OA18326A

OA18326A - Schiff-base complex for enhancing urease inhibition

Info

Publication number: OA18326A
Application number: OA1201600486
Authority: OA
Inventors: Hugo OPPERMAN; Leon Lubbe
Original assignee: Kimleigh Technologies (Pty) Ltd
Priority date: 2015-12-14
Filing date: 2016-12-14
Publication date: 2018-10-16

Abstract

A chemical composition, which includes any one of a granular and liquid nitrogenous compound a Schiff-base organic acid urease inhibitor active to the nitrogenous compound a first organic additive for making the Schiff-base inhibitor water soluble and, if the nitrogenous compound is granular, a second binder to bind the Schiff-base inhibitor and organic additive to granular nitrogenous compounds.

Description

FIELD OF THE INVENTION

This invention relates to a Schiff-base complex for enhancing urease inhibition in urea fertiliser or urea containing substances. In particular, the invention relates to a urease inhibitor composition ând a method of preparing such a urease inhibitor composition.

BACKGROUND OF THE INVENTION

In attempts to reduce losses through NH3 volatilization, various coatings and treatments have been applied to urea and other N fertilizers include polymers (Biaise and Prasad 1995; Rochette et al. 2009a), elemental S (Prasad 1976; Knight et al. 2007), urease and nitrification inhibitors (Gioacchini et al. 2002; Asing et al. 2008, Zaman et al. 2008).^7,910 ¹⁴ The high concentrations of ammonia arising from the biochemical reactions of urease, as well as pH élévation, resuit into important négative implications in medicine and agriculture.¹⁰

High concentrations of urease cause significant environmental and économie problems by releasing abnormally large amounts of ammonia into the atmosphère during the breakdown of urea in urea particulates, urea containing aqueous solutions and exposure of animal wastes/manure which contain urea substances. When urea is used as a fertilizer, négative effects on plant growth is induced by primarily depriving plants of their essential nutrients and secondarily by ammonia toxicity which resuit in pH increase ofthe soil.¹¹

When animais and especially chickens are kept in confined breeding pens, the volatile ammonia causes damage to nasal mucosa, air passages and web surrounding the eyes of the birds. In addition, the increase in pH as a resuit ofthe ammonium formation causes significant damage to the feet of smaller birds, due to the corrosive effect of the ammonium, ρζ

The structure of urease has been studied extensively. Urease has a trimer of alpha (a), beta (β) and gamma (y) units in a triangular arrangement with three active sites per enzyme. These subunits include a 60.3KD a, a 11.7KD β and a 11.1 KD γ subunit.¹⁵ The interactions between the individual subunits of ail three a subunits make extensive contacts with each other to build a trimer. Each a subunit packs between two β and two γ subunits to form the side of the triangle arrangement. Each β subunit packs between the two of the adjacent a subunits at each corners of the triangle. The remaining γ subunit interacts with two γ subunits at the crystallographic three-fold axis. Because of the high degree of interaction, it is not obvious which α, β and γ chains make up the primary units. High conservation ofsequences and the extensive interactions ofthe trimer suggest a similar trimer structure in ail the ureases which is further subdivided into subunits. The γ subunit consists of an α - β domain with four helices and two antiparallel stands. Two helices (b and c) and the two strands. pack together with a righthanded, up-down-up-down topological order. One of the remaining helices (a) is lined up peripherally and interacts at the three-fold axis with the other two γ subunits, tightly packing against itself and to other (a) helices in an orthogonal manner.¹⁵ The last hélix (d) expériences a single turn. The first fifteen residues of the β subunits form two antiparallel β sheets with the amino-terminal residues of the a subunits. The core of the β subunit forms an imperfect six-stranded antiparallel β sheet. This subunit stabilizes the trimer by interacting with domain two of its own a subunits and domain one of a symmetry related a subunit. The a subunit consists of two domains, an (α, β) 8 barrel domain and a primarily β domain.¹⁵ The (α, β) 8 barrel is the only domain that contributes to the active site. Its barrel is rather elliptical with the long axis connecting strands 1 and 7. Strands 9 and 10 extend the lower portion of strands 1 and 3. The active site is located at the carboxyl terminal of the strands. A flap is formed that covers the active site by a helical excursion between strand 7 and hélix 7. Mixed four-strands and eightstranded β sheets form the wall of U-shaped canyon which makes up the second β domain. The walls are connected by the long strands 5 and 6 together with strands 10 and 11 which go down on one side, and up on the other side.¹⁵ Of more interest to the inventors, is the active site proposed in literature.¹⁵^/

