Purification and properties of the major pectinesterases in lemon fruits (Citrus limon)

Pectinesterase from lemon was separated into seven fractions by chromatography on CM-Sephadex C-50. Purification was hindered by the presence of pectin, for which the pectinesterase had a high affinity. Two major pectinesterases were purified: one was located solely in the peel and the other in the endocarp. They possessed similar molecular weights (35 kDa and 33 kDa, respectively) and high isoelectric points (> 11, and c 9). They required the presence of cations for optimal activity, the peel pectinesterase requiring a higher concentration of cations than the endocarp enzyme. Both enzymes were completely inactivated at temperatures above 88°C. Although neither enzyme destabilised the cloud of lemon juice or of comminuted lemon beverages, they caused cloud destabilisation of orange juice.     

Purification and Properties of Apple Pectinesterase

Pectinesterase from apple was separated into two fractions by chromatography on Sephadex G-75. The major pectinesterase was purified by cation-exchange chromatography on CM-Sephadex C-50. It had a molecular mass of 36 kDa, and isoelectric point of about 9, similar to one of the major pectin-esterases of lemon. The enzyme required the presence of cations for optimum activity and was completely inactive at temperatures above 75°C. Although the major pectinesterase had no effect on the cloud stability of apple juice, a fraction containing a minor pectinesterase caused cloud clarification.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF PEACH PECTINESTERASE

Pectinesterase (EC 3.1.1.11) was extracted from peaches (Prunus persica) and partially purified by preparative free solution isoelectric focusing. On SDS-PAGE gels, protein bands at 36.3 and 33.9 kilodaltons represented the major bands; minor bands were observed at 108.4, 40.7, and 17.0 kilodaltons. The pH optimum for pectinesterase activity in the partially purified extract was 8.0. The enzyme was stable at 30°C for 30 min between pH values of 5 and 8. Peach pectinesterase is stable when heated at 55°C for 5 min in 0.1 M NaCl, 50 mM sodium phosphate, pH 7, buffer. However, residual activity decreased to 23% of 65°C for 5 min and was inactivated at 70°C for 5 min. The energy of activation of peach pectinesterase was determined to be 34, 600 J/mol °K. The Q₁₀ between 30°C and 60°C was estimated to be 1.5–1.6.     

Purification and characterisation of carrot (Daucus carota L) pectinesterase

A pectinesterase isoform with an alkaline isoelectric point of over 8.66 was detected in crude extracts of carrot. The enzyme was purified by ion exchange and molecular exclusion chromatography. The molecular weight of the isoform was 25 kDa, determined in native conditions by filtration through Sephadex G‐75 SF. The enzyme showed a high affinity for its substrate, with K_m and V_max values of 0.031 mg ml^?1 and 6.77 units respectively for apple pectin. The pectinesterase activity exhibited an optimum around pH 7.4 and was activated by metallic ions, with optimum activities at NaCl concentrations between 130 and 330 mM and at CaCl₂ concentrations between 15 and 50 mM . The enzyme was activated most by Ca²⁺ and exhibited a greater tolerance of high concentrations of Na⁺. Comparison of its heat stability with other pectinesterases of vegetable origin indicated that the purified isoform was very thermolabile, being rendered inactive by heating for 5 min at 70 °C. The enzyme was inhibited by high concentrations of polygalacturonic acid and competitively inhibited by D ‐galacturonic acid, with a K_i value of 1 mM . Copyright © 2003 Society of Chemical Industry     

PECTINESTERASE-CATALYZED FIRMING EFFECTS DURING PRECOOKING OF VEGETABLES

Many vegetables exhibit a firming effect after precooking at a temperature between 50 and 70C. This effect has generally been attributed to the action of endogenous pectinesterase which hydrolyzes the methyl ester linkages in pectin molecules. The resulting free carboxyl groups then form Ca-bridges between pectin molecules. We have shown, by using the pectinesterases of pea sprouts, that the enzymes catalyzed not only the hydrolysis of the methoxyl groups of pectin molecules, but also a tranacylation reaction of the galacturonic acyl groups from methanol to other hydroxyl groups of pectin. The latter reaction results in the formation of new ester linkages between pectin molecules, which also contributes to the firming of the tissue. The pectinesterases have been separated into four isozymes, PE1, PE2, PE3, and PE5. The isozymes exhibited similar transacylation activity, with the exception of PE5 that did not catalyze transacylation.     

