in three places. The cultivation and fertilization management measures were consistent with the routine management of local farmers. The yield was calculated at the time of crop harvest.

[0024] This study found that N-methylglycine and its salts have significant urease inhibitory activity, with an IC50 value of [missing value]. 50 With a concentration of 0.63 µM to 0.95 µM, it can be used as a new type of nitrogen fertilizer enhancer in crop cultivation, reducing the amount of nitrogen fertilizer used while ensuring that crop yields are not reduced. Detailed Implementation

[0025] Example 1: Add 25 μL of urease (Giant Bean) solution (10 U / mL) and 25 μL of N-methylglycine solution (0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, 40 µM, prepared with DMSO-phosphate buffer) to a 96-well plate. Co-incubate at 37 °C for 30 min. Add 50 μL of phosphate buffer containing 25 mmol of urea and co-incubate at 37 °C for 30 min. Then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl). Co-incubate at 37 °C for 30 min and measure the absorbance at 620 nm. Each concentration was repeated 5 times. The blank sample was without urea and the test compound, and everything else was the same as above; the control sample was without the test compound, and everything else was the same as above. The inhibition rates of N-methylglycine against urease at concentrations of 0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, and 40 µM were measured to be 12.83%, 42.35%, 59.50%, 74.61%, 93.62%, 95.71%, and 96.33%, respectively. The half-maximal inhibitory concentration (IC50) was calculated. 50 The concentration was 0.68 µM, and the IC50 of acetoxyxamic acid in the positive control group was [missing value]. 50 It is 17.6 µM.

[0026] Example 2: Add 25 μL of urease (Giant Bean) solution (10 U / mL) and 25 μL of N-methylglycine hydrochloride solution (0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, 40 µM, prepared with DMSO-phosphate buffer) to a 96-well plate. Co-incubate at 37 °C for 30 min. Add 50 μL of phosphate buffer solution containing 25 mmol of urea and co-incubate at 37 °C for 30 min. Then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl). Co-incubate at 37 °C for 30 min and measure the absorbance at 620 nm. Each concentration is repeated 5 times. The blank sample is without urea and the test compound, and everything else is the same as above; the control sample is without the test compound, and everything else is the same as above. The inhibition rates of N-methylglycine hydrochloride against urease at concentrations of 0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, and 40 µM were measured to be 9.50%, 34.46%, 51.25%, 67.76%, 91.32%, 94.37%, and 96.68%, respectively. The half-maximal inhibitory concentration (IC50) was calculated. 50The concentration was 0.95 µM, and the IC50 of acetoxyxamic acid in the positive control group was [missing value]. 50 It is 17.6 µM.

[0027] Example 3: Add 25 μL of urease (Giant Bean) solution (10 U / mL) and 25 μL of N-methylglycine phosphate solution (0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, 40 µM, prepared with DMSO-phosphate buffer) to a 96-well plate. Co-incubate at 37 °C for 30 min. Add 50 μL of phosphate buffer solution containing 25 mmol of urea and co-incubate at 37 °C for 30 min. Then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl). Co-incubate at 37 °C for 30 min and measure the absorbance at 620 nm. Each concentration was repeated 5 times. The blank sample was without urea and the test compound, and everything else was the same as above; the control sample was without the test compound, and everything else was the same as above. The inhibition rates of N-methylglycine phosphate against urease at concentrations of 0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, and 40 µM were measured to be 10.53%, 37.05%, 54.02%, 70.10%, 92.07%, 94.92%, and 96.55%, respectively. The half-maximal inhibitory concentration (IC50) was calculated. 50 The concentration was 0.85 µM, and the IC50 of acetyloxyoxime in the positive control group was [missing value]. 50 It is 17.6 µM.

[0028] Example 4: Add 25 μL of urease (Giant Bean) solution (10 U / mL) and 25 μL of N-methylglycine sodium salt solution (0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, 40 µM, prepared with DMSO-phosphate buffer) to a 96-well plate. Co-incubate at 37 °C for 30 min. Add 50 μL of phosphate buffer solution containing 25 mmol of urea and co-incubate at 37 °C for 30 min. Then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl). Co-incubate at 37 °C for 30 min and measure the absorbance at 620 nm. Each concentration is repeated 5 times. The blank sample is without urea and the test compound, and everything else is the same as above; the control sample is without the test compound, and everything else is the same as above. The inhibition rates of N-methylglycine sodium salt against urease at concentrations of 0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, and 40 µM were measured to be 13.72%, 44.25%, 61.34%, 76.02%, 94.05%, 95.95%, and 97.25%, respectively. The half-maximal inhibitory concentration (IC50) was calculated. 50 The concentration was 0.63 µM, and the IC50 of acetyloxyoxime in the positive control group was [missing value]. 50 It is 17.6 µM.

