Characterization of a salt-independent pectin methylesterase purified from valencia orange peel

The pectin methylesterase (PME; EC 3.1.1.11) present in a commercial orange peel enzyme preparation was characterized to establish its identity among the multiple PME isozymes present in Valencia orange (Citrus sinensis L.) peel. We show the commercial enzyme corresponds to the major peak 2 PME previously separated by heparin-Sepharose chromatography (Cameron et al., J. Food Sci. 1998, 63, 253). Both PMEs have comparable elution profiles on cation-exchange and hydrophobic-interaction perfusion chromatography columns, molecular weights (ca. 34 kDa) and pI (pH 9.2), and biochemical properties, including a broad pH activity range and activity in the absence of added cations. An identical partial amino terminal peptide sequence was also obtained for the PMEs, which further demonstrated a structural identity with other plant PMEs. The biochemical and structural properties readily distinguish this Valencia orange PME from salt-dependent isozymes and further suggest that it is an ortholog to the salt-independent fruit-specific isozyme of tomato. This work provides a well-defined, enzymatically homogeneous, salt-independent (type 1) plant PME isozyme that is suitable for studying details of the enzyme's mode of action and for use in modifying methylester patterns for studying the structure-functional property relationships in pectin.     

Partial purification,characterization, and thermal and high-pressure inactivation of pectin methylesterase from carrots (Daucus carrota L.)

Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model.     

Inactivation of orange pectinesterase by combined high-pressure and -temperature treatments: a kinetic study

Pressure and/or temperature inactivation of orange pectinesterase (PE) was investigated. Thermal inactivation showed a biphasic behavior, indicating the presence of labile and stable fractions of the enzyme. In a first part, the inactivation of the labile fraction was studied in detail. The combined pressure-temperature inactivation of the labile fraction was studied in the pressure range 0.1-900 MPa combined with temperatures from 15 to 65 degrees C. Inactivation in the pressure-temperature domain specified could be accurately described by a first-order fractional conversion model, estimating the inactivation rate constant of the labile fraction and the remaining activity of the stable fraction. Pressure and temperature dependence of the inactivation rate constants of the labile fraction was quantified using the Eyring and Arrhenius relations, respectively. By replacing in the latter equation the pressure-dependent parameters (E(a), k(ref)(T)()) by mathematical expressions, a global model was formulated. This mathematical model could accurately predict the inactivation rate constant of the labile fraction of orange PE as a function of pressure and temperature. In a second part, the stable fraction was studied in more detail. The stable fraction inactivated at temperatures exceeding 75 degrees C. Acidification (pH 3.7) enhanced thermal inactivation of the stable fraction, whereas addition of Ca(2+) ions (1 M) suppressed inactivation. At elevated pressure (up to 900 MPa), an antagonistic effect of pressure and temperature on the inactivation of the stable fraction was observed. The antagonistic effect was more pronounced in the presence of a 1 M CaCl(2) solution as compared to the inactivation in water, whereas it was less pronounced for the inactivation in acid medium.     

Effect of intrinsic and extrinsic factors on the interaction of plant pectin methylesterase and its proteinaceous inhibitor from kiwi fruit

A proteinaceous pectin methylesterase inhibitor (PMEI) was isolated from kiwi fruit (Actinidia chinensiscv. Hayward) and purified by affinity chromatography on a cyanogen bromide (CNBr) Sepharose 4B-orange PME column. The optimal pH of banana PME activity was 7.0, whereas that for carrot and strawberry PME activity was 9.0. The optimal pH for the binding between kiwi fruit PMEI and these PMEs was 7.0. The kiwi fruit PMEI has a different affinity for PME depending on the plant source. The inhibition kinetics of kiwi fruit PMEI to banana and strawberry PME followed a noncompetitive type, whereas that to carrot PME followed a competitive type. The kiwi fruit PMEI was mixed with banana, carrot, and strawberry PME to obtain PMEI-PME complexes, which were then subjected to thermal (40-80 degrees C, atmospheric pressure) or high-pressure (10 degrees C, 100-600 MPa) treatment. Experimental data showed that the PMEI-PME complexes were easily dissociated by both thermal and high-pressure treatments.     

Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.     

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.     

