Retarding Hydrolysis Research Articles

Detecting a quasi-stable imine species on the reaction pathway of SHV-1 β-lactamase and 6β-(hydroxymethyl)penicillanic acid sulfone.

For the class A β-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6β position by a CH2OH group (6β-(hydroxymethyl)penicillanic acid). The 6β substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a β-aminoacrylate) in 1 h for sulbactam and in 4 h for 6β-(hydroxymethyl) sulbactam. Rapid mix–rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6β-(hydroxymethyl) sulbactam formed longer-lived acyl–enzyme intermediates that are a mixture of imine and enamines. Single crystal Raman studies, soaking in and washing out unreacted substrates, revealed stable populations of imine and trans-enamine acyl enzymes. The corresponding X-ray crystallographic data are consonant with the Raman data and also reveal the role played by the 6β-hydroxymethyl group in retarding hydrolysis of the acyl enzymes. The 6β-hydroxymethyl group sterically hinders approach of the water molecule as well as restraining the side chain of E166 that facilitates hydrolysis.

The Effect of Different Cold Storage Conditions on the Compositions of Extra Virgin Olive Oil

AbstractStorage conditions can affect the stability and quality of extra virgin olive oil (EVOO). While many studies have reported the influence of high temperature and light exposure during storage, little is known on the influence of the cold storage. The aim of this study was to evaluate the effect of different storage conditions (25, 4.5 and −27 °C) on the various compositions of EVOO and to determine if cold storage will prolong shelf‐life by retarding hydrolysis and oxidation. The changes of quality indices (FFA, PV, and UV) and natural antioxidants such as α‐tocopherol and phenolic compounds were evaluated periodically during storage. The characterization and quantification of phenolics were achieved by ultra‐performance liquid chromatography—diode array detector (UPLC‐DAD). In addition, 1, 2‐diacylglycerols (DAGs), pyropheophytin A (PPP) were measured to indicate thermal degradation during storage.

Antioxidant effects of pH-regulating agents on the thermal deterioration of vegetable oils.

pH-Regulating agents, such as sodium tartrate, disodium succinate, and trisodium citrate, were investigated for their antioxidant activities during the thermal deterioration of vegetable oils. Refined rapeseed and rice bran oils, supplemented with pH-regulating agents and α-tocopherol (0.1%) were heated at 180℃. After heating, acid values (AVs), carbonyl values (CVs), polar material contents, and color (absorbance at 420 nm) of each sample were measured. All pH-regulating agents gave rise to reduced AVs, CVs, and polar material contents of vegetable oils during heating relative to samples not containing a pHregulating agent. Rapeseed and rice bran oils supplemented with sodium tartrate showed the lowest AVs, CVs, polar material contents and absorbances at 420 nm after heating. Sodium tartrate not only retarded the hydrolysis, thermal oxidation, polymerization, and coloration of both oils while heating at high temperatures, but it also showed antioxidant activity at the supplementation level of 0.01%. The antioxidant activity of sodium tartrate was higher than that of α-tocopherol during the deterioration of vegetable oils. Sodium tartrate was particularly effective retarding hydrolysis while heating at high temperatures, resulting in increase of AVs of vegetable oils. Sodium tartrate is therefore expected to be an effective antioxidant for the thermal deterioration of fats and oils during deep-fat frying.

Importance of Protease Cleavage Sites within and Flanking Human Immunodeficiency Virus Type 1 Transframe Protein p6* for Spatiotemporal Regulation of Protease Activation

The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors.

Hydrolysis and acidogenesis of particulate organic material in mesophilic and thermophilic anaerobic digestion