In soil or other urease containing substrates, urea is hydrolysed by urease, a nickel-dependent enzyme that catalyses the hydrolysis of urea to form ammonia and carbon dioxide.²⁴

NH/, which has négative side effects in agriculture and plant health. Thus, in surface applications, losses of gaseous NH3 can occur and can constitute up to 50% ofthe fertilizer nitrogen appiied, especially in soils with low buffer.capacity, in calcareous soils, and in soils with high organic carbon content. The other négative 10 effect of urea hydrolysis is the accumulation of NO2' and NH3, which can damage germinating seeds, seedlings, and young plants. One approach to overcome the problems associated with the use of urea fertilizers is to find compounds that would inhibit the urease activity and thereby retard urea hydrolysis when appiied to soils together with the fertilizer. The possibility of controlling thé rate of the 15 enzymatic urea hydrolysis using urease inhibitors is an important goal to pursue.

Ureases hâve been isolated from a wide variety of organisms, including plants, fungi, and bacteria.²⁵ ²⁷ At présent, crystallographic structures are available for only some of the bacterial ureases.²⁸ However, a highly conserved 20 amino acid sequences is observed in ail known ureases and the constant presence of two Nickel (Ni) ions, which are bridged by the carboxylate group of the carbamylated lysine (LysR220)is seen throughout.. Furthermore, these are coordinated by some surrounding histidine and aspartic residues. The structure of native Bacillus pasteurii urease (BPU) reveals an analogous αβγ quaternary 25 structure and a very similar active site geometry to other ureases.²⁹

Among the known inhibitors of urease, the most efficient are phosphorodiamide and phosphorotriamide dérivatives. This group includes the following: N-n-butylthiophosphorictriamide (NBPT), which has been shown to form 30 stable complexes with urease and is among the most efficient inhibitors of the enzyme. Furthermore, phenylphosphorodiamidate (PPD), N-nbutylphosphorictriamide (NBPTO), and N-diaminophosphoryl-4-fluorobenzamide (Flurofamide).²⁹ Agrotain® is a commercially available urease inhibitor based on a NBTP containing formulation. The half maximal inhibitory (IC50) value is a / measure ofthe effectiveness of a compound in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process or component of a process. This value for urease inhibitors is standardized by acethydroxamic acid with an IC₅o value of 42.5μΜ.

The inventors are of the opinion that the method and composition of preparing the urease inhibitor compound provides new combinations of components that are bénéficiai as unique urease inhibiting compositions.

In this spécification reference will be made to the following documents:

1. Harrison R, Webb J (2001) A review of the ëffect of N fertilizer type on . gaseous émissions. Advances in Agronomy 71,65-108.

2. Sommer SG, Schjoerring JK, Denmead OT (2004) Ammonia émission from minerai fertilizers and fertilized crops. Advances in Agronomy 82, 557-622.

3. Draaijers GPJ, Ivens WPMF, Bos MM, Bleuten W (1989) The contribution of ammonia émissions from agriculture to the déposition of acidifÿing and eutrophying compounds onto forests. Environmental Pollution 60, 55-66.

4. Watson CJ (2000) Urease activity and inhibition - principles and practice. In ‘Proceedings ofthe International FertilizerSociety.

5. Fenn LB, Hossner IR (1985) Ammonia volatilisation from ammonium or ammonium forming nitrogen fertilizers. Advances in Soil Science 1, 771776

6. Gameh MA, Angle JS, Axley JH (1990) Effects of urea-potassium chloride and nitrogen transformations on ammonia volatilization from urea. Soil Science Society ofAmerica Journal 54, 1768-1772. ·

7. Gioacchini P, Nastri A, Marzodori C, Giovannini C, Antisari LV, Gassa C (2002) Influence of urease and nitrification inhibitors on N losses from soil fertilized with urea. Biology and Fertility of Soils 36,125-131.

