Thermal stability of alpha-amylase from malted jowar (Sorghum bicolor)

Malted cereals are rich sources of alpha-amylase, which catalyzes the random hydrolysis of internal alpha-(1-4)-glycosidic bonds of starch, leading to liquefaction. Amylases play a role in the predigestion of starch, leading to a reduction in the water absorption capacity of the cereal. Among the three cereal amylases (barley, ragi, and jowar), jowar amylase is found to be the most thermostable. The major amylase from malted jowar, a 47 kDa alpha-amylase, purified to homogeneity, is rich in beta structure ( approximately 60%) like other cereal amylases. T(m), the midpoint of thermal inactivation, is found to be 69.6 +/- 0.3 degrees C. Thermal inactivation is found to follow first-order kinetics at pH 4.8, the pH optimum of the enzyme. Activation energy, E(a), is found to be 45.3 +/- 0.2 kcal mol(-)(1). The activation enthalpy (DeltaH), entropy (DeltaS*), and free energy change (DeltaG) are calculated to be 44.6 +/- 0.2 kcal mol(-)(1), 57.1 +/- 0.3 cal mol(-)(1) K(-)(1), and 25.2 +/- 0.2 kcal mol(-)(1), respectively. The thermal stability of the enzyme in the presence of the commonly used food additives NaCl and sucrose has been studied. T(m) is found to decrease to 66.3 +/- 0.3, 58.1 +/- 0.2, and 48.1 +/- 0.5 degrees C, corresponding to the presence of 0.1, 0.5, and 1 M NaCl, respectively. Sucrose acts as a stabilizer; the T(m) value is found to be 77.3 +/- 0.3 degrees C compared to 69.6 +/- 0.3 degrees C in the control.     

Inactivation of heat-resistant pectinmethylesterase from orange by manothermosonication

Pectinmethylesterase of navel oranges shows two fractions greatly differing in thermostability. The most thermostable fraction accounts for approximately 10% of total activity. The thermal inactivation of this fraction follows first-order kinetics both in 5 mM, pH 3.5, citrate buffer and in orange juice at the same pH, showing a z value of 5.1 degrees C and an activation energy (E(a)) of 435 kJ mol(-)(1) K(-)(1). The heat resistance of the enzyme is approximately 25-fold higher in the juice than in citrate buffer. When ascorbic acid, sucrose, glucose, and fructose are added to the citrate buffer at the concentrations found in orange juice, the heat resistance of the enzyme increases 3-fold. The addition of pectin at 0.01% concentration multiplies it by a factor of 50. Manothermosonication (MTS), the simultaneous application of heat and ultrasound under moderate pressure (200 kPa), at 72 degrees C, increases the inactivation rate 25 times in buffer and >400 times in orange juice. MTS inactivation shows a higher z value (35.7 degrees C) and lower E(a) (56.9 kJ mol(-)(1) K(-)(1)) than simple heating.     

Protein binding in deactivation of ferrylmyoglobin by chlorogenate and ascorbate

Kinetics of reduction of iron(IV) in ferrylmyoglobin by chlorogenate in neutral or moderately acidic aqueous solutions (0.16 M NaCl) to yield metmyoglobin was studied using stopped flow absorption spectroscopy. The reaction occurs by direct bimolecular electron transfer with (2.7 +/- 0.3) x 10(3) M(-)(1).s(-)(1) at 25.0 degrees C (DeltaH( )(#) = 59 +/- 6 kJ.mol(-)(1), DeltaS(#) = 15 +/- 22 J. mol(-)(1).K(-)(1)) for protonated ferrylmyoglobin (pK(a) = 4.95) and with 216 +/- 50 M(-)(1).s(-)(1) (DeltaH( )(#) = 73 +/- 8 kJ. mol(-)(1), DeltaS( )(#) = 41 +/- 30 J.mol(-)(1).K(-)(1)) for nonprotonated ferrylmyoglobin in parallel with reduction of a chlorogenate/ferrylmyoglobin complex by a second chlorogenate molecule with (8.6 +/- 1.1) x 10(2) M(-)(1).s(-)(1) (DeltaH( )(#) = 74 +/- 8 kJ.mol(-)(1), DeltaS( )(#) = 59 +/- 28 J.mol(-)(1).K(-)(1)) for protonated ferrylmyoglobin and with 61 +/- 9 M(-)(1).s(-)(1) (DeltaH( )(#) = 82 +/- 12 kJ.mol(-)(1), DeltaS( )(#) = 63 +/- 41 J. mol(-)(1).K(-)(1)) for nonprotonated ferrylmyoglobin. Previously published data on ascorbate reduction of ferrylmyoglobin are reevaluated according to a similar mechanism. For both protonated and nonprotonated ferrylmyoglobin the binding constant of chlorogenate is approximately 300 M(-)(1), and the modulation of ferrylmyoglobin as an oxidant by chlorogenate (or ascorbate) leads to a novel antioxidant interaction for reduction of ferrylmyoglobin by ascorbate in mixtures with chlorogenate.     