Thermal inactivation of pectin methylesterase,polygalacturonase, and peroxidase in tomato juice

Thermal inactivation kinetics have been determined for pectin methylesterase (PME), polygalacturonase (PG), and peroxidase (POD) in tomato juice. Two parameters, the inactivation rate constant (k) at a reference temperature and the activation energy for inactivation (E(a)), were determined for each enzyme. For PME and PG, the k and E(a) values reported here do not agree with those in several previously published reports. These differences can be explained either by the differences in pH values used for inactivation determinations or by inadequacies in the heating methods used in some previous studies. POD showed simple first-order inactivation kinetics and was less thermally stable than either PME or PG. When different cultivars of tomatoes were evaluated, there was no difference in the thermal inactivation kinetics of these enzymes.     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

Effects of thermal treatments and storage on pectin methylesterase and peroxidase activity in freshly squeezed orange juice

A specific indicator of freshness, allowing routine distinction between freshly squeezed orange juices (FSOJs) and FSOJ-like products, was to be identified. Using the Actijoule unit of a tubular heater at a flow rate of 60 L/h, FSOJs from Citrus sinensis (L.) Osbeck cv. Valencia Late were continuously heated on a pilot plant scale at six different temperatures (42-92 degrees C), followed by continuous cooling to ambient temperature and subsequent filling into sterilized glass jars. The cloud stability and residual activities of pectin methylesterase (PE) and peroxidase (POD) were monitored over the storage at 4 degrees C for up to 62 days, thus considering the storage conditions of FSOJs in retail markets. As shown by the viable microbial counts throughout storage, microbial activity was insignificant due to good sanitary practice, thus proving that the enzyme activities detected were of plant origin. The juices processed at temperatures > or =62 degrees C were characterized by minor residual activities. When exposed to temperatures <62 degrees C in the genuine acidic matrix of the juices, the heat stability of PE exceeded that of POD. Compared with the aforementioned samples, the juice processed at 52 degrees C with a residual PE activity of 33.8% was hardly inferior in terms of cloud stability within the first 14 days. After the juice was processed at 42 degrees C, rapid clarification occurred within the first 8 days, consistent with undetectable PE deactivation. Hence, only the range of approximately 50-60 degrees C is relevant in minimal heat-processing for the retention of cloud stability within the short turnover period of FSOJ-like products, with partial PE and POD deactivation being already sufficient to distinguish those juices from FSOJs. Irrespective of the previous thermal treatment, the total PE activity remained nearly constant during storage, whereas the POD activity rapidly declined to minor levels after 20 days. Consequently, as to the future analysis of samples with unknown processing history, PE was suggested as an indicator enzyme for the freshness of FSOJs, allowing their unambiguous distinction from minimally heat-processed juices.     

Partial purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectin methylesterase (PME) is present in acerola fruit and was partially purified by gel filtration on Sephadex G-100. The results of gel filtration showed different PME isoforms. The total PME (precipitated by 70% salt saturation) and one of these isoforms (fraction from Sephadex G-100 elution) that showed a molecular mass of 15.5 +/- 1.0 kDa were studied. The optimum pH values of both forms were 9.0. The total and the partially purified PME showed that PME specific activity increases with temperature. The total acerola PME retained 13.5% of its specific activity after 90 min of incubation at 98 degrees C. The partially purified acerola (PME isoform) showed 125.5% of its specific activity after 90 min of incubation at 98 degrees C. The K(m) values of the total PME and the partially purified PME isoform were 0.081 and 0.12 mg/mL, respectively. The V(max) values of the total PME and the partially purified PME were 2.92 and 6.21 micromol/min/mL/mg of protein, respectively.     

Cloning and expression of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

Pectin methylesterase (PME) is the key enzyme responsible for the gelation of jelly curd in the water extract of jelly fig (Ficus awkeotasang) achenes. The jelly fig PME extracted from achenes was isoelectrofocused at pH 2.5 and subjected to N-terminal amino acid sequencing. A cDNA fragment encoding the mature protein of this acidic PME was obtained by PCR cloning using a poly(T) primer and a degenerate primer designed according to the N-terminal sequence of the purified PME. The complete cDNA sequence of its precursor protein was further obtained by PCR using the same strategy. The PME clone was overexpressed in Escherichia coli, and its expressed protein was immunologically recognized as strongly as the original antigen using antibodies against purified PME. Fractionation analysis revealed that the overexpressed PME was predominantly present in the pellet and thus presumably formed insoluble inclusion bodies in E. coli cells.     