8. Sigunga DO, Janssen BH, Oenema O (2002) Ammonia volatilization from vertisols. European Journal of Soil Science 53,195-202. ç(

9. Zaman M, Nguyen Ml, Blennerhassett JD, Quin BF (2008) Reducing NH3, N2O and NO3^--N losses from a pasture soil with urease or nitrification inhibitors and elemental S-amended nitrogenous fertilizers. Biology and Fertility of Soils 44, 693-705.

10. Biaise D, Prasad R (1995) Effect of blending urea with pyrite or coating urea with polymer on ammonia volatilization loss from surface-applied prilled urea. Biology and Fertility of Soils 20, 83-85.

11. Rochette P, MacDonald JD, Angers DA, Chantigny MH, Gasser M, Bertrand N (2009a) Banding of urea increased ammonia volatilisation in a dry acidic soil. Journal of Environmental Quality 38,1383-1390.

12. Prasad, M (1976) Gaseous loss of ammonia from sulfur-coated urea, ammonium sulfate, and urea applied to a calcareous soil (pH 7.3). Soil Science Society of America Journal 40,130-134.

13. —Knight EC, Guertal, EA, WoodCW (2007) Mowing and nitrogen source effects on ammonia volatilisation from turfgrass. Crop Science 47,1628^34.

14. Asing J, Saggar S, Singh J, Nolan NS (2008) Assessment of nitrogen losses from urea and an organic manure with and without nitrification inhibitor, dicyandiamide, applied to lettuce under glasshouse conditions. Australian Journal of Soil Research 46, 535-541.

15. Muhammad Raza Shah and Zahid Hussain Soomro (2012). Urease Inhibition, Enzyme Inhibition and Bioapplications, Prof. Rakesh Sharma (Ed.), ISBN: 978-953-51-0585-5, InTech, Available from: http://www.intechopen.com/books enzyme-inhibition-and-bioapplications/ureaseinhibition

16. Christians, Nick (2004). Fertilization. Fundamentals of Turfgrass Management (2nd ed.). John Wiley & Sons. pp. 137-138,142-143.

17. Website: http://www.wengfuaustralia.com/sustain.html. Date accessed

12/06/2013. '

18. Lawrence, J. R. and Germida, J. J. 1991. Enumération of sulphur-oxidizing populations in Saskatchewan agricultural soils. Can. J. Soil Sci. 71: 127— 136.

19. Grayston, S. J. and Germida, J. J. 1991. Sulfur-oxidizing bacteria as plantgrowth promoting rhizobacteria for canola. Can. J. Microbiol. 37:521-529.

20. Chapman, S. J. 1990. Thiobacillus populations in some agricultural soils. Soil Biol. Biochem. 22:479-482.

21. Lawrence, J. R. and Germida, J. J. 1991. Enumération of sulphur-oxidizing populations in Saskatchewan agricultural soils. Can. J. Soil Sci. 71: 127136.

22. Suzuki, I., Lee, D., Mackay, B., Harahuc, L. and Oh, J. K. 1999. Effect of various ions, pH and osmotic pressure on oxidation of elemental sulphur by Thiobacillus thiooxidans. Appl. Environ. Microbiol. 65: 5163-5168.

23. Takeuchi, T.L., and I. Suzuki. 1997. Cell Hydrophobicity and Sulphur Adhesion of Thiobacillus thiooxidans. Applied and Environmental Microbiology. 63(5):2058-2061.

24. Sahrawat, K. L. Control of urea hydrolysis. and nitrification in soil by chemicals. Prospects and problems. Plant Soil 1980, 57, 335-352.

— 25. Bacanamwo, M.; Witte, C. P.; Lubbers, M. W.; Polacco, J. C. Activation of the urease of Schizosaccharomyces pombe by the UreF accessory protein from soybean. Mol. Genet. Genomics 2002, 268, 525-534.

26. Takishima, K.; Suga, T.; Mamiya, G. The structure of jack bean urease. The complété amino acid sequence, limited proteolysis and reactive cysteine residues. Eur. J. Biochem. 1988, 175,151-165.