ESR/spin probe study of ice cream

Spin probes based on the 1,1,3,3-tetramethylisoindolin-2-yl structure have been used, in conjunction with electron spin resonance spectroscopy (ESR), to study the physical changes occurring in ice cream during freezing and melting. The ESR measurements allowed the rotational correlation times, tau(B), of the spin probes to be determined. Two probes were used together in a given sample of ice cream, namely, 1,1,3,3-tetramethylisoindolin-2-yl (TMIO), which samples the fat phase, and the sodium salt of 1,1,3,3-tetramethylisoindolin-2-yloxyl-5-sulfonate (NaTMIOS), which samples the aqueous phase. Data from the TMIO probe showed that when ice cream is cooled, the fat phase is a mixture of solid and liquid fat until a temperature of approximately -60 degrees C is reached. The water-soluble probe NaTMIOS showed that the aqueous phase changes completely from liquid to solid within 1 degrees C of -18 degrees C. On cooling further to -24.7 degrees C and then allowing it to warm to +25.0 degrees C, the rotational correlation times of the NaTMIOS were slow to recover to their previous values. For the lipid phase, tau(B)(298) was found to be 65.7 +/- 2.0 ps and the corresponding activation enthalpy, DeltaH, was 32.5 +/- 0.9 kJ mol(-)(1): These values are typical of those expected to be found in the type of fat used to make ice cream. The water phase gave corresponding values of 32.2 +/- 0.5 ps and 24.5 +/- 0.4 kJ mol(-)(1) values, which are those expected for a sucrose concentration of 24%.     

Pseudoperoxidase activity of myoglobin: kinetics and mechanism of the peroxidase cycle of myoglobin with H2O2 and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) as substrates

Using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate, it has been shown that the increased peroxidase activity for decreasing pH of myoglobin activated by hydrogen peroxide is due to a protonization of ferrylmyoglobin, MbFe(IV)=O, facilitating electron transfer from the substrate and corresponding to pK(a) approximately 5.2 at 25.0 degrees C and ionic strength 0.16, rather than due to specific acid catalysis. On the basis of stopped flow absorption spectroscopy with detection of the radical cation ABTS(.+), the second-order rate constant and activation parameters for the reaction between MbFe(IV)=O and ABTS were found to have the values k = 698 +/- 32 M(-1) s(-1), DeltaH# = 66 +/- 4 kJ mol(-1), and DeltaS# = 30 +/- 15 J mol(-1) K(-1) at 25.0 degrees C and physiological pH (7.4) and ionic strength (= 0.16 M NaCl). At a lower pH (5.8) corresponding to the conditions in meat, values were found as follows: k = 3.5 +/- 0.3 x 10(4) M(-1) s(-1), DeltaH# = 31 +/- 6 kJ mol(-1), and DeltaS# = -53 +/- 19 J mol(-1) K(-1), indicative of a shift from outersphere electron transfer to an innersphere mechanism. For steady state assay conditions, this shift is paralleled by a shift from saturation kinetics at pH 7.4 to first-order kinetics for H2O2 as substrate at pH 5.8. In contrast, the activation reaction between myoglobin and hydrogen peroxide was found at 25.0 degrees C to be slow and independent of pH with values of 171 +/- 7 and 196 +/- 19 M(-1) s(-1) found at physiological and meat pH, respectively, as determined by sequential stopped flow spectroscopy, from which a lower limit of k = 6 x 10(5) M(-1) s(-1) for the reaction between perferrylmyoglobin, .MbFe(IV)=O, and ABTS could be estimated. As compared to the traditional peroxidase assay, a better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.     