Isolation, characterization, and pectin-modifying properties of a thermally tolerant pectin methylesterase from Citrus sinensis var. Valencia

The thermally tolerant pectin methylesterase (TT-PME) was isolated as a monocomponent enzyme from sweet orange fruit (Citrus sinensis var. Valencia). It was also isolated from flower and vegetative tissue. The apparent molecular weight of fruit TT-PME was 40800 by SDS-PAGE and the isoelectric point estimated as pI 9.31 by IEF-PAGE. MALDI-TOF MS identified no tryptic-peptide ions from TT-PME characteristic of previously described citrus PMEs. TT-PME did not absolutely require supplemented salt for activity, but salt activation and pH-dependent activity patterns were intermediate to those of thermolabile PMEs. Treatment of non-calcium-sensitive pectin with TT-PME (reducing the degree of methylesterification by 6%) increased the calcium-sensitive pectin ratio from 0.01 to 0.90, indicating a blockwise mode of action. TT-PME produced a significantly lower end-point degree of methylesterification at pH 7.5 than at pH 4.5. Extensive de-esterification with TT-PME did not reduce the pectin molecular weight or z-average radius of gyration, as determined by HPSEC.     

Effect of high-pressure processing at elevated temperatures on thiamin and riboflavin in pork and model systems

High-pressure/high-temperature properties of vitamins in food are important with respect to the new pressure-assisted thermal sterilization method utilizing pressure-induced adiabatic temperature changes. Riboflavin, thiamin, and thiamin monophosphate (TMP) stabilities were assayed in the temperature range from 25 to 100 degrees C under normal pressure (0.1 MPa) and high pressure (600 MPa) in acetate-buffered (pH 5.5) model solutions, some with added fructose, hemoglobin, or ascorbic acid. Thiamin and riboflavin stabilities were also assayed in minced fresh pork fillet and in rehydrated pork reference material with and without pressure treatment at 600 MPa in the temperature range from 20 to 100 degrees C. In pork, the vitamins proved to be sufficiently stabile for high-pressure/high-temperature processing. Under similar conditions, vitamin decay in model solutions was up to 30 times faster, especially that of TMP. Thus, it appears that it may not be possible to draw conclusions for the pressure behavior of real food matrices from the results of investigations in food models. A further consequence is that caution is necessary when supplementing foods with synthetic B vitamins preceding high-pressure/high-temperature processing.     

Purification and glycosylation analysis of an acidic pectin methylesterase in jelly fig (Ficus awkeotsang) achenes

An acidic pectin methylesterase (PME) is responsible for the gelation of water extract from jelly fig (Ficus awkeotasang) achenes. A new, fast and efficient, method has been developed to purify this acidic PME. The method includes preparing jelly curd by traditional hand washing, extracting proteins from the curd, and separating PME by anion-exchanger. The purified PME exists as a monomer of 38 kDa determined by gel filtration, and exerts enzymatic activity over a broad pH range, particularly in acidic environments where most known PME enzymes from various species are inactivated. Chemical staining and enzymatic cleavage suggest that the jelly fig PME is an N-linked glycoprotein. Fluorophore-assisted carbohydrate electrophoresis reveals that the polysaccharide of this glycoprotein putatively consists of 22 hexoses including 16 mannose, 4 N-acetylglucosamine, and 2 galactose residues.     

Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk

Changes in the activity and structure of alkaline phosphatase (ALP) and L-lactate dehydrogenase (LDH) were investigated after high pressure processing (HPP). HPP treatments (206-620 MPa for 6 and 12 min) were applied to ALP and LDH prepared in buffer, fat-free milk, and 2% fat milk. Enzyme activities were measured using enzymatic assays, and changes in structure were investigated using far-ultraviolet circular dichroism (CD) spectroscopy and dynamic light scattetering (DLS). Kinetic data indicated that the activity of ALP was not affected after 6 min of pressure treatments (206-620 MPa), regardless of the medium in which the enzyme was prepared. Increasing the processing time to 12 min did significantly reduce the activity of ALP at 620 MPa (P < 0.001). However, even the lowest HPP treatment of 206 MPa induced a reduction in LDH activity, and the course of reduction increased with HPP treatment until complete inactivation at 482, 515, and 620 MPa. CD data demonstrated a partial change in the secondary structure of ALP at 620 MPa, whereas the structure of LDH showed gradual denaturation after exposure at 206 MPa for 6 min, leading to a random coil structure at both 515 and 620 MPa. DLS results indicated aggregation of ALP only at HPP treatment of 206 MPa and not above and enzyme precipitation as well as aggregation at 345, 415, 482, and 515 MPa. The loss of LDH activity with increasing pressure and time treatment was due to the combined effects of denaturation and aggregation.     