27. Davis, G. S.; Mobley, H. L. Contribution of dppA to urease activity in Hélicobacter pylori 26695. Hélicobacter 2005,10, 416-423.

28. Manunza, B.; Deiana, S.; Pintore, M.; Gessa, C. The binding mechanism of urea, hydroxamic acid and N-(n-butyl)phosphorictriamide to the urease active site. A comparative molecular dynamics study. Soil Biol. Biochem. 1999, 31,789-796.

29. Dom'inguez, M.J., Sanmart’in, C, Font, M, Palop, J.A., San Francisco, S. Urrutia, O., Houdusse, F. and Garc'ia-Mina, J.M., Design, Synthesis, and Biological Evaluation of Phosphoramide Dérivatives as Urease Inhibitors, J. Agric. Food Chem. 2008, 56, 3721-3731.

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6.1. SADABS, version 2.03, Broker AXS Inc., Madison, 2000.

SUMMARY OF THE INVENTION

- According to one aspect of the invention, there is provided a chemical composition, which includes;

any one of a granular and liquid nitrogenous compound;

a Schiff-base organic acid urease inhibitor active to the nitrogenous compound;

a first organic additive for making the Schiff-base inhibitor water soluble; and if the nitrogenous compound is granular, a second binder to bind the Schiffbase inhibitor and organic additive to granular nitrogenous compounds.

The nitrogenous compound may be in the form of a Nitrogen containing fertilizer.

The Nitrogen containing fertilizer may be selected from any one of urea, mono-ammonium phosphate (MAP), di-ammonium phosphate (DAP), or the like.

The urea may be in the form of animal/human waste. The animal/human waste is in the form of any one of manure and urine.

The Schiff-base inhibitor may be: in the case of a granular nitrogenous compound, in the form of a coating around the granular nitrogenous compound and; in the case of a liquid nitrogenous compound, a liquid additive.

The Schiff-base inhibitor may be in the range of 0.1% to 5% by weight of the nitrogenous compound.

The Schiff-base inhibitor may include a coordinated métal. The coordinated métal may originate from a métal sait. The métal sait may be selected from any one of a sulphate, nitrate, chloride, or the like.

The coordinated métal may be in the form of a transition métal selected from any one of copper, iron, nickel, zinc, manganèse, or the like.

The Schiff-base inhibitor may include a second binder. The Schiffbase inhibitor may include an organic acid.

The Schiff-base inhibitor may include an organic ligand constituent. The organic ligand constituent may be formed by reacting an amine with any one of an aryl and alkyl functional group. The aryl and alkyl functional group may include any one of the following: salicylaldéhyde, phenolenediamine, 5diethylamino-2-[(pyridin-2-ylmethylimino)methyl]phenol, - 2-[1-(2-piperidin-1ylethylimino)ethyl]phenol, furan-2-carbaldehyde and benzohydrazide, dihydrohyacetic acid, or the like.

The amine may include one or more of the following: propylamine, butylamine, ethanolamine, pentylamine. hexylamine, benzenepropanamine, methylbenzoamine, or the like.

The Schiff-base inhibitor may be mixed with the first organic additive to bind the Schiff-base inhibitor to the nitrogenous compound.

The invention extends to a method of preparing a urease inhibitor composition, which can be used in granular or liquid fertilizer coatings or the treatment of urea containing animal/human waste, the method including: selecting a granular or liquid nitrogenous compound;

formulating a Schiff-base organic acid urease inhibitor active to inhibit the urease activity of said nitrogenous compound; and if the nitrogenous compound is granular, mixing a second binder with the Schiff-base inhibitor and; if the nitrogenous compound is a liquid, adding an organic additive to the nitrogenous compound.

The step of selecting the granular nitrogenous compound may include making a sélection from a group of Nitrogen containing fertilizers.

The step of formulating the Schiff-base inhibitor may include dissolving a métal sait in water and an ethanolamine based solvent.

The step of formulating the Schiff-base inhibitor may include the formation of ligands -by mixing an aromatic acid and amine in an ethanolamine based solvent.