[0029] Example 5: Add 25 μL of urease (Giant Bean) solution (10 U / mL) and 25 μL of N-methylglycine potassium salt solution (0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, 40 µM, prepared with DMSO-phosphate buffer) to a 96-well plate. Co-incubate at 37 °C for 30 min. Add 50 μL of phosphate buffer solution containing 25 mmol of urea and co-incubate at 37 °C for 30 min. Then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl). Co-incubate at 37 °C for 30 min and measure the absorbance at 620 nm. Each concentration is repeated 5 times. The blank sample is without urea and the test compound, and everything else is the same as above; the control sample is without the test compound, and everything else is the same as above. The inhibition rates of N-methylglycine potassium salt against urease at concentrations of 0.1 µM, 0.5 µM, 1.0 µM, 2.0 µM, 10 µM, 20 µM, and 40 µM were measured to be 13.43%, 43.48%, 60.57%, 75.43%, 93.71%, 95.86%, and 96.90%, respectively. The half-maximal inhibitory concentration (IC50) was calculated. 50The concentration was 0.65 µM, and the IC50 of acetyloxyoxime in the positive control group was [missing value]. 50 It is 17.6 µM.

[0030] Example 6: A paddy field in Jingzhou, Hubei Province, was selected as the experimental site. The rice variety was "Quanyou 607". The experiment was conducted from May to September 2025, and three treatment groups were set up (each treatment group had an area of ​​1 fen (66.67 m²)). 2 ): (1) Apply urea (20 kg / mu, of which 10 kg / mu is used as basal fertilizer and tillering fertilizer). (2) Apply urea (16 kg / mu, of which 8 kg / mu is used as basal fertilizer and tillering fertilizer) + N-methylglycine, with the proportion of N-methylglycine added being 0.5% of the urea quality; (3) Apply urea (14 kg / mu, of which 7 kg / mu is used as base fertilizer and tillering fertilizer) + N-methylglycine, with the proportion of N-methylglycine added being 0.5% of the urea quality.

[0031] Each treatment had three replicates. Cultivation and fertilization management practices were consistent with the routine management practices of local farmers. (Phosphorus fertilizer (superphosphate, P2O5≥12%) and potassium fertilizer (potassium chloride, K2O≥60%) were applied as base fertilizer in one application, with a total nutrient content of 5 and 6 kg / mu, respectively.) The yield per mu was calculated at rice harvest: Group 1 average yield 605.5±4.8 kg / mu, Group 2 average yield 628.3±6.7 kg / mu, and Group 3 average yield 609.0±5.1 kg / mu.

[0032] Example 7: A paddy field in Jingzhou, Hubei Province, was selected as the experimental site. The rice variety was "Quanyou 607". The experiment was conducted from May to September 2025, and three treatment groups were set up (each treatment group had an area of ​​1 fen (66.67 m²)). 2 ): (1) Apply urea (20 kg / mu, of which 10 kg / mu is used as basal fertilizer and tillering fertilizer). (2) Apply urea (16 kg / mu, of which 8 kg / mu is used as basal fertilizer and tillering fertilizer) + N-methylglycine phosphate, with the N-methylglycine phosphate added at 0.3% of the urea content; (3) Apply urea (14 kg / mu, of which 7 kg / mu is used as base fertilizer and tillering fertilizer) + N-methylglycine phosphate, with the N-methylglycine phosphate addition ratio being 0.3% of the urea quality.

[0033] Each treatment had three replicates. Cultivation and fertilization management practices were consistent with the routine management practices of local farmers. (Phosphorus fertilizer (superphosphate, P2O5≥12%) and potassium fertilizer (potassium chloride, K2O≥60%) were applied as base fertilizer in one application, with a total nutrient content of 5 and 6 kg / mu, respectively.) The yield per mu was calculated at rice harvest: Group 1 average yield 605.5±4.8 kg / mu, Group 2 average yield 617.5±5.6 kg / mu, and Group 3 average yield 607.1±4.5 kg / mu.

