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- Urease Inhibitors of Agricultural Interest Inspired by Structures of Plant Phenolic Aldehydes
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- Improving Efficiency of Urea Fertilizers by Inhibition of Soil Urease Activity
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Then, in vitro assays were performed with pure jack bean urease to check the potential of synthesized compounds as inhibitors of ureolytic activity and disclose the mechanism of action of promising molecules. The effect of such phenolic aldehyde derivatives on soil ureases was addressed to confirm the potential of synthesized compounds for use as additives in urea-based fertilizers. An ethanolic mixture containing protocatechuic aldehyde PA , syringaldehyde SA or vanillin VA individually 1 mmol; Sigma-Aldrich , ethyl acetoacetate 1.
After then, the mixture was filtered and the phenolic aldehyde derivative formed was recrystallized using ethanol. The phenolic aldehyde derivatives synthesized based on urea 2A7 or thiourea 2A9 , 2B10 and 2D2 structure were characterized by 1 H and 13 C nuclear magnetic resonance NMR , infrared, melting point and elemental analysis and the data compared to those reported elsewhere.
Urease Inhibitors of Agricultural Interest Inspired by Structures of Plant Phenolic Aldehydes
Initially, the phenolic aldehydes and derivatives 2A7 , 2A9 , 2B10 and 2D2 were screened for the ability to inhibit in vitro the ureolytic activity of purified Canavalia ensiformis jack bean type III urease Sigma, St. Each reaction medium containing 20, 1 and 10 mmol L -1 of phosphate buffer pH 7. Reactions were stopped by adding 0. Three independent experiments were performed, each with four replicates.
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Effect of phenolic aldehyde derivatives on the kinetic parameters of jack bean urease. Jack bean urease kinetic parameters such as initial velocity V o , K M Michaelian constant and maximum velocity V max were obtained using Hyper32 software.
The effect of phenolic aldehyde derivatives on the activity of soil ureases was assessed by the salicylate method as described elsewhere, 36 with some modifications. Sieved soil samples 0. A supernatant aliquot was taken after soil decantation and added to a solution containing 3.
Then, assays using different concentrations from 0. Independent experiments were performed, each with at least five replicates. The in vitro assay with purified jack bean type III urease showed that, among the natural products tested, only protocatechuic aldehyde PA; at 1. Results are representative of three independent experiments, each with four replicates.
Urease is categorized as a Michaelian enzyme since the graph of initial velocity V o versus urea concentration exhibits a typical hyperbolic behavior Figure 3. The average urea K M Michaelian constant and urease maximum velocity V max in reactions free of urease inhibitor were, respectively, 3. The addition of 2A7 , 2A9 , 2B10 or 2D2 to the reaction medium caused a concentration-dependent increment of urea K M apparent Michaelis constant: K M app and decrease of urease V max apparent maximum velocity: V max app Figure 3.
All the phenolic aldehyde derivatives tested behaved as mixed inhibitors, as attested by the lines intersection in the second quadrant of Lineweaver-Burk plots Figure 3 ; right column. A V o versus urea concentration plot obtained from data of assays with 2A7 is shown to exemplify the Michaelian behavior of urea catalysis.
Improving Efficiency of Urea Fertilizers by Inhibition of Soil Urease Activity
The derivative 2A7 was the most potent mixed inhibitor since the K i and K' i values for complexes formed with this compound were the lowest in comparison with the others inhibitors Table 1. In general, K i values were lower than the K' i values for complexes related to the same inhibitor by 2.
When tested at 3. Results are representative of independent experiments, each with at least five replicates. There was not a pattern in the behavior of results observed for the replicates of independent experiments performed with 2B10 and NBPT, which did not allowed for determining the IC 50 values for such inhibitors.
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Thus, the plant natural products protocatechuic aldehyde PA , vanillin VN and syringaldehyde SA were selected as prototypes for the design of new urease inhibitors based on urea or thiourea scaffolds, urease substrate and inhibitor respectively. Besides methyl gallate and related derivatives, 37 other plant phenolic compounds, such as iso quercitrin, avicularin, guaijaverin, flavonoid glucosides and shoreaphenol, were also reported to inhibit jack bean urease.
Under our experimental conditions, the novel urease inhibitors 2A7 and 2B10 were as potent as hydroxyurea HU; known urease inhibitor , while 2D2 also novel and 2A9 was less effective than HU Figure 2.
The potential of the derivative 2A9 as an inhibitor of one of the jack bean ureases was recently reported, 40 although the experimental conditions were different from the one reported herein. Thus, the outstanding performance of these phenolic derivatives, compared to the natural products they originated from, might be attributed to the combination of a catechol skeleton with urea or thiourea core.
We performed kinetic experiments varying urea concentration in urease-catalyzed reactions containing each inhibitor at fixed concentrations Figure 3. The urea K M value obtained from reactions carried out in 20 mmol L -1 phosphate buffer pH 7. Other studies with jack bean urease, under experimental conditions distinct from the one used here, reported urea K M values ranging from 1.
The kinetic behavior of jack bean urease in the presence of the phenolic aldehyde derivatives is consistent with the one expected for an enzyme in the presence of a mixed inhibitor. Mixed inhibitors are known to be capable of binding both the free enzyme forming an enzyme-inhibitor complex and the enzyme-substrate complex forming an enzyme-inhibitor-substrate complex. Or, email Ken Tarlow at: Tarlow Design is your one stop solution for consumer product design, development, prototype manufacturing, product manufacturing and licensing.
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