Kinetics of reduction of ferrylmyoglobin by (-)-epigallocatechin gallate and green tea extract

The hypervalent heme pigment ferrylmyoglobin, a potential prooxidant in muscle tissue and meat, is efficiently reduced by epigallocatechin gallate (EGCG) from green tea and by green tea polyphenol extract (GTP) in neutral or moderately acidic aqueous solution (0.16 M NaCl) to yield metmyoglobin in two parallel processes. The second-order rate constant for direct reduction at pH 7.4 and 25 degrees C was found to have the value 1170 +/- 83 M(-1).s(-1) and activation parameters DeltaH(#) = 70.6 +/- 7.2 kJ.mol(-1) and DeltaS(#) = 50.7 +/- 24.1 J.mol(-1).K(-1) for EGCG and the value 2300 +/- 77 M(-1).s(-1) and parameters DeltaH(#) = 60.6 +/- 2.6 kJ.mol(-1) and DeltaS(#) = 23 +/- 9 J.mol(-1).K(-1) for GTP (based on EGCG concentration). For decreasing pH, the rate increased moderately due to a parallel reduction of protonated ferrylmyoglobin. At physiological pH, EGCG is more efficient in deactivating ferrylmyoglobin than other plant phenols investigated, and the relatively high enthalpy and positive entropy of activation suggest an outer-sphere electron transfer mechanism. The interaction between EGCG and other tea catechins in GTP could be responsible for the even stronger ability for GTP to deactivate ferrylmyoglobin.     

Immobilization of recombinant invertase (re-INVB) from Zymomonas mobilis on D-sorbitol cinnamic ester for production of invert sugar

The recombinant invertase (re-INVB) from Zymomonas mobilis was immobilized by adsorption onto the totally cinnamoylated derivative of D-sorbitol. The polymerization and cross-linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of re-INVB on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. Enzyme concentration, immobilization time, and irradiation time were important parameters affecting the immobilization efficiency. The optimum reaction pH of immobilized enzyme was 5, and the optimal reaction temperature was 40 degrees C. The apparent Michaelis constant and the apparent catalytic constant of re-INVB immobilized on the SOTCN derivative acting on sucrose was 78+/-5 mM and 5x10(4)+/-3x10(2) s(-1), respectively, while for the free enzyme, it was 98.0+/-4 mM and 1.2x10(4)+/-2.5x10(2) s(-1), respectively, suggesting a better apparent affinity of the enzyme for the substrate and a better hydrolysis rate when immobilized than when in solution. Immobilized re-INVB also showed good thermal stability and good operational stability (40% of the initial activity remaining after 45 cyles of 1 min duration and 90.6 mg of sucrose being hydrolyzed in 45 min per 2.5 mg of immobilized protein). The results showed that cinnamic carbohydrate esters of D-sorbitol are an appropriate support for re-INVB immobilization and the production of invert sugar.     

Characterization of the temperature activation of pectin methylesterase in green beans and tomatoes

Low-temperature blanching of vegetables activates the enzyme pectin methylesterase (PME), which demethylates cell wall pectins and improves tissue firmness. This temperature activation of PME has been investigated by measuring the formation of methanol in intact tissue of green beans and tomatoes. Rates of methanol formation at temperatures of 35-65 degrees C were obtained by measuring the release of methanol from thin slices of tomato pericarp or green bean pod material. Activation energies of 112 and 97 kJ mol(-1) were calculated for PME activity in green beans and tomatoes, respectively. These activation energies indicate that the rate of pectin demethylation at 65 degrees C will be nearly 100 times that at 25 degrees C. PME activity was also determined titrimetrically using a solubilized form of the enzyme and purified pectin at temperatures from 30 to 60 degrees C. Under these conditions, much lower activation energies of 37 and 35 kJ mol(-1) were obtained for green beans and tomatoes, respectively. Methanol accumulation during heating of whole intact green beans was also determined and yielded an activation energy similar to that obtained with sliced beans. Whole green beans held at room temperature did not accumulate any methanol, but sliced or homogenized beans did. If whole beans were first heated to 45 degrees C and then cooled, methanol accumulation was observed at room temperature. These results indicate that two factors contribute to the observed high rate of pectin de-esterification during low-temperature blanching: (1) An irreversible change, causing PME to become active, occurs by heating to > or = 45 degrees C. (2) The high activation energy for pectin de-esterification means that the rate of de-esterification increases substantially with increasing temperature.     