The purpose of this study was to evaluate the effect of pH and inorganic nutrient supplementations for anaerobic hydrolysis and addogenesis of paniculate organic materials at both mesophilic (35 °C) and thermophilic (55 °C) temperatures. Hydrolysis and addogenesis of a synthetic sludge was observed in batch operation for the evaluation of the pH effect. pH was uncontrolled in one reactor and controlled at 4.5, 5.5, and 6.5 in the other three reactors at both temperatures. The greatest degree of hydrolysis and addogenesis occurred when the pH was controlled at 6.5. The pH of the uncontrolled reactor dropped to 3.4 at both temperatures severely retarding hydrolysis and addogenesis. Concentrations of acetic and n‐butyric adds predominated with lower concentrations of propionic add at both temperatures in all reactors. Lactic add was produced as the earliest intermediate but as the reaction proceeded, short chain VF As were produced as final end products with a decrease in lactic add. The higher the pH, the earlier this trend was observed. For the controlled reactors at pH 6.5, the soluble COD production and the VSS reduction peaked in 4 days at 55 °C whereas it took about 11 days at 35 °C to obtain the same result. During the linear SCOD production period at a pH of 6.5 the hydrolysis rate of the thermophilic reactor was greater than that for mesophilic. Thermophilic conditions appeared to be more sensitive to pH than mesophilic ones for both hydrolysis and addogenesis. Additional experiments were conducted to establish the effect of inorganic nutrient (Ca, Fe, Co, and Ni) supplementation on hydrolysis and addogenesis at both temperatures. It has, prior to this, been assumed that only methanogenesis benefited from trace metal supplementation. However, the results demonstrated the importance of inorganic nutrient supplementation to optimize hydrolysis and addogenesis at both temperatures.

ANA and GIS zeolite synthesis directly from alumatrane and silatrane by sol-gel process and microwave technique

Alumatrane and silatrane were successfully used as precursors to produce aluminosilicate via the sol-gel process. Due to their ability in retarding hydrolysis process, forming meso-porous material was easier. Both NaCl and NaOH can be used as hydrolysis agents, however, NaOH had highly influenced crystalline formation. The higher NaOH concentration, the better crystalline formation was observed. Gel transformation was an endothermic reaction. The maximum transformation occurred at 106 °C, as determined by differential scanning calorimetry (DSC). By using NaOH/H2O as a hydrolysis agent and treating amorphous metal oxide gel by microwave technique, the crystalline aluminosilicate was formed and narrow particle size distribution was obtained. By fixing the ratio of SiO2, Al2O3, Na2O and H2O at 1:0.25:3:410, GIS was synthesized at hydrothermal treatment of 3 h at 110 °C, while analcium (ANA) was produced at 130 °C for 8 h. GIS obtained had 4.55 μm in size while ANA's size was 9.96 μm.

Efficiency of N-(n-butyl) thiophosphoric triamide in retarding hydrolysis of urea and ammonia volatilization losses in a flooded sandy loam soil amended with organic materials

Using a forced-draft chamber technique, the suppression of NH3 volatilization losses by applying N-(n-butyl) thiophosphoric triamide (NBPT) was studied in an alkaline sandy loam soil amended with green manure or wheat straw. Applied urea was completely hydrolysed in 12, 8 and 6 days in unamended, green manure and wheat straw amended soil, respectively. By applying 0.5% (w/w of urea) NBPT, complete hydrolysis of urea was delayed up to 16 days in the unamended soil, whereas in wheat straw amended soil urea hydrolysis was completed by the 12th day even when it was treated with 2% NBPT. Applied at 1 or 2% level, NBPT delayed the NH3 volatilization to the 4th day after application of urea in green manure or wheat straw amended soil. Hydrolysis of urea was more effectively retarded by applying NBPT in the unamended soil than in soil amended with green manure or wheat straw. In the unamended soil, 7.1% of the applied urea was lost through NH3 volatilization. The losses were reduced to 1.2 and 0.7% by applying 0.5 and 1% NBPT, respectively. Enhanced NH3 volatilization caused by the green manure or wheat straw was counteracted by applying NBPT.

Ligand-Induced Conformational Changes of Thymidylate Synthase Detected by Limited Proteolysis

Limited tryptic proteolysis was used to investigate conformational changes of thymidylate synthase from Lactobacillus casei induced by ligand binding. Most of the identified sites of proteolysis were between R72 and R178, a region that includes a large loop containing residues 90-139 that is absent in thymidylate synthase from most other sources. Hydrolysis at both ends of this region was affected by the presence of dUMP. With dUMP, the preference of initial hydrolysis at the N-terminus of this region was switched from R78 to R72, and hydrolysis at R178 was retarded; the latter effect may be primarily a consequence of steric hinderance since R178 is involved in binding the phosphate moiety of dUMP. Orthophosphate had an effect similar to that of dUMP, not only in retarding hydrolysis at the phosphate binding site (R178) but also in retarding hydrolysis at R78 in favor of R72. Alkylation of the catalytically essential sulfhydryl group of thymidylate synthase by iodoacetamide also resulted in R72 being favored over R78 as a site of initial proteolysis. Its effect on hydrolysis at R178 was, as expected, less than that of dUMP or phosphate. These results indicate that dUMP binding induces conformational changes in thymidylate synthase. Phosphate binding and sulfhydryl alkylation also induce conformational changes similar to those resulting from dUMP binding. While the similarity of the proteolytic behavior of thymidylate synthase in the presence of dUMP or phosphate agrees with the report by Finer-Moore et al.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent research on problems in the use of urea as a nitrogen fertilizer