Influence of precooking on the firmness and pectic substances of three stem vegetables

Edible portions of the stems of sprouting broccoli, asparagus lettuce, and large-stem mustard were compared for total pectin contents, amounts of different pectin fractions, pectinesterase activities and changes during cooking to investigate effects on the textural changes during cooking. Slices precooked for 30min at temperatures below 60°C (broccoli) or 70°C (lettuce, mustard) were firmer after 15 min recooking in boiling water than those directly cooked without precooking. Optimum temperatures for this firming effect of precooking were 50, 60 and 60°C, respectively, and coincided with the optimum temperatures of activity of pectinesterases extracted from the fresh tissues. Analysis of pectin fractions revealed that the firming effect of precooking is related to the shift from the cold water-soluble fraction to sodium hexametaphosphate-soluble and hot water-soluble fractions of pectins.     

Pectinesterase Activity, Pectin Methylation, and Texture Changes During Storage of Blanched Cucumber Slices

Pectin methylation in blanched cucumber slices after 6 months’storage in acid brine (pH 3.7) ranged from 9% (no blanch) to 48% (99°C, 3 min blanch). An 81°C blanch caused complete pectinesterase inactivation, but 15 - 20% reactivation occurred during storage. After a 99°C blanch, only slight reactivation was observed. Pectinesterase was not inactivated at 66°C or less, but up to 85% of the activity was lost during storage. Firmness changes were complex. A clear relationship between pectin methylation and firmness changes was not observed. A 66 or 81°C blanch resulted in best firmness retention. Calcium ion was very effective in prevention of firmness loss regardless of the extent of pectin methylation.     

The effect of processing on the texture of canned mung bean (Phaseolus aureus) shoots

The effect of various processing regimes on the pectic substances and the final texture of canned mung bean shoots has been studied. A blanching temperature of 75°C for 30 s was optimal in activating the native pectinesterase of the shoots and a holding temperature of 55°C for an optimal 30 min led to maximal deesterification of pectin. This treatment resulted in a canned product that was superior to a sample blanched at 100°C.; the addition of calcium ions did not improve the product.     

Purification and properties of pectinesterase from papaya

Pectinesterase (PE) was partially purified from papaya pulp, and its biochemical properties were studied. The enzyme was eluted in a single peak after DEAE-cellulose and Sephadex G-100 chromatography. The PE had a molecular weight of 53000 and showed an optimum pH of 8.0. Its activity was dependent on an NaCl concentration of 0.2M . The enzyme was heat stable: approximately 80% of the original activity remained after 60 min of heating at 50°C but completely inactivated by incubation at 80°C for 1 min. The activity was linear with time and protein concentration. The maximum reaction in 3 min was found at 60°C and the initial rate increased 9-fold from 20 to 60°C. The estimated K_m was 0.12g litre^?1 with citrus pectin as the substrate. The kinetic study revealed that polygalactur-onic acid is a competitive inhibitor, and a K_i value of 0.07 g litre^?1 was determined. On the basis of this study, papaya PE properties resembled those of pectinesterase from other sources.     

Thermal and Calcium Pretreatment Affects Texture, Pectinesterase and Pectic Substances of Frozen Sweet Cherries

Freezing causes loss of turgidity and firmness in sweet cherries. Thermal pretreatment at 50°C for 10 min followed by immersion in 100 mM CaCl₂ and thermal pretreatment at 70°C/2 min with or without immersion in 100 mM CaCl₂ prevented freezing-induced loss of firmness. Thermal pretreatments increased the pectin fraction soluble in EDTA, reduced the degree of pectin esterification, and increased both the concentration of divalent cations in the cell wall and the pectinesterase activity bounded to the cell wall. Immersion in CaCl₂ increased the concentration of Ca²⁺ cations in the cell wall and enhanced the effect of thermal pretreatments on pectinesterase activity.     