The ethanolamine based solvent may be one of mono-ethanolamine (MEA), di-ethanolamine (DEA), tri-ethanolamine (TEA), or the like.

The step of formulating the Schiff-base inhibitor may further include slowly adding the ligands to the dissolved métal sait solution to form a water insoluble Schiff-base complex.

The Schiff-base complex may further be mixed with a second binder and an organic acid to form the Schiff-base inhibitor.

The invention is now described, by way of non-limiting example, with reference to the accompanying drawings:

FIGURE(S)

In the figure(s):

Figure 1 shows a flow diagram of the method of preparing the Schiffbase organic acid complex.

Figure 2 shows a structural représentation ofthe Schiff-base organic acid complex.

Figure 3 shows a line graph of the change in absorbance for different concentrations of ammonium in Example 1.

Figure 4 shows a line graph of the change in.average ammonium concentrations at different urease concentrations in Example-1.

Figure 5 shows a table portraying the results obtained for each of the nitrogen treatments used in Example 2.

Figure 6 shows a bar graph portraying the yield of grass obtained for each of the nitrogen treatments used in Example 2.

EMBODIMENT OF THE INVENTION

The invention provides a method of preparing a chemical composition which includes a granular or liquid nitrogenous compound; a Schiffbase organic acid urease inhibitor and a first organic additive to inhibit urease.

In a further embodiment of the invention, the Schiff-base inhibitor can also be used to treat urea substances in human and animal waste in a solid or dissolved form.

Figure 1 shows a method 10 of preparing the Schiff-base organic acid urease inhibitor which includes a métal sait, an organic ligand constituent, a second binder and an organic acid.

According to-the method shown in Figure 1, the métal sait 12 is dissolved in a mixture of water and an ethanolamine based solvent at 14.

The organic ligand constituent is formed by mixing an aromatic acid together with an amine in an ethanolamine based solvent at 16. The aromatic acid and amine mixed together form a functional group connected to an aryl/alkyl group at 18 with a general formula R¹R²C=NR³, where R is an organic side chain.

In one embodiment the organic ligand constituents are in the form of the formula -(C_XH_XNO)2.

The organic ligand constituents_are slowly added to the dissolved métal sait to form a water insoluble Schiff-base complex at 20. The Schiff-base complex at 22 may be further mixed with the second binder and an organic acid 24 at 26 to form the Schiff-base inhibitor at 28.

Figure 2 shows a structural drawing representing a Schiff-base organic acid complex, in which ‘M’represents a métal derived from the métal sait, ‘L’the organic ligand constituent and ΌΑ’ the organic acid. In one embodiment the métal is in the form of a transition métal.

As shown in Figure 2, the organic ligand constituées are specifically coordinated.

The method of preparing a urease inhibitor composition in Figure 1 further includes applying the second binder to urea containing the nitrogenous compound. The second binder is mixed with the Schiff-base inhibitor to improve the binding action to the nitrogenous compound. In one embodiment a granular nitrogenous compound in the form of urea was used and the Schiff-base inhibitor is in the form of a coating around the urea. In Figure 1, the first organic additive is added to the Schiff-base complex to form a Schiff-base organic acid complex (shown in Figure 2), rendering the compound water soluble to use in aqueous and non-aqueous urea containing liquids orsolids.

Example 1

METHOD

Glycine max seeds were soaked in deionised water for 72h. The seeds were wet, grinded and sieved through acheese cloth to extract the urease enzyme from the seeds. Urease activity was measured using a spectrophotometric method. Most of the tests¹ used to détermine urease activity monitor the products obtained from enzyme catalysed hydrolysis of urea into carbon dioxide and ammonia, where the ammonia formed interacts with an agent to form a coloured product. An indophenol assay (Berthelot method) was used, in which ammonium ions reacted with NaCIO and monochloramine was formed. Addition of a phenolic solution-to monochloramine resulted in quinonechlorimine. The imine interacted with the phénol and indophenol was formed. In an acidic solution, indophenol is a yellow colour, but after alkalization ofthe solution a blue product was formed. The absorbance of the resulting blue solutions are shown in Figure 3. The intensity of the colouring strongly dépends on ammonium ions concentration up to 5mg/ml, then the intensity enhances more gradually. The absorbance of the blue colour was measured via UV-VIS at 636nm. The absorbance is directly proportional to the amount of ammonium présent, which is directly proportional to the activity and amount of urease.