[0034] Example 8: A rapeseed field in Dangyang, Hubei Province, was selected as the experimental site. The rapeseed variety was "Zhongyou 516". The experiment was conducted from October 2024 to May 2025, and three treatment groups were set up (each treatment group had an area of ​​1 mu, i.e., 66.67 m²). 2 ): (1) Apply urea (22 kg / mu, of which 11 kg / mu is used as basal fertilizer and top dressing during the greening period). (2) Apply urea (17.6 kg / mu, of which 8.8 kg / mu is used as basal fertilizer and top dressing during the greening period) + N-methylglycine, with the proportion of N-methylglycine being 0.3% of the urea quality; (3) Apply urea (15.4 kg / mu, of which 7.7 kg / mu is used for basal fertilizer and topdressing during the greening period) + N-methylglycine, with the N-methylglycine addition ratio being 0.3% of the urea quality. Each treatment was set up with 3 replicates. The cultivation and fertilization management measures were consistent with the routine management of local farmers (the phosphate fertilizer (superphosphate), potassium fertilizer (potassium chloride), and boron fertilizer (boric acid) for each treatment were 15, 10, and 1 kg / mu, respectively, with basal fertilizer and topdressing each accounting for half). The rapeseed was not removed from the buds. The rapeseed yield was calculated at the time of rapeseed harvest: the average yield of Group 1 was 200.1±2.3 kg / mu, the average yield of Group 2 was 218.2±3.5 kg / mu, and the average yield of Group 3 was 203.4±2.6 kg / mu.

[0035] Example 9: A rapeseed field in Dangyang, Hubei Province, was selected as the experimental site. The rapeseed variety was "Zhongyou 516". The experiment was conducted from October 2024 to May 2025, and three treatment groups were set up (each treatment group had an area of ​​1 mu, i.e., 66.67 m²). 2 ): (1) Apply urea (22 kg / mu, of which 11 kg / mu is used as basal fertilizer and top dressing during the greening period). (2) Apply urea (17.6 kg / mu, of which 8.8 kg / mu is used as basal fertilizer and top dressing during the greening period) + N-methylglycine potassium salt, with the N-methylglycine potassium salt added at 0.6% of the urea quality; (3) Apply urea (15.4 kg / mu, of which 7.7 kg / mu are used for basal fertilizer and topdressing during the greening period) + N-methylglycine potassium salt. The proportion of N-methylglycine potassium salt added is 0.6% of the urea quality. Each treatment has 3 replicates. The cultivation and fertilization management measures are consistent with the routine management of local farmers (the phosphate fertilizer (superphosphate), potassium fertilizer (potassium chloride), and boron fertilizer (boric acid) for each treatment are 15, 10, and 1 kg / mu, respectively, with basal fertilizer and topdressing each accounting for half). The rapeseed is not harvested. The rapeseed yield is calculated at the time of rapeseed harvest: the average yield of Group 1 is 200.1±2.3 kg / mu, the average yield of Group 2 is 223.8±3.6 kg / mu, and the average yield of Group 3 is 206.5±2.2 kg / mu.

Claims

1. A urease inhibitor, characterized in that, The active ingredient of the urease inhibitor is N-methylglycine.

2. The urease inhibitor according to claim 1, characterized in that, The urease inhibitor comprises a pharmaceutically acceptable or agriculturally acceptable salt of N-methylglycine, the salt being selected from one or more of the following: hydrochloride, phosphate, citrate, malate, salicylate, fumarate, sulfonate, methanesulfonate, potassium salt, sodium salt, magnesium salt, zinc salt, or calcium salt.

3. The use of the urease inhibitor according to any one of claims 1-2 in inhibiting urease activity.

4. The application according to claim 3, characterized in that, The application involves adding the urease inhibitor to urea-containing fertilizers to inhibit the catalytic hydrolysis of urea by urease in the soil.

5. A nitrogen fertilizer synergist, characterized in that, It includes the urease inhibitor according to any one of claims 1-2.

6. The nitrogen fertilizer synergist according to claim 5, characterized in that, The synergist is used in combination with nitrogen-containing fertilizers, which contain urea.

7. A fertilizer composition, characterized in that, It includes nitrogen-containing fertilizers and the urease inhibitors as described in any one of claims 1-2.

8. The fertilizer composition according to claim 7, characterized in that, The nitrogen-containing fertilizer is urea or a compound fertilizer containing urea. Based on the mass of the urea, the mass percentage of the urease inhibitor is 0.01% to 10%. In a preferred embodiment, the mass percentage of the urease inhibitor is 0.1% to 5%.

9. A method for improving nitrogen fertilizer utilization or reducing nitrogen fertilizer application, characterized in that, When applying nitrogen-containing fertilizers to soil or crops, the urease inhibitor described in any one of claims 1-2 or the nitrogen fertilizer synergist described in any one of claims 5-6 shall be applied simultaneously.

10. The method according to claim 9, characterized in that, The nitrogen-containing fertilizer is urea or a compound fertilizer containing urea, and the application amount of the urease inhibitor is 0.01% to 10% based on the mass of the urea.

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