Effect of cooking on banana and plantain texture

The effect of temperature and duration of cooking on plantain and banana fruit texture and cytpoplasmic and cell wall components was investigated. The firmness of both banana and plantain pulp tissues decreased rapidly during the first 10 min of cooking in water above 70 degrees C, although plantain was much firmer than banana. Cooking resulted in pectin solubilzation and middle lamella dissolution leading to cell wall separation (as observed by SEM). Dessert banana showed more advanced and extensive breakdown than plantain. Although dessert banana had a higher total pectin content than plantain, the former had smaller-sized carboxyethylenediaminetetraacetic acid (CDTA) soluble pectic polymers which are associated with plant tissues that have a propensity to soften. Plantain had higher levels of starch and amylose than banana but this was associated with a firmer fruit texture rather than a softening due to cell swelling during starch gelatinization. Different cooking treatments showed that cooking in 0.5% of CaCl(2) solution and temperatures below 70 degrees C had significant effects on maintenance of pulp firmness.     

Photodegradation of the acaricide abamectin: a kinetic study

The acaricide abamectin is a mixture of two colorless homologues in a molar ratio of at least 4:1 with the same structure of macrocyclic lactone. The kinetics of its degradation under direct (254 nm) and dye-sensitized (>400 nm) photoirradiation in methanol solution has been studied by UV-vis spectrophotometry, potentiometric detection of dissolved oxygen, stationary fluorescence, laser flash photolysis, and time-resolved detection of singlet molecular oxygen (O2((1)Delta(g))) phosphorescence. The results indicate that the degradation is very efficient under direct irradiation with UV light (254 nm), with a quantum yield of 0.23. On the contrary, under visible-light irradiation, using the natural pigment riboflavin or the synthetic dye rose bengal as sensitizers, the degradation is very inefficient and proceeds through a O2((1)Delta(g))-mediated mechanism, with a bimolecular rate constant for the overall O2((1)Delta(g)) quenching (the sum of physical and chemical quenching) of 5.5 x 10(5) M(-1) s(-1). This value is similar to those reported for the rate constants of the reactions of O2((1)Delta(g)) with isolated double bonds or conjugated dienes and points to similar processes in the case of abamectin.     

Enzymatic modification of a model homogalacturonan with the thermally tolerant pectin methylesterase from Citrus: 1. Nanostructural characterization, enzyme mode of action, and effect of pH

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ～10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).     

Inactivation of soybean trypsin inhibitors and lipoxygenase by high-pressure processing

Trypsin inhibitors (TIA), one of the antinutritional factors of soy milk, are usually inactivated by heat treatment. In the current study, high-pressure processing (HPP) was evaluated as an alternative for the inactivation of TIA in soy milk. Moreover, the effect of HPP on lipoxygenase (LOX) in whole soybeans and soy milk was studied. For complete LOX inactivation either very high pressures (800 MPa) or a combined temperature/pressure treatment (60 degrees C/600 MPa) was needed. Pressure inactivation of TIA was possible only in combination with elevated temperatures. For TIA inactivation, three process parameters, temperature, time, and pressure, were optimized using experimental design and response surface methodology. A 90% TIA inactivation with treatment times of <2 min can be reached at temperatures between 77 and 90 degrees C and pressures between 750 and 525 MPa.     

Effect of high-pressure treatment on lipoxygenase activity

Solutions of commercial soybean lipoxygenase (100 microgram/ML in 0.2 M citrate-phosphate and 0.2 M Tris buffer were subjected to pressures of 0.1, 200, 400, and 600 MPa for 20 mm. The enzyme was stable at atmospheric pressure (0.1 MPa) over a wide pH range (5-9). In citrate phosphate buffer, the enzyme had maximum stability over the pH range 58 in untreated samples and after treatment at 200 MPa, but with increasing pressure, the pH stability range become narrower and centered around pH 78. The enzyme was more sensitive to acid than alkali, and at pH 9, it lost virtually all activity after pressurization at 600 MPa for 20 mm in both buffers. The activity of the crude enzyme extracted from tomatoes treated at 200 and 300 MPa for 10 mm was not significantly different from that of the untreated tomatoes, while a pressure of 400 MPa for 10 mm caused a significant decrease in activity and treatment at 600 MPa led to complete and irreversible activity loss. Compared to unpressurized tomatoes, treatment at 600 MPa gave significantly reduced levels of hexanal, cis-3-hexenal, and trans-2-hexenal, which are important contributors to "fresh" tomato flavor, and this was attributed to the inactivation of lipoxygenase.