Characterization Of Pectin Methylesterase Research Articles

Characterization of Pectin Methylesterase from Aspergillus niger

The present study was undertaken to evaluate the characteristics of pectin methylesterase produced from Aspergillus niger. The enzyme had a molecular weight of 59.668 kDa. The enzyme exhibited maximum activity at 45°C. The enzyme lost most of its initial activity after 20 min of incubation at 75°C. Optimal activity and stability of the enzyme occurred at pH 4, with more than 90 % of retained activity after 10 min at pH 5. Michaelis constant (Km) was found equal to (1) mg/ml, whereas, maximum velocity (Vmax) was (1000) μmol/min. The catalyzed reaction by pectin methylesterase had activation energy equal to 9.66 Kcal/mol. The enzyme lost 24% and 13% of its initial activity during two months of storage at4°C and -20 °C, respectively. The enzyme was activated by NaCl, KCl and CaCl2 at concentration of 25 mM and inhibited by MgSO4, polygalacturonic acid, methanol, IAA and EDTA.

Extraction and characterization of pectin methylesterase from muskmelon biowaste for pectin remodeling.

Pectin methylesterase (PME) extracted from muskmelon was purified by anion exchange chromatography. The specific activity of purified enzyme was 152.01U/mg and relative molecular weight was ~69,000Da. Methylesterase was characterized for various physicochemical factors to designate its suitability in the food industry applications. The optimum temperature of the enzyme was 30°C and is thermally stable between the temperature ranges of 15-65°C with critical temperature for stability being >65°C. Thermal inactivation first order kinetics and thermodynamic parameters in temperature range (45-65°C) favors stability of PME and at 75°C complete inactivation of enzyme was observed indicating the unstable nature of enzyme over >65°C. Activation energy (Ea ) and Z values of thermal inactivation were found to be 100.108kJ/mol and 2.05°C, respectively. About 0.1M NaCl is essential for enzyme to attain the maximum activity. The enzyme lost activity in presence of divalent calcium (Ca2+ ) and magnesium (Mg2+ ) ions. PRACTICAL APPLICATIONS: Pectin methylesterase (EC3.1.1.11) are an important class of enzymes expressed in plants and microbes and they bring about the de-methylesterification on pectin substrate. Up to ~13% degree of esterification of pectin was observed with muskmelon PME enzyme treatment. The de-methylesterified pectin thus prepared was subjected for gelation in presence of Ca2+ ions and above 0.5% of demethylesterified pectin stable calcium pectate gels were produced. The study demonstrates the suitability of muskmelon PME extracted from biowaste in food applications with good gelling property.

Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum.

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2kDa on SDS-PAGE gels. The molecular mass, 35.5kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5mg/mL). CtPME showed maximum activity (18.1U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50°C, respectively. The enzyme was stable in pH range 8.0-9.0 and thermostable between 45 and 70°C. CtPME activity was increased by 40% by 5mM Ca2+ or Mg2+ ions. Protein melting curve of CtPME gave a peak at 80°C. The peak was shifted to 85°C in the presence of 5mM Ca2+ ions, and the addition of 5mM EDTA shifted back the melting peak to 80°C. CtPME can be potentially used in food and textile industry applications.

Extraction and characterization of pectin methylesterase from Alyanak apricot (Prunus armeniaca L).

This study was carried out to determine some of the biochemical properties of pectin methylesterase (PME) from Alyanak apricot which is an important variety grown in Malatya region of Turkey. The enzyme had high activity in a pH range of 7.0-8.0 with the maximal activity occurring at pH7.5. However, the enzyme activity at high and low pH values was very low. The optimum temperature for maximal PME activity was found to be 60°C. The activity of PME has been enhanced by NaCl, particularly at 0.15M. Km and Vmax values for Alyanak apricot PME using apple pectin as substrate were found to be 1.69mg/mL (r(2) = 0.992) and 3.41units/mL, respectively. The enzyme was stable at 30-45°C/10min whereas it lost nearly all of its activity at 80°C/10min. Ea and Z values were found to be 206.1kJ/mol (r(2) = 0.993) and 10.62°C (r(2) = 0.992), respectively.