Microbial and enzymatic changes in natural soursop puree during storage

Microbial and enzymatic comparisons were made on natural soursop puree stored at 15, 4 and −20°C without pasteurisation and with pasteurisation at 79°C for 69 s. Results showed that pasteurisation caused significant decrease in microbial count in soursop puree from 6.4×10³ to 8.5×10¹ cfu per g. Generally, storage at −20°C exhibited greater stability of samples as compared to those kept at 4 and 15°C. In contrast to microbial reduction, pasteurisation caused significant increase in cloud from 0.318 to 0.529 and completely inactivated the pectinesterase enzyme. The puree packed into foils kept at 4°C exhibited decreased cloud loss as compared to others packed in cans and bottles kept at 15 and −20°C. No pectinesterase enzyme regeneration was observed for pasteurised puree in this study during 12 weeks of storage time.     

Heat Inactivation of Pectinesterase in Orange Juice Pulp

Heat inactivation of pectinesterase in juice pulp was nonlinear suggesting the presence of fractions of pectinesterase with differing heat stabilities. The residual activity rapidly decreased to 4% upon heating for 19 sec at 80°C and decreased very little upon subsequent heating for up to 180 sec at 80°C. The D values (time to inactivate 90% of the enzyme) at 90°C of the heat sensitive and heat stable fractions were estimated to be 0.225 and 32 sec, respectively. The Z values (change in temperature to achieve a 10-fold change in time of inactivation) were estimated to be 10.8°C and 6.5°C for the sensitive and stable fractions, respectively.     

Changes in physico-chemical properties of pectin from jelly fig (Ficus awkeotsang Makino) seeds during extraction and gelling

Degree of esterification of pectin from jelly fig (Ficus awkeotsang Makino) achene seeds and the pH value decreased rapidly during extraction, while apparent reduction of free calcium content in the pectin extract was observed at the gelling stage. Compared to those of the native pectin, total ester linkages and methyl ester linkages of pectin extract decreased, and the bound calcium content increased during pectin gelling. However, non-methyl ester linkages (the difference between the total ester linkage and the methyl ester linkage) increased by approximately 40% during pectin gelling, revealing esterification reaction between C₆ carboxyl groups and hydroxyl groups in the presence of pectinesterase. Scanning electron microscopy showed that pectin fragments from jelly curds were large with flake-like structure, while those from hot (85°C) ethanol-treated achenes were small and porous.     

Activity,localisation and thermal inactivation of deteriorative enzymes in Australian carrot (Daucus carota L) varieties

Four Australian carrot varieties have been selected for a study of deteriorative enzymes. The varieties included those commonly used for table and also for processing purposes (Red Hot Original, Red Count, Top Pak and Red Hot Carotene 100). The activities of three deteriorative enzymes (peroxidase, catechol oxidase and pectinesterase) have been assayed in juices prepared from whole carrot as well as superficial tissues, core, root tip and stem end. The levels and relative distribution vary for the different enzymes and varieties studied. Thermal inactivation was assessed over a range of temperatures. Catechol oxidase was found to be the least stable and pectinesterase the most stable in each of the varieties. In most cases, effective inactivation was achieved within 2 min at 85 °C. The enzymes of Top Pak variety showed greater thermal stability. In this variety, pectinesterase required treatment at 90 °C to ensure rapid inactivation. It is concluded that pectinesterase should be used as the indicator enzyme in the assessment of blanching sufficiency for processing of carrots. © 1999 Society of Chemical Industry     

Temperature–time relationships for thermal inactivation of pectinesterases in orange juice

The first‐order kinetic model of a two‐component system was applied to calculate the necessary parameters using the commercial Mathcad program package during the experimental thermal pasteurisation of Valencia orange juice. Thermostable and thermolabile pectinesterases, accounting for 6.6 and 93.4% of the total enzyme population in the juice respectively, showed a 5.63 °C Z_s value and a 9.54 °C Z_l value respectively from the thermal destruction curves at temperatures ranging from 75 to 90 °C. The calculated Z_s values were consistent with the literature values for Valencia orange juice. The calculated and experimental non‐linear thermal pasteurisation curves for orange juice were also in good agreement at temperatures between 75 and 90 °C. © 2003 Society of Chemical Industry     

Some Pectinolytic and Cellulolytic Enzyme Activities of Fungi Causing Rots of Cocoyams

Representative isolates of Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum and F solani recovered from rotten cocoyams were studied for the production of pectinolytic and cellulolytic enzymes. All the isolates elaborated high levels of hydrolase, lyase and pectinesterase in cocoyam tissue medium and lyase and pectinesterase in pectin medium. There were no significant differences in the overall levels of lyase and pectinesterase activities produced by all the isolates in both media. The level of hydrolase, lyase and pectinesterase activities individually produced by A niger, B theobromae and C rolfsii in both media was significantly higher than that of any other isolate. The highest hydrolase activity was produced by C rolfsii in cocoyam medium while A niger produced the highest lyase activity in pectin medium. Maximum pectinesterase activity was obtained from B theobromae and C rolfsii in pectin medium. All the test isolates produced cellulase in a medium containing carboxymethyl cellulose with C rolfsii showing significantly high activity followed by F oxysporum and A niger. © 1997 SCI.     