Kinetics of chlorophyll degradation and color loss in heated broccoli juice

Degradation of chlorophyll in broccoli juice occurred at temperatures exceeding 60 degrees C. Chemical analysis revealed that degradation of chlorophyll a and b to pheophytin a and b, respectively, followed first-order kinetics and that chlorophyll a was more heat sensitive than chlorophyll b. Temperature dependencies of chlorophyll a and b degradation rate constants could be described by Arrhenius equations with activation energies (E(a)) of 71.04 +/- 4.89 and 67.11 +/- 6.82 kJ/mol, respectively. Objective greenness measurements, using the -a value as the physical property, together with a fractional conversion kinetic analysis, indicated that green color degradation followed a two-step process. Kinetic parameters for the first degradation step were in accordance with the kinetic parameters for pheophytinization of the total chlorophyll content, as determined by chemical analysis (E(a) approximately 69 kJ/mol). The second degradation step, that is, the subsequent decomposition of pheophytins, was characterized by an activation energy of 105.49 +/- 4.74 kJ/mol.     

Effect of temperature and moisture on the degradation and sorption of florasulam and 5-hydroxyflorasulam in soil

The degradation rate and sorption characteristics of the triazolopyrimidine sulfonanilide herbicide florasulam and its principal degradation product 5-hydroxyflorasulam (5-OH-florasulam) were determined as a function of temperature and moisture in three different soils. The half-life for degradation of florasulam ranged from 1.0 to 8.5 days at 20-25 degrees C and from 6.4 to 85 days at 5 degrees C. The half-life for degradation of 5-OH-florasulam ranged from 8 to 36 days at 20-25 degrees C and from 43 to 78 days at 5 degrees C. The degradation rate of both compounds was strongly influenced by temperature, with activation energies ranging from 57 to 95 kJ/mol for florasulam and from 27 to 74 kJ/mol for 5-OH florasulam. Soil moisture content had negligible impact on the degradation rate. Apparent (nonequilibrium) sorption coefficients for florasulam and 5-OH-florasulam at 0 days after treatment (DAT) were 0.1-0.6 L/kg and increased linearly with time for both florasulam and 5-OH-florasulam (r(2) > 0.90) to levels as high as 12-23 L/kg. Heats of adsorption were calculated on one soil as a function of time. Heat of adsorption values for both florasulam and 5-OH-florasulam increased as incubation time increased and the amount of each compound decreased; values were near 0 kJ/mol initially and increased to a maximum of 91 and 66 kJ/mol for florasulam and 5-OH-florasulam, respectively.     

Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.     

Kinetics and mechanism of cymoxanil degradation in buffer solutions

The kinetics and mechanism(s) of the hydrolytic degradation of a compound are needed to evaluate a compound's abiotic degradation in the environment. In this paper, the hydrolysis of cymoxanil [2-cyano-N-[(ethylamino)carbonyl]-2-(methoxyimino) acetamide] was investigated in dark sterile aqueous solutions under a variety of pH conditions (pH 2.8-9.2) and temperatures (15-50 degrees C). Hydrolysis of cymoxanil was described by first-order kinetics, which was dependent on pH and temperature. Cymoxanil degraded rapidly at pH 9 (half-life = 31 min) and relatively slowly at pH 2.8 (half-life = 722 days). The effect of temperature on the rate of cymoxanil degradation was characterized using the Arrhenius equation with an estimated energy of activation of 117.1 kJ mol(-)(1). An increase in temperature of 10 degrees C resulted in a decrease in half-life by a factor of approximately 5. Three competing degradation pathways are proposed for the hydrolysis of cymoxanil, with two of the pathways accounting for approximately 90% of cymoxanil degradation. These two pathways involved either initial cyclization to 1-ethyldihydro-6-imino-2,3,5(3H)-pyrimidinetrione-5-(O-methyloxime) (1, Figure 1) or direct cleavage of the C-1 amide bond to form cyano(methoxyimino) acetic acid (7). The third pathway of degradation involved initial cyclization to 3-ethyl-4-(methoxyimino)-2,5-dioxo-4-imidazolidinecarbonitrile (8), which rapidly degrades into 1-ethyl-5-(methoxyimino)-2,4-imidazoline-2,4-dione (9). All three pathways eventually lead to the formation of the polar metabolite oxalic acid.     

