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At least 883 records · Page 49Linked to original sources
PubMed2006

Separation of pectin methylesterases and polygalacturonases on monolithic columns.

The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; M(r) 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with M(r) 82 kDa) and a basic protein with M(r) 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with M(r) 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH(3)CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.

Carboxylic Ester Hydrolases↗
PubMed1992

Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonas marginalis N6301.

A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonas marginalis N6301 was cloned and expressed in Escherichia coli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2. 1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5 alpha. A plasmid thus obtained was designated as pPNL6301. E. coli DH5 alpha harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pn1 gene in the plasmid pPNL6301 encoding PNL from P. marginalis N6301 was determined. The structural gene of pn1 consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P. marginalis N6301 determined. The nucleotide sequence of the 5'-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichia coli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pn1 gene between P. marginalis N6301 and E. carotovora Er were 60.8% and 57.2%, respectively.

Amino Acid Sequence↗
PubMed2002

Studies on the glycosylation of wild-type and mutant forms of Aspergillus niger pectin methylesterase.

Pectin methylesterase (PME) is one of a number of enzymes released by the fungus Aspergillus niger that are involved in the degradation of specific plant cell-wall structures. PME is a glycoprotein with three potential sites for N-linked glycosylation. The glycosylation may affect the hydrolytic activity or the substrate specificity of PME. In this work, we investigate first the structures and the attachment sites of the glycans present on recombinant wild-type PME. Further, a series of PME mutants was created in which the three potential N-linked glycosylation sites were eliminated in all possible combinations. The glycosylation of the mutants and their activities were then studied. Mass spectrometric techniques tailored for carbohydrate analysis were applied to both characterize the glycan structures and to determine the specific sites of attachment. High mannose structures with variable numbers of mannose were found on the wild-type, as well as the mutant forms. Studies using the mutants suggest that glycosylation does not strongly influence the activity. Whether it may affect the substrate specify of the enzyme is unknown, and that aspect will be explored in future work.

Amino Acid Sequence↗
PubMed2003

Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis.

Pectin methylesterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell wall. Previous work indicated that plant PMEs may be subject to post-translational regulation. Here, we report the analysis of two proteinaceous inhibitors of PME in Arabidopsis thaliana (AtPMEI1 and 2). The functional analysis of recombinant AtPMEI1 and 2 proteins revealed that both proteins are able to inhibit PME activity from flowers and siliques. Quantitative RT-PCR analysis indicated that expression of AtPMEI1 and 2 mRNAs is tightly regulated during plant development with highest mRNA levels in flowers. Promotor::GUS fusions demonstrated that expression is mostly restricted to pollen.

Arabidopsis↗
PubMed1999

Pectin and psyllium decrease the susceptibility of LDL to oxidation in guinea pigs.

These studies were undertaken to determine whether pectin (PE) and psyllium (PSY) intake affect the circulating levels of alpha-tocopherol and the susceptibility of low density lipoprotein (LDL) to oxidation. For that purpose, male Hartley guinea pigs were fed 19 g/100 g of a fat mix with a 2:1:1 ratio of saturated:polyunsaturated:monounsaturated fatty acids and 35 g/100 g total carbohydrate with 80% of the carbohydrate energy contributed by sucrose. Diets were identical in composition except for the fiber source: cellulose (control diet), PE, or PSY. Guinea pigs fed PE or PSY had 36% and 67% lower plasma cholesterol concentrations, respectively, compared with controls (P < 0.001). This plasma cholesterol lowering was associated with both very low density lipoproteins and LDL cholesterol fractions. Intake of PE or PSY resulted in 54% lower plasma triacylglycerol (TAG) concentrations compared with the control group (P < 0.001). LDL from PE and PSY fed guinea pigs contained fewer molecules of cholesteryl ester, and alpha-tocopherol concentrations in this particle were 49% and 66% higher, respectively, compared with controls. In addition, LDL from guinea pigs fed soluble fiber exhibited less susceptibility to oxidation than those from the control group, as determined by thiobarbituric acid-reactive substances formation. Hepatic free and esterified cholesterol were 32% lower and hepatic TAG was 25% lower in guinea pigs fed PE and PSY compared with controls. The data from these studies confirm that PE and PSY reverse the hyperlipidemia associated with high fat-sucrose diets and demonstrate a potential antioxidant effect of soluble fiber on circulating LDL.