The activity was measured using the above described Berthelot method. Phenolic solution - phénol (7g) and sodium nitroprusside (disodium pentacyanonitrosylferrate, 34mg) were dissolved in deionised water (50ml) and then madetip to 100ml. This reagent was stored in a dark-coloured bottle at 4°C. The buffered hypochloride reagent was prepared by dissolving 2.96g NaOH in 140ml of deionised water, adding 22.29g Na2HPO4-7H2O, and dissolving it completely. Then NaCIO (12% v/v, 16.6ml) solution was added. The pH was adjusted to 12.0 and the deionised water was added to complété the final volume of 200ml. This reagent was stored in a dark-coloured bottle at room température.

Standard ranges of ammonium were measured as follows: NH4CI (40μΙ, 7pg/ml - 7mg/ml) was pipetted to glass test tubes. Thereafter deionised water (1960μΙ), the phenolic solution (200μΙ) and the buffered hypochloride reagent (400μΙ) were added. The mixture was vortexed for 5 minutes using Vortex - 2 Genie (Scientific Industries, New York, USA) and stored for 20 minutes at 50°C. The solutions were measured by a spectrophotometer (Helios, Thermo Fisher Scientific, USA) at 636nm against a blank sample, contaîning deionised water (2ml), the phenolic solution (200μΙ) and the buffered hypochloride reagent (400μΙ). The blank sample was also stored for 20 minutes at 50°C prior to measurements. The results ob'tained are shown in Figure 3. The procedure was repeated but the NH₄CI solutiomwas replaced by 2ml of a 50mM solution. To this a range of urease amounts (5, W, 20, 40 and 80μΙ) were added and incubated for 1h at 30°C. The colour change was once again measured using UV-VIS at o(

636nm. The blank sample contained only the urea solution without any urease. Each concentration of urease was repeated 6 times and the average ofthe results are shown in Figure 4. The procedure was once again repeated but this time 1 μΙ of the preferred urease inhibitor was added to each tube. This was also repeated 6 times and the averages of the results are shown in Figure 4.

Example 2

METHOD

Buchloe dactyloides (Nutt.) Engelm. (Buffalo forage grass) seeds were sown over a 12Ha area in 24 X 0.5Ha plots (6 réplications for each treatment). The average day/night température was 27’C and 19’C respectively. No irrigation was provided and watering depended exclusively on rainfall. Average rainfall during the growth period was 65mm per month.

It can be seen from the table in Figure 5 and the graph in Figure 6 that ail nitrogen treatments had a significant (p^0.05) positive effect on the yield at harvest time. The fresh weight of the harvested grass also differed significantly (p£0.05) between some of the treatments. Both the Schiffbase acid urease inhibitor treated urea and ammonium sulphate treatments showed significant (p<0.05) yield increases compared to the untreated urea. No significant (p<0.05) différence was seen between the Schiff-base acid urease inhibitor treated urea and ammonium sulphate treatments.

The inventors believe that the invention provides new fertilizer compositions that has increased urease inhibition efficiency, which prevents the release of high amounts of ammonia into the atmosphère. The inventors also believe that the increased urease inhibition will increase efficiency.by preventing ammonia volatilization of urea substances found in human and animal waste. c/

Claims

CLAIMS:

1. A chemical composition, which includes;

any one of a granular and liquid nitrogenous compound;

a Schiff-base organic acid urease inhibitor active to the nitrogenous compound;

_ a first organic additive for making the Schiff-base inhibitor water soluble; and if the nitrogenous compound is granular, a second binder to bind the Schiffbase inhibitor and organic additive to granular nitrogenous compounds.
2. A chemical composition as claimed in claim 1, in which the nitrogenous compound is in the form of a Nitrogen containing fertilizer.
3. A chemical composition as claimed in claim 2, in which the Nitrogen containing fertilizer is selected from any one of urea, mono-ammonium phosphate (MAP) and di-ammonium phosphate (DAP).
4. A chemical composition as claimed in claim 3, in which the urea is in the form of animal/human waste.
5. A chemical composition as claimed in claim 4, in which the animal/human waste is in the form of any one of manure and urine.
6. A chemical composition as claimed in claim 1, in which the Schiffbase inhibitor is: in the case of a granular nitrogenous compound, in the form of a coating around the granular nitrogenous compound and; in the case of a liquid nitrogenous compound, a liquid additive.