Deactivation of ferrylmyoglobin by vanillin as affected by vanillin binding to β-lactoglobulin

Vanillin was found to be efficient as a deactivator of ferrylmyoglobin with a second-order rate constant of k(2) = 57 ± 1 L mol(-1) s(-1) for reduction to metmyoglobin with ΔH(?) = 58.3 ± 0.3 kJ mol(-1) and ΔS(?) = -14 ± 1 J mol(-1) K(-1) in aqueous pH 7.4 solution at 25 °C. Binding to β-lactoglobulin (βLG) was found to affect the reactivity of vanillin at 25 °C only slightly to k(2) = 48 ± 2 L mol(-1) s(-1) (ΔH(?) = 68.4 ± 0.4 kJ mol(-1) and ΔS(?) = 17 ± 1 J mol(-1) K(-1)) for deactivation of ferrylmyoglobin. Binding of vanillin to βLG was found to have a binding stoichiometry vanillin/βLG > 10 with K(A) = 6 × 10(2) L mol(-1) and an apparent total ΔH° of approximately -38 kJ mol(-1) and ΔS° = -55.4 ± 4 J mol(-1) K(-1) at 25 °C and ΔC(p, obs) = -1.02 kJ mol(-1) K(-1) indicative of increasing ordering in the complex, as determined by isothermal titration microcalorimetry. From tryptophan fluorescence quenching for βLG by vanillin, approximately one vanillin was found to bind to each βLG far stronger with K(A) = 5 × 10(4) L mol(-1) and a ΔH° = -10.2 kJ mol(-1) and ΔS° = 55 J mol(-1) K(-1) at 25 °C. The kinetic entropy/enthalpy compensation effect seen for vanillin reactivity by binding to βLG is concluded to relate to the weakly bound vanillin oriented through hydrogen bonds on the βLG surface with the phenolic group pointing toward the solvent, in effect making both ΔH(?) and ΔS(?) more positive. The more strongly bound vanillin capable of tryptophan quenching in the βLG calyx seems less or nonreactive.     

Thermodynamic parameters of beta-lactoglobulin-pectin complexes assessed by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) was used to determine the binding constant, stoichiometry, enthalpy, and entropy of beta-lactoglobulin/low- and high-methoxyl pectin (beta-lg-LM- and HM-pectin) complexes at 22 degrees C and at pH 4. The binding isotherms revealed the formation of soluble intrapolymer complexes (C1) further followed by their aggregation in interpolymer complexes (C2). The interaction between beta-lg and LM- or HM-pectin in C1 and C2 occurred spontaneously with a Gibbs free energy around -10 kcal/mol. The C1 were enthalpically driven, whereas enthalpic and entropic factors were involved in the C2 formation. Because ITC did not allow the dissociation of different enthalpic contributions, the values measured as pectin and beta-lg interacted could partially be attributed to conformational changes. The C1 had a binding stoichiometry of 8.3 and 6.1 beta-lg molecules complexed per LM- or HM-pectin molecule, respectively. The C2 had about 16.5 and 15.1 beta-lg molecules complexed per LM- and HM-pectin, respectively.     

Studies on kinetics and thermostability of a novel acid invertase from Fusarium solani

The present investigation deals with purification and thermal characterization of an acid invertase produced by Fusarium solani in submerged culture. The maximum enzyme activity (9.90 U mL(-1)) was achieved after 96 h of cultivation at pH 5.0 and 30 degrees C in a basal medium containing molasses (2%) as the carbon and energy source supplemented with 1% peptone. Invertase was purified by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephadex G-200. The purified enzyme was proven to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass of the enzyme was 65 kDa. The optimum pH and temperature for activity were 2.6 and 50 degrees C, respectively. The Km value for sucrose was 3.57 mM with an activation energy of 4.056 kJ mol(-1). Enthalpies of activation (DeltaH) were decreased while entropies (DeltaS) of activation increased at higher temperatures. The effects of alpha-chymotrypsin and 4 M urea were tetraphasic with periodic gain and loss of enzyme activity. A possible explanation for the thermal inactivation of invertase at higher temperatures is also discussed.     