Journal Article↗
PubMedDate not supplied

Strawberry pectin methylesterase (PME): purification, characterization, thermal and high-pressure inactivation.

Pectin methylesterase (PME) was extracted from strawberries (Fragaria ananassa, cv Elsanta) 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 molecular mass, pI, and kinetic parameters of strawberry 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 strawberry PME could be described by a fractional-conversion model. Purified strawberry PME is much more stable toward high-pressure treatments in comparison to those from oranges and bananas.

Carboxylic Ester Hydrolases↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2001

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.

Bioreactors↗
PubMed2002

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.

Acetylglucosamine↗
PubMed2002

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.

Carboxylic Ester Hydrolases↗
PubMed2002

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.

Carboxylic Ester Hydrolases↗
PubMed2002

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.

Beverages↗
PubMed2003

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.

Carboxylic Ester Hydrolases↗
PubMed2004

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.

Carboxylic Ester Hydrolases↗
PubMed2004

Pectin methylesterase from kiwi and kaki fruits: purification, characterization, and role of pH in the enzyme regulation and interaction with the kiwi proteinaceous inhibitor.

Pectin methylesterase was purified from kiwi (Actinidia chinensis) and kaki fruit (Diospyros kaki). The pH values of the fruit homogenates were 3.5 and 6.2, respectively. The kiwi enzyme is localized in the cell wall and has a neutral-alkaline pI, whereas the kaki enzyme is localized in the soluble fraction and has a neutral-acidic pI. The molecular weights of the kiwi and kaki enzymes were 50 and 37 kDa, respectively. The two enzymes showed a similar salt and pH dependence of activity, and a different pH dependence of the inhibition by the kiwi proteinaceous inhibitor.

Actinidia↗
PubMed2005

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.

Carboxylic Ester Hydrolases↗
PubMed2003

The plant invertase inhibitor shares structural properties and disulfide bridges arrangement with the pectin methylesterase inhibitor.

Attempts to purify the inhibitor of pectin methylesterase (PMEI) from the soluble extract of ripe apricot (Prunus armeniaca) fruit led to isolation of a protein (Pa-INH) similar to PMEI, but having invertase inhibitory activity against vacuolar invertase from tomato. The molecular charge, the native and SDS-PAGE molecular weights were similar to those of PMEI. Partial amino acid sequence indicated a high level of identity with invertase inhibitors and a significant identity with PMEI. Circular dichroism analysis showed a mainly alpha-helix secondary structure for both the inhibitors and a higher thermostability of Pa-INH. Four Cys residues forming disulfide bridges in PMEI were conserved in Pa-INH. Similarly to PMEI, these residues were linked by disulfide bridges (first to second and third to fourth). The free Cys139 of PMEI is substituted by Ala in Pa-INH. The results reported in this study suggest a common structural arrangement of the two inhibitors.

Amino Acid Sequence↗
PubMed2003

Partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of Aspergillus niger.

Investigations on the mode of action of Aspergillus niger pectin methylesterase (PME) towards differently C(6)- and C(1)-substituted oligogalacturonides (oligoGal p A) are described. De-esterification of methyl-esterified (un)saturated oligoGal p A proceeds via a specific pattern, depending on the degree of polymerization. Initially, a first methyl ester of the oligomer is hydrolysed, resulting in one free carboxyl group. Subsequently, this first product is preferred as a substrate and is de-esterified for a second time. This product is then accumulated and hereafter de-esterified further to the final product, i.e. oligoGal p A containing one methyl ester located at the non-reducing end residue for both saturated and unsaturated oligoGal p A, as found by post-source decay matrix-assisted laser-desorption/ionization-time-of-flight MS. The saturated hexamer is an exception to this: three methyl esters are removed very rapidly, instead of two methyl esters. When unsaturated oligoGal p A were used, the formation of the end product differed slightly, suggesting that the unsaturated bond at the non-reducing end influences the de-esterification process. In vivo, PME prefers methyl esters, but the enzyme appeared to be tolerant for other C(6)- and C(1)-substituents. Changing the type of ester (ethyl esterification) or addition of a methyl glycoside (C(1)) only reduced the activity or had no effect respectively. The specific product pattern was identical for all methyl- and ethyl-esterified oligoGal p A and methyl-glycosidated oligoGal p A, which strongly indicates that one or perhaps two non-esterified oligoGal p A are preferred in the active-site cleft.

Aspergillus niger↗