A chemical composition as claimed in claim 6, in which the Schiffbase inhibitor is in the range of 0.1% to 5% by weight of the nitrogenous compound.
8. A chemical composition as claimed in claim 1, in which the Schiffbase inhibitor includes a coordinated métal.
9. A chemical composition as claimed in claim 8, in which the coordinated métal originates from a métal sait.
10. A chemical composition as claimed in claim 9, in which the métal sait is selected from any one of a sulphate, nitrate and chloride.
11. A chemical composition as claimed in claim 8, in which the coordinated métal is in the form of a transition métal selected from any one of copper, iron, nickel, zinc or manganèse.
12. A chemical composition as claimed in claim 1, in which the Schiffbase inhibitor includes a second binder.
13. A chemical composition as claimed in claim 9, in which the Schiffbase inhibitor includes an organic acid.
14. A chemical composition as claimed in claim 1, in which the Schiffbase inhibitor includes an organic ligand constituent.
15. A chemical composition as claimed in claim 14, in which the organic ligand constituent is derived by reacting an amine with any one of an aryl and alkyl functional group.
16. A chemical composition as claimed in claim 15, in which the aryl and alkyl functional groups includes any one of the following: salicylaldéhyde, phenolenediamine, 5-diethylamino-2-[(pyridin-2-ylmethylimino)methyl]phenol, 2[1-(2-piperidin-1-ylethylimino)ethyl]phenol, furan-2-carbaldehyde and benzohydrazide, dihydrohyacetic acid.
17. A chemical composition as claimed in claim 15, in which the amine includes one or more of the following: propylamine, butylamine, ethanolamine, pentylamine, hexylamine, benzenepropanamine and methylbenzoamine.
18. A chemical composition as claimed in claim 1, in which the Schiffbase inhibitor is mixed with the first organic additive to bind the Schiff-base inhibitor to the nitrogenous compound.
19. A method of preparing a urease inhibitor composition, which can be used in granular or liquid fertilizer coatings or the treatment of urea containing animal/human waste, the method including:

selecting a granular or liquid nitrogenous compound;

formulating a Schiff-base organic acid urease inhibitor active to inhibit the urease activity of said nitrogenous-compound; and if the nitrogenous compound is granular, mixing a second binder with the Schiff-base inhibitor and; if the nitrogenous compound is a liquid, adding an organic additive to the nitrogenous compound.
20. A method of preparing a urease inhibitor composition as claimed in claim 19, in which the step of selecting the granular nitrogenous compound includes making a sélection from a group of Nitrogen containing fertilizers.
21. A method of preparing a urease inhibitor composition as claimed in claim 19, in which the step of formulating the Schiff-base inhibitor includes dissolving a métal sait in water and an ethanolamine based solvent.
22. A method of preparing a urease inhibitor composition as claimed in claim 19, in which the step of formulating the Schiff-base inhibitor includes the formation of ligands by mixing an aromatic acid and amine in an ethanolamine based solvent.
23. A method of preparing a urease inhibitor composition as claimed in claim 22, in which the ethanolamine based solvent is one of mono-ethanolamine (MEA), di-ethanolamine (DEA) and tri-ethanolamine (TEA).
24. A method of preparing a urease inhibitor composition as claimed in claim 22, in which the step of formulating the Schiff-base inhibitor further includes slowly adding the ligands to the dissolved métal sait solution to form a water

5 insoluble Schiff-base complex.
25. A method of preparing a urease inhibitor composition as claimed in claim 24, in which the Schiff-base complex is further mixed with a second binder and an organic acid to form the Schiff-base inhibitor.