Kinetic study of the oxidation of quercetin by mushroom tyrosinase

The kinetic behavior of mushroom tyrosinase in the presence of the flavonol quercetin was studied. This flavonol was oxidized by mushroom tyrosinase and the reaction was followed by recording spectral changes over time. The spectra obtained during the reaction showed two isosbectic points, indicating a stable o-quinone. When quercetin was oxidized by tyrosinase in the presence of cysteine and 3-methyl-2-benzothiazolone hydrazone (Besthorn's hydrazone, MBTH) isosbestic points were also observed indicating a definite stoichiometry. From the data analysis of the initial rate in the presence of MBTH, the kinetic parameters: = (16.2 +/- 0.6) microM/min, = (0.12 +/- 0.01) mM, (/) = (V(max)/K(S)(')()) = (13.5 +/- 1.4) x 10(-)(2) min(-)(1), = (6.2 +/- 0.6) s(-)(1) were determined. We propose that quercetin acts simultaneously as a substrate and a rapid reversible inhibitor of mushroom tyrosinase, depending on how it binds to the copper atom of the enzyme active site. Thus, if the binding occurs through the hydroxylic groups at the C3' and C4' positions, quercetin acts as a substrate, while if it occurs through the hydroxylic group at the C3 position of the pyrone ring, quercetin acts as an inhibitor.     

Purification and characterization of ferulic acid esterase from malted finger millet (Eleusine coracana, Indaf-15)

Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5'-OH group of arabinosyl residues of arabinoxylans and is known to modulate their functional properties. In this study, ferulic acid esterase from 96 h finger millet malt was purified to apparent homogeneity by three-step purification with a recovery of 3% and a fold purification of 22. The substrate p-nitrophenylferulate (PNPF) was synthesized and used to assay this enzyme spectrophotometrically. The products liberated from ragi and wheat water-soluble polysaccharides by the action of purified ragi ferulic acid esterase were identified by ESI-MS. The pH and temperature optima of the enzyme were found to be 6.0 and 45 degrees C, respectively. The pH and temperature stabilities of the enzyme were found to be in the range of 5.5-9.0 and 30 degrees C, respectively. The activation energy of the enzymatic reaction was found to be 4.08 kJ mol(-1). The apparent K m and V max of the purified ferulic acid esterase for PNPF were 0.053 microM and 0.085 unit mL(-1), respectively. The enzyme is a monomer with a molecular mass of 16.5 kDa. Metal ions such as Ni(2+), Zn(2+), Co(2+), and Cu(2+) and oxalic and citric acids enhanced the enzyme activity. The enzyme was completely inhibited by Fe(3+). Group specific reagents such as p-chloromercuric benzoate and iodoacetamide inhibited the enzyme, indicating the possible presence of cysteine residues in the active site pocket.     

Use of differential scanning calorimetry to study lipid oxidation. 1. Oxidative stability of lecithin and linolenic acid

The oxidation of linolenic acid (LNA) and soy lecithin was studied by differential scanning calorimetry (DSC) with linear programmed heating rates (non-isothermal mode). The interpretation of the shape of DSC curves is discussed, and it has been concluded that temperatures of the extrapolated start of heat release are the most reliable data for the rapid estimation of the oxidative stability of lipid materials. The Ozawa-Flynn-Wall method was used to calculate the kinetic parameters of the process: for LNA autoxidation the activation energy, Ea, and pre-exponential factor, Z, are 66 +/- 4 kJ/mol and 1.5 x 10(7) s(-1), respectively, and the autoxidation of lecithin is described by Ea = 98 +/- 6 kJ/mol and Z = 9.1 x 10(10) s(-1). Values of Ea and Z can be applied for calculation of the overall first-order rate constant of autoxidation at various temperatures, k(T). For the two studied lipids the comparison of k(T) values shows the inversion of their oxidative stabilities; that is, below 167 degrees C lecithin is more stable than LNA, k(T)lecithin < k(T)LNA, and above that temperature (termed the isokinetic temperature) k(T)lecithin > k(T)LNA. The calculated inversion of oxidative stabilities can be an explanation of similar observations for other pairs of lipids if the results of accelerated tests measured at temperatures above 100 degrees C are compared with the results obtained at temperatures below 100 degrees C.     

