Functional expression in pichia pastoris of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

A cDNA fragment encoding an acidic pectin methylesterase (PME) of jelly fig achene was successfully expressed in Pichia pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase promoter. The recombinant PME was produced as a secretory protein by N-terminal fusion of a cleavable prepropeptide for signal trafficking, and thus easily harvested from the culture medium. Compared with native N-glycosylated PME (38 kDa) purified from jelly fig achenes, this recombinant PME (45 kDa) appeared to be hyperglycosylated. Activity staining indicated that the recombinant PME was functionally active. Yet the hyperglycosylated recombinant PME possessed thermostability and enzymatic capability over a broad pH range equivalent to those of the native PME. The success of functional production of this acidic jelly fig PME in P. pastoris has significantly broadened its applications in industry.     

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.     

Characterization of pectinesterase inhibitor in jelly fig (Ficus awkeotsang Makino) achenes

Pectinesterase inhibitor (PEI) extract prepared from intact jelly fig (Ficus awkeotsang Makino) achenes was separated by membrane (MWCO 3 and 10 kDa) and fractionated by a Sepharose G-50 gel permeation chromatography. Results from Sepharose G-50 gel permeation chromatography and concanavalin A Sepharose chromatography revealed PEI as polypeptides with molecular weights ranging from 3.5 to 4.5 kDa. Incubation of a PE (1 unit/mL)-PEI (2 mg/mL) mixture for 1 min decreased the PE activity by approximately 50%. On the basis of the results of Lineweaver-Burk double-reciprocal plots, Michaelis constant (K(m)) and V(max) values for jelly fig achenes PE (pH 6.0, 30 degrees C) were 0.78 mM -OCH3 and 1.09 microequiv of -COOH/min, respectively. In addition, PEI competitively inhibited both citrus and jelly fig achenes PEs.     

Purification, cloning, and identification of two thaumatin-like protein isoforms in jelly fig (Ficus awkeotsang) Achenes

Jelly curd used for a popular summer drink in Taiwan is prepared by extracting the pericarpial portion of jelly fig (Ficus awkeotsang Makino) achenes. The two most abundant proteins found in jelly curd have been identified as a pectin methylesterase and a chitinase. A method was developed to purify the next abundant protein by 40% ammonium sulfate precipitation and flowing through Mono Q chromatography. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the purified protein migrated as a polypeptide of 20 kDa in the absence of beta-mercaptoethanol but split into a minor polypeptide of 20 kDa and a major polypeptide of 27 kDa in the presence of this reducing agent. Two cDNA fragments encoding precursor polypeptides of two putative thaumatin-like protein isoforms were obtained by polymerase chain reaction cloning and subsequently overexpressed in Escherichia coli to generate recombinant proteins for antibody preparations. Immunological detection and mass spectrometric analyses indicated that the two split polypeptides were thaumatin-like protein isoforms encoded by the two cloned cDNA fragments.     

Pectinesterase inhibitor from jelly-fig (Ficus awkeotsang Makino) achenes reduces methanol content in carambola wine

Crude pectinesterase (PE) inhibitor (PEI) extracted from jelly-fig achenes (JFA) (Ficus awakeosang Makino) was added to carambola (Averrhoa carambola L.) puree to determine the change in methanol production during fermentation. Addition of pectin or microbial pectic enzyme to puree increased dose-dependently the methanol content in fermented products. Decreasing ratio (from 1:0 to 1:19, v:v) of pectic enzyme to diluted crude PEI solution in the puree-enzyme mixture decreased the PE activity remarkably. Except for transmittance (%T), addition of crude PEI to puree did not affect apparently the physical and chemical properties of wine; however, it reduced methanol content in the control from 256 to 58 ppm. The degree of esterification (DE) of pectin in starting puree was approximately 70%. It decreased to approximately 27% in the control group and reduced slightly to approximately 67% in fermented puree with crude PEI added after 14 days of fermentation. This reveals that crude PEI solution was potent in inhibiting intrinsic carambola PE activity and appeared to be a potential alternative for methanol reduction in wines.     

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.     

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.     

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.     

Tomato (Lycopersicon esculentum) pectin methylesterase and polygalacturonase behaviors regarding heat- and pressure-induced inactivation.

The combined high pressure/thermal (HP/T) inactivation of tomato pectin methyl esterase (PME) and polygalacturonase (PG) was investigated as a possible alternative to thermal processing classically used for enzyme inactivation. The temperature and pressure ranges tested were from 60 degrees C to 105 degrees C, and from 0.1 to 800 MPa, respectively. PME, a heat-labile enzyme at ambient pressure, is dramatically stabilized against thermal denaturation at pressures above atmospheric and up to 500-600 MPa. PG, however, is very resistant to thermal denaturation at 0.1 MPa, but quickly and easily inactivated by combinations of moderate temperatures and pressures. Selective inactivation of either PME or PG was achieved by choosing proper combinations of P and T. The inactivation kinetics of these enzymes was measured and described mathematically over the investigated portion of the P/T plane. Whereas medium composition and salinity had little influence on the inactivation rates, PME was found less sensitive to both heat and pressure when pH was raised above its physiological value. PG, on the other hand, became more labile at higher pH values. The results are discussed in terms of isoenzymes and other physicochemical features of PME and PG.     