Characterization of pectin methylesterase and polygalacturonase extracted from fresh-cut melons

Characterization of pectin methylesterase and polygalacturonase extracted from fresh-cut melons

Partial purification and characterization of pectinmethylesterase from ripening guava (Psidium guajava L.) fruits

Employing the techniques of (NH4)2SO4 fractionation, ion exchange chromatography on DEAE cellulose and gel filtration through Sephadex G-100, pectinmethylesterase (EC 3.1.1.11) was purified from guava (Psidium guajava L.) fruits var. Hisar Safeda harvested at turning stage of maturity to 129-fold with 28% recovery. Molecular weight as determined by gel filtration was found to be 51 kDa and the enzyme preparation exhibited the same molecular weight under native (Native-PAGE) and denaturating conditions (SDS-PAGE) indicating that the enzyme was a monomer. With pectin as the substrate, it exhibited the Michaelis Menten kinetics with Km value of 3.1 g l−1. The enzyme was found to be stimulated by Ca++ and Na+ and inhibited competitively by d-galacturonic acid with Ki value of 1.97 mM. The enzyme was completely inactivated by iodine while with diethyl pyrocarbonate and N-acetylimidazole, the enzyme was inhibited up to the extent of 56 and 45%, respectively. However, DTNB had no inhibitory effect whatsoever precluding the participation of any –SH group in the active centre. It is tentatively proposed that the enzyme has tyrosine and histidine residues at its active centre.

Purification and characterization of pectinmethylesterase from Aspergillus repens isolated from cultivated soil

Aspergillus repens isolated from cultivated soils released pectinmethylesterase (PME) into the liquid culture medium during growth. The enzyme preparation was partially purified by ammonium sulphate precipitation and dialysed. The ammonium sulphate-dialysate fraction of the enzyme was separated by molecular exclusion and ion exchange chromatography. The molecular weight of the enzyme was found to be 141,300 daltons. The optimum temperature for PME activity was 30oC and most active at pH 6.5. The activity of the enzyme was stimulated by Na+, K+, Ca2+, Mg2+ and Zn2+, while EDTA, PbCl2, HgCl2 and IAA inhibited enzyme activity. The activity of the enzyme increased with increase in substrate concentration reaching maximum at 4 mg/ml. The Lineweaver-Burk plot for the hydrolysis of pectin indicated approximately 1.3 mg/ml. These qualities could be explored during the industrial applications of this enzyme.

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

AbstractThe enzyme pectinmethylesterase (PME) from acerola was extracted and purified by gel anion‐exchange chromatography (Q Sepharose) and filtration on Sephadex G‐100. The results showed two different PME isoforms (PME1 and PME2), with molecular masses of 25.10 and 5.20 kDa, respectively. PME1 specific activity increased by 9.63% after 60 min incubation at 98 °C, while PME2 retained 66% of its specific activity under the same conditions. The Km values of PME1, PME2 and concentrated PME were 0.94, 0.08 and 0.08 mg mL−1, respectively. The Vmax value of PME1, PME2 and concentrated were 204.08, 2, 158.73 and 2.92 µmol min−1 mg−1 protein, respectively. Copyright © 2007 Society of Chemical Industry

Partial Purification and Characterization of Pectin Methylesterase from Green Beans (PhaseolusvulgarisL.)

Pectin methylesterase (PE) is present in green beans and may have an influence on the texture of green beans after commercial sterilization. Two fractions of PE were partially purified from the pods and the seed hulls of green beans (cv. Odessa). PE-1 and PE-2 had molecular weights of 45 and 29 kD, respectively, under native conditions and of 42 and 33.5 kD under denaturating conditions. PE-1 and PE-2 both contained two isoforms of PE activity with isoelectric points of 8.4 and 9.8 and 10.5 and >11, respectively. The active fractions showed a difference in salt dependency, and PE-2 was less heat stable than PE-1. Keywords: Isoforms; pectin methylesterase; Phaseolus vulgaris; purification; texture