Recent research on the NH3 volatilization, NO 2 - accumulation, and phytotoxicity problems encountered in the use of urea fertilizer is reviewed. This research has shown that the adverse effects of urea fertilizers on seed germination and seedling growth in soil are due to NH3 produced through hydrolysis of urea by soil urease and can be eliminated by addition of a urease inhibitor to these fertilizers. It also has shown that the leaf burn commonly observed after foliar fertilization of soybean with urea results from accumulation of toxic amounts of urea in soybean leaves rather than formation of toxic amounts of NH3 through hydrolysis of urea by leaf urease. It further showed that this leaf burn is accordingly increased rather than decreased by addition of a urease inhibitor to the urea fertilizer applied. N-(n-butyl)thiophosphoric triamide (NBPT) is the most effective compound currently available for retarding hydrolysis of urea fertilizer in soil, decreasing NH3 volatilization and NO 2 - accumulation in soils treated with urea, and eliminating the adverse effects of urea fertilizer on seed germination and seedling growth in soil. NBPT is a poor inhibitor of plant or microbial urease, but it decomposes quite rapidly in soil with formation of its oxon analog N-(n-butyl) phosphoric triamide, which is a potent inhibitor of urease activity. It is not as effective as phenylphosphorodiamidate (PPD) for retarding urea hydrolysis and ammonia volatilization in soils under waterlogged conditions, presumably because these conditions retard formation of its oxon analog. PPD is a potent inhibitor of urease activity but it decomposes quite rapidly in soils with formation of phenol, which is a relatively weak inhibitor of urease activity. Recent studies of the effects of pesticides on transformations of urea N in soil indicate that fungicides have greater potential than herbicides or insecticides for retarding hydrolysis of urea and nitrification of urea N in soil.

Problems in the use of urea as a nitrogen fertilizer

Abstract. N‐(n‐butyl) thiophosphoric triamide (NBPT) is the most effective compound currently available for retarding hydrolysis of urea fertilizer in soil and for decreasing ammonia volatilization and nitrite e accumulation in soils treated with urea. It is a poor inhibitor of plant or microbial urease, but decomposes quite rapidly in soil with formation of N‐(n‐butyl) phosphoric triamide, which is a potent inhibitor of urease activity.The adverse effects of urea fertilizers on seed germination and seedling growth in soil are due to ammonia produced through hydrolysis of urea by soil urease. They can be eliminated by addition of a urease inhibitor to these fertilizers.The leaf‐burn commonly observed after foliar fertilization of soybeans with urea results from accumulation of toxic amounts of urea in the soybean leaves rather than formation of toxic amounts of ammonia through urea hydrolysis by leaf urease. Leaf‐burn is accordingly increased rather than decreased by addition of a urease inhibitor to the urea fertilizer applied.

Evaluation of ammonium thiosulfate as a soil urease inhibitor

Interest in use of ammonium thiosulfate (ATS) in conjunction with urea as a fertilizer has been stimulated by recent reports that this compound retards hydrolysis of urea by soil urease and thereby reduces volatilization of urea N as ammonia from soils fertilized with urea. We evaluated ATS as a soil urease inhibitor by studying its effects on urea hydrolysis, seed germination, and early seedling growth in soil. We found that ATS significantly retarded urea hydrolysis only when applied at rates as high as 2,500 or 5,000µg g−1 soil, whereasN-(n-butyl) thiophosphoric triamide (NBPT) (a patented inhibitor of urea hydrolysis in soil) caused substantial retardation of urea hydrolysis when applied at rates as low as 1µg g−1 soil. We also found that ATS had an adverse effect on germination of corn or wheat seeds in soil when applied at the rate of 2,500 or 5,000µg g−1 soil and caused a dramatic reduction of early seedling growth of corn or wheat when applied at the rate of 1,000, 2,500, or 5,000µg g−1 soil. These findings indicate that ATS has little, if any, potential value for retarding hydrolysis of urea fertilizer in soil.