Nutrition of fig (Ficus carica L.) under hydroponics and greenhouse conditions

In this investigation the extraction curve of macronutrients (N, P, K, Ca, Mg) and micronutrients (Fe, Cu, Zn and Mn) were determined in the cultivation of fig. A system of intensive production of fig in greenhouse and hydroponics was established with 1.25 plants m^?2. The determination of the nitrogen content was done by the micro-Kjeldahl method. The P was by the yellow molybdovanadate method throughon a spectrophotometer. The K was determined by flamometry and the Ca, Mg, Fe, Cu, Zn and Mn were determined by atomic absorption spectrophotometry. Of the organs analyzed, the stem was the that accumulated more dry matter, then, the leaves and finally the fruits. The nutrient extraction dynamics presented similar upward behavior in all nutrients. The demand for macronutrients in decreasing order was N?>?K?>?P?>?Ca?>?Mg and for the micronutrients Cu?>?Fe?>?Mn?>?Zn.     

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.     

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.     

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.     

Seasonal and diurnal photosynthetic behaviour of fig (Ficus carica L.) under semi-arid climatic conditions

Abstract

Diurnal and seasonal variations in net photosynthetic (P_N) and transpiration (E) rates and water use efficiency (WUE) of the fig tree (Ficus carica L.) were investigated under semi-arid climatic conditions. The two types of leaves from southern and northern parts of trees experienced natural air temperature and irradiance conditions, but differ particularly in water use efficiency. The obtained data reveal that leaf temperature, because of decrease in stomatal conductance, is the major factor limiting the gas exchange capacity of fig trees grown under rain-fed conditions. Stomatal conductance is the major control mechanism, particularly in the northern parts of the trees; however, P_N was most probably decreased by both stomatal and non-stomatal resistance mechanisms such as photoinhibition under severe drought and high irradiance conditions in the southern parts of the trees.     

Antioxidant activities and anthocyanin content of fresh fruits of common fig (Ficus carica L.)

Fig fruit has been a typical component in the health-promoting Mediterranean diet for millennia. To study the potential health-promoting constituents of fig fruits, six commercial fig varieties differing in color (black, red, yellow, and green) were analyzed for total polyphenols, total flavonoids, antioxidant capacity, and amount and profile of anthocyanins. Using reversed-phase liquid chromatography (RP-LC), various concentrations of anthocyanins but a similar profile was found in all varieties studied. Hydrolysis revealed cyanidin as the major aglycon. Proton and carbon NMR confirmed cyanidin-3-O-rhamnoglucoside (cyanidin-3-O-rutinoside; C3R) as the main anthocyanin in all fruits. Color appearance of fig extract correlated well with total polyphenols, flavonoids, anthocyanins, and antioxidant capacity. Extracts of darker varieties showed higher contents of phytochemicals compared to lighter colored varieties. Fruit skins contributed most of the above phytochemicals and antioxidant activity compared to the fruit pulp. Antioxidant capacity correlated well with the amounts of polyphenols and anthocyanins (R2 = 0.985 and 0.992, respectively). In the dark-colored Mission and the red Brown-Turkey varieties, the anthocyanin fraction contributed 36 and 28% of the total antioxidant capacity, respectively. C3R contributed 92% of the total antioxidant capacity of the anthocyanin fraction. Fruits of the Mission variety contained the highest levels of polyphenols, flavonoids, and anthocyanins and exhibited the highest antioxidant capacity.     

Effect of mild-heat and high-pressure processing on banana pectin methylesterase: a kinetic study

Pectin methylesterase (PME) was extracted from bananas and purified by affinity chromatography. The thermal-high-pressure inactivation (at moderate temperature, 30-76 degrees C, in combination with high pressure, 0.1-900 MPa) of PME was investigated in a model system at pH 7.0. Under these conditions, the stable fraction was not inactivated and isobaric-isothermal inactivation followed a fractional-conversion model. At lower pressure (< or =300-400 MPa) and higher temperature (> or =64 degrees C), an antagonistic effect of pressure and heat was observed. Third-degree polynomial models (derived from the thermodynamic model) were successfully used to describe the heat-pressure dependence of the inactivation rate constants.     

Cloning and expression of desoxyhemigossypol-6-O-methyltransferase from cotton (Gossypium barbadense)

Terpenoids play an important role in defense against insects and pathogens in cotton. These terpenoids contain phenolic groups. Metabolites in which the phenolic group has been converted to a methoxy group are less toxic to most insects and pathogens and thus may alter resistance. Here is reported the cloning of a gene from Gossypium barbadense that encodes the enzyme that methylates the phenolic group of desoxyhemigossypol (dHG) exclusively at the 6-position, dHG-6-O-methyltransferase (dHG-6-OMT). Partial peptide sequences from digests of purified dHG-6-OMT were used to design primers for RT-PCR amplification of cDNA fragments from poly(A) mRNA. Fragments were extended to full length using 5' and 3' RACE. The resulting cDNA codes for a 365-residue polypeptide with a calculated molecular weight of 40.6 kDa, in agreement with the molecular mass of purified dHG-6-OMT. When expressed in Escherichia coli, the bacterial lysates showed a high specificity for the methylation of desoxyhemigossypol, differentiating the cloned gene from other pathogen-induced methyltransferases.     

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 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.