In vitro inhibition of the activation of Pro-matrix Metalloproteinase 1 (Pro-MMP-1) and Pro-matrix metalloproteinase 9 (Pro-MMP-9) by rice and soybean Bowman-Birk inhibitors

The in vitro inhibitory activity of the rice Bowman-Birk inhibitor (rBBI) or soybean Bowman-Birk inhibitor (sBBI) against trypsin-catalyzed activation of pro-matrix metalloproteinase 1 or 9 (pro-MMP-1 or pro-MMP-9), respectively, was investigated using electrophoresis with silver staining, heparin-enhanced zymography, biotinylated gelatin, Biotrak assay, and fluorescence quenched substrate hydrolysis. rBBI at concentrations of 0.08-0.352 mg/mL dose-dependently inhibited the in vitro activation of 45 microg/mL pro-MMP-1 by trypsin. Heparin-enhanced zymography analysis of pro-MMP-1, trypsin-activated MMP-1, and a mixture of pro-MMP-1-trypsin-rBBI showed clear zones associated with trypsin-activated MMP-1 and the absence of clear zones in lanes containing pro-MMP-1 or a mixture of pro-MMP-1, trypsin, and rBBI. The results of the Biotrak assay also indicated that rBBI dose-dependently suppressed the activation of pro-MMP-1 by trypsin. sBBI dose-dependently inhibited the activation of 100 microg/mL of pro-MMP-9 by trypsin. Biotinylated gelatin assays demonstrated that pro-MMP-9 or pro-MMP-9 in the presence of trypsin and BBI did not hydrolyze gelatin, whereas p-aminophenylmercury acetate (APMA)-activated MMP-9 and trypsin-activated MMP-9 caused significant hydrolysis of gelatin. Quenched fluorescence substrate hydrolysis for total MMP activity showed that pro-MMP-1 or pro-MMP-9 did not hydrolyze the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; active MMP-1 or MMP-9 hydrolyzed the substrate, but lower substrate hydrolysis was obtained when pro-MMP-1 or pro-MMP-9 was incubated with trypsin in the presence of increasing concentrations of rBBI. The results are discussed in light of the role of MMP-1 and MMP-9 in the process of angiogenesis and the potential of rBBI or sBBI as a functional food ingredient.     

Kinetics and hydrolysis of fenamiphos, fipronil, and trifluralin in aqueous buffer solutions.

Hydrolyses of fenamiphos, fipronil, and trifluralin were studied in aqueous buffer solutions of pH 4.1, 7.1, and 9.1 at different temperatures, 5, 22 +/- 1, 32 +/- 1, and 50 +/- 1 degrees C. Fenamiphos, fipronil, and trifluralin were found to be more stable in acidic and neutral buffer solutions at temperatures of 5 and 22 +/- 1, and dissipation is rapid at 50 +/- 1 degrees C. In basic buffer and at higher temperature, degradation of fenamiphos was found to be very rapid when compared with fipronil and trifluralin. The rate constants calculated at 32 degrees C for fenamiphos were 2349.4 x 10(-)(8) (pH 4.1), 225.2 x 10(-)(8) (pH 7.1), and 30476.0 x 10(-)(8) (pH 9.1); for fipronil 1750.0 x 10(-)(8) (pH 4.1), 3103.0 x 10(-)(8) (pH 7.1), and 3883.0 x 10(-)(8) (pH 9.1); and for trifluralin 2331.0 x 10(-)(8) (pH 4.1), 2360.0 x 10(-)(8) (pH 7.1), and 3188.0 x 10(-)(8) (pH 9.1). On the basis of rate constant values, these pesticides appeared to be more susceptible to hydrolysis than synthetic organophosphorus compounds such as chlorpyriphos, diazinon, malathion, and ronnel. DT(50) values calculated at 32 degrees C were 228 (pH 4.1), 5310.24 (pH 7.1), and 37.68 (pH 9.1) h for fenamiphos; 608.6 (pH 4.1), 373.9 (pH 7.1), and 270.2 (pH 9.1) h for fipronil; and 502.1 (pH 4.1), 496.8 (pH 7.1), and 355.7 (pH 9.1) h for trifluralin.