Formation of phosphoryl triamide by decomposition of thiophosphoryl triamide in soil

Thiophosphoryl triamide inhibits soil urease activity when added to soil and is under consideration as a fertilizer amendment for retarding hydrolysis of urea fertilizer by soil urease. Work reported showed that this compound is a poor inhibitor of jackbean urease and that its ability to retard soil urease activity is due to its decomposition in soil with formation of phosphoryl triamide, which is a potent inhibitor of urease activity. This conclusion was supported by studies showing a close relationship between the peak area of the 31P-nuclear magnetic resonance (NMR) signal from phosphoryl triamide in thiophosphoryl triamide solutions that had been incubated with soil for various times and the ability of these solutions to inhibit jackbean urease activity.

Effects of phosphoroamides on ammonia volatilization and nitrite accumulation in soils treated with urea

We compared the effects of N-(n-butyl) thiophosphoric triamide (NBPT), N-(diaminophosphinyl)-cyclohexylamine (DPCA), phenylphosphorodiamidate (PPD), and hydroquinone on transformations of urea N in soils. The ability of these urease inhibitors to retard urea hydrolysis, ammonia volatilization, and nitrite accumulation in soils treated with urea-decreased in the order NBPT > DPCA ≫ PPD > HQ. When five soils were incubated at 30°C for 14 days after treatment with urea (1 mg urea N g−1 soil), on average, the gaseous loss of urea N as ammonia and the accumulation of urea N as nitrite were decreased from 52 to 5 % and from 11 to 1%, respectively, by addition of NBPT at the rate of 10 μg g−1 soil (0.47 parts of NBPT per 100 parts of urea). The data obtained support previous evidence that NBPT is more effective than PPD for reduction of the problems encountered in using urea as a fertilizer and deserves consideration as a fertilizer amendment for retarding hydrolysis of urea fertilizer in soil.

Effects of phosphoroamides on nitrification, denitrification, and mineralization of organic nitrogen in soil

Abstract Recent work has shown that several phosphorodiamides and phosphorotriamides are potent inhibitors of soil urease activity and have potential value for retarding hydrolysis of fertilizer urea in soil. The effects of nine of these compounds on nitrification, denitrification, and mineralization of organic nitrogen in soil were studied by determining the influence of different amounts of each compound on (1) the amounts of (nitrate + nitrite)‐N produced when soils were incubated aerobically after treatment with ammonium sulfate, (2) the amounts of nitrate‐N lost and the amounts of (nitrite + N2O + NO + N2)‐N produced when soils were incubated anaerobically after treatment with potassium nitrate, and (3) the amounts of inorganic N produced when soils were incubated aerobically after treatment with alanine. The compounds studied were phenylphosphorodiamidate, N‐(diamino‐phosphinyl)‐cyclohexylamine, N‐benzyl‐N‐methyl phosphoric triamide, trichloroethyl phosphorodiamidate, diethyl phosphoric triamide, dimethyl phosphoric triamide, N‐butyl phosphorothioic triamide, N‐fluoro‐N‐(diaminophosphinyl)benzamide, and N‐phenylphosphoric triamide. The data obtained showed that only trichloroethyl phosphorodiamidate had a significant inhibitory effect on nitrification when applied at the rate of 5 or 10 μg g‐1 soil and that only trichloroethyl phosphorodiamidate and N‐butyl phosphorothioic triamide had an appreciable inhibitory effect when applied at the rate of 50 or 100 μg g‐1 soil. None of the phosphoroamides studied inhibited denitrification of nitrate or mineralization of organic nitrogen when applied at the rate of 10 or 50 μg g‐1 soil.

Evaluation of N‐butyl phosphorothioic triamide for retardation of urea hydrolysis in soil

Abstract Work reported showed that N‐butyl phosphorothioic triamide (NBPT) is considerably more effective than phenylphosphorodiamidate (PPD) as a soil urease inhibitor and merits consideration as a fertilizer amendment for retarding hydrolysis of urea fertilizer in soil. Studies to determine the factors influencing the effectiveness of NBPT for retardation of urea hydrolysis in soil showed that the inhibitory effect of NBPT on hydrolysis of urea by soil urease increased markedly with the amount of NBPT added and decreased markedly with time and with increase in temperature from 10 to 40°C. They also showed that the ability of NBPT to retard urea hydrolysis in 13 surface soils selected to obtain a wide range in properties was significantly correlated with organic C content (r = ‐0.70**), total N content (r = ‐0.76**), cation‐exchange capacity (r = ‐0.67* ), sand content (r = 0.61*), clay content (r = ‐0.63*), and surface area (r = ‐0.66*), but was not significantly correlated with pH, silt content, urease...