Isolation and Characterization of Pectin Methylesterase from Papaya

Pectin methylesterase (PME) (EC 3.1.1.11) has been purified to apparent homogeneity from ripe papaya fruits. The purification protocol consisted of ammonium sulphate precipitation (60-80%) and cation exchange chromatography in CM Sepharose CL-6B and Mono S. Papaya PME consists of two components (PME 1 and PME 2), which have been shown to be isoenzymes by Ferguson plot analysis. The molecular weight of the enzyme is 27,000 while its isoelectric point is greater than pH 9.0. The N-terminal sequences of PME 1 and PME 2 are SVVTPNAVVADDGVFXFKTG. Both PME 1 and PME 2 showed optimum activities at pH 8.0 and 35°C. The average K m s of PME 1 and PME 2 are 0.0071 and 0.0166 g/liter pectin respectively, while the corresponding average V maxs are 741 and 800 μmol methanol/min/mg protein, respectively. Papaya pectin methylesterase is activated by cations, but the effect is more pronounced for divalent than monovalent cations. Inhibition studies showed that sucrose is a noncompetitive inhibitor while p-chloromercuribenzoic acid has no significant effect on its activity.

Purification and Characterization of Pectinmethylesterase from Ficus awkeotsang Makino Achenes

Pectinmethylesterase from the pericarp of jelly fig (Ficus awkeotsang) achenes was extracted and purified to a specific activity of 289 micromole proton produced per minute per milligram protein. Pectinmethylesterase, a major protein with high specific activity in the crude extract, was monomeric with a molecular weight of 38,000. The enzyme preparation was stable in distilled water at 4 degrees C for at least 6 months, and at 60 degrees C for at least 10 minutes. This enzyme functioned optimally at pH 6.5 to 7.5 when the assay mixture contained no NaCl or at low NaCl concentration. The pH optimum shifted to lower pH as the NaCl concentration was increased. The K(m) value for pectin was 0.75 milligram per milliliter pectin, corresponding to a V(max) value of 310 micromoles per minute per milligram protein. Inhibition studies with antibodies indicated that jelly fig achene pectinmethylesterase and the two other pectinmethylesterases from orange and tomato were similar in their active site conformation; however, the surface determinants may be very different because no precipitation between anti-jelly fig pectinmethylesterase immune serum and the pectin methylesterase from orange and tomato could be observed in the double immunodiffusion analysis. Specific antisera raised against jelly fig achene pectinmethylesterase in a Western blot experiment also showed low similarity between jelly fig pectinmethylesterase with that from orange and tomato. This observation was also supported by the very low isoelectric point (pH 3.5) of jelly fig pectinmethylesterase, compared with high isoelectric points reported for most of the pectinmethylesterases. Amino acid composition and N-terminal sequence have been obtained. High homology of the N-terminal amino acid residues between jelly fig and tomato pectinmethylesterase (O Markovic, H Jornvall [1986] Eur J Biochem 158: 455-462) was observed. Pectinmethylesterase activity causes the release of protons from the deesterification of pectin such that a low pH environment is created, and this may be related to the cell growth. Pectinmethylesterase is not needed for jelly fig seed germination, however the gel formed from pectin and pectinmethylesterase may insure a water source for the germinating jelly fig seeds.

Isolation and Characterization of Pectin Methylesterase from Apple Fruit

ABSTRACTTwo forms of the enzyme pectin methylesterase are evidenced in the apple (Malus communis). They differ both in their charge and molecular weight. The two enzymes were separated by DEAE‐cellulose chromatography. Their molecular weights, determined by gel‐filtration, were 55000 and 28000 daltons. The heavier form has been purified at homogeneity and subjected to investigations regarding its activity as a function of the pH and temperature, and determination of its kinetic parameters. The enzyme has a Km value of 1.05 mg/mL for citrus pectin and an optimum activity in the pH range between 6.5‐7.5. The enzyme was stable up to 40°C. Incubation for 1 min at 90°C leads to its complete inactivation.