Fractionation of fungal pectic enzymes by immobilised metal ion affinity chromatography

A juice-clarifying fraction that will not produce methanol was obtained from a commercial pectic enzyme by separating the pectin lyase (EC4.2.2.10) from pectinesterase (EC3.1.1.11). This was achieved by immobilised metal ion affinity chromatography (IMAC) on Sepharose—iminodiacetic acid—Cu(II). Pectin lyase did not bind to the chromatographic matrix at pH 8.0, while pectinesterase was retained and only eluted when the pH of the buffer was brought down to 3.0. Polygalacturonase activity (EC3.2.1.15) was found in both fractions thus suggesting that IMAC can discriminate between two forms of that enzyme Both fractions have the ability to clarify apple juice, the first without producing methanol. The behaviour of each of the above enzymes in IMAC suggests that pectin lyase lacks accessible histidine residues, while pectinesterase could have one or more of them.     

Interactions of (β-Lactoglobulin and High-methoxyl Pectins in Acidified Systems

The interactions that occur when β-lactoglobulin (β-lg) is mixed with a high-methoxyl pectin (HMP) and a modified pectin (mHMP, modified using plant pectinesterase) were examined at pH 3.8. Whereas soluble aggregates formed in β-lg-HMP, β-lg-mHMP precipitated upon mixing. β-lg-HMP mixtures showed soluble aggregates with larger hydrodynamic diameters when heated at 65°C than when heated at 90°C. β-lg-HMP mixtures adjusted to pH 6.0 after heating showed that the aggregates formed at 65°C could be dissociated, but the complexes were not reversible after heating at 90°C. A similar effect also was observed when resuspending the°-lg-mHMP precipitates at pH 6.0. The behavior of the 2 pectins was attributed to their differences in charge distribution.     

A study of factors affecting in-situ De-esterification of mango (Mangifera indica) pectin

De-esterification of mango peel pectin in situ by the action of the enzyme pectinesterase (PE) has been investigated. The rate of enzymic deesterification was highest between pH 8.5 and 9.5 with chemical deesterification also being important. In-situ PE activity declined with fruit ripening but remained relatively constant when assayed on citrus pectin. The most likely explanation for this difference is a change in suitability of the pectin substrate within the fruit during ripening. A reduction in the degree of esterification (DE) of the pectin to 36-42% was obtained following incubation of the mango peels at pH 8.5 for 90 min. A crude preparation of exogenous PE from mango peel and lime pulp was incubated at pH 8.5 with heat-treated (PE inactive) peel as a means of increasing the rate and extent of de-esterification. The PE activity of both these exogenous enzyme preparations was lower on the peel than that of the in-situ mango enzyme, but considerably higher on a hot-water-soluble fraction extracted from the peel. It is suggested that enzyme solubility and pectin accessibility are the major factors affecting in-situ de-esterification of pectin in mango peel.     

Pressure Induced Inactivation of Selected Food Enzymes

Pectinesterase, lipase, polyphenol oxidase, lipoxygenase, peroxidase, lactoperoxidase, phosphatase and catalase have been examined at distinct conditions within a pressure range of 0.1 to 900 MPa, temperatures from 25°C to 60°C, pH 3 to 7, and time of treatment of 2 min to 45 min. Results in model buffers made it possible to rank the enzymes according to their pressure induced inactivation in the following order: lipoxygenase, lactoperoxidase, pectinesterase, lipase, phosphatase, catalase, polyphenol oxidase, peroxidase. A combination of pressure with moderate temperature increased the degree of enzyme inactivation. Pressure treatment of real food systems showed a protective effect of food ingredients on the pressure inactivation of most enzymes evaluated. For example sucrose protected pectinesterase from inactivation by pressure while lactoperoxidase and lipoxigenase were as stable in milk as in buffer.