Pectinesterase Activity Research Articles

Molecular cloning and sequencing of a pectinesterase gene from Pseudomonas solanacearum

Two pectinesterase-positive Escherichia coli clones, differing in expression levels, were isolated from a genomic library of Pseudomonas solanacearum. Both clones contained a common DNA fragment which included the pectinesterase-encoding region. The different expression levels found with the two clones could be ascribed to different positioning of the pectinesterase gene with respect to a vector promoter. Restriction analysis, subcloning, and further exonuclease deletion mapping revealed that the genetic information for pectinesterase was located within a 1.3 kb fragment. A protein of 41 to 42 kDa was expressed from this fragment. Nucleotide sequence analysis of the respective region disclosed an open reading frame of 1188 bp. The deduced polypeptide had a calculated molecular mass of 41,004 Da, which is consistent with the determined size of the pectinesterase protein. The predicted amino acid sequence showed significant homology to pectinesterases from Erwinia chrysanthemi and tomato. In cultures of E. coli clones up to 30% of total pectinesterase activity was transported into the medium. However, no significant pectinesterase activity could be detected in the periplasm.

Characteristics of pectinesterase isolated from bramley apple waste

AbstractPectinesterase was isolated from Bramley apple waste (peel, cores and offcuts) and its characteristics were determined. Optimum pectinesterase activity was obtained between pH 7.0 and 9.5 and activity was still detectable at 5.0. The enzyme was stable over a wide pH range in the presence of substrate, with over 90% of the activity retained after 5 min at pH 2.5. Pectinesterase activity increased with temperature up to 60°. Activation energy was calculated at 32 kJ mol−1 K−1 between 20 and 50°. Optimum activity was obtained in sodium chloride concentrations between 0.025 and 0.20 M with activity declining at higher salt concentrations. A Km of 0.044% apple pectin and a Vmax of 9.52 units g−1 DM were obtained for one specific enzyme extract.

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

SummaryEdible 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.

Partial characterization of the in situ activity of pectinesterase in Bramley apple

SummaryThe characteristics of pectinesterase (PE) have been examined in waste material (peel, cores and offcuts) obtained from Bramley seedling apples barn stored for 2, 4 and 12 weeks. A crude enzyme extract was prepared by suspending the dried and milled apple waste in 0.1 m NaCl at pH 8.5. The activity of PE under standard assay conditions of 0.5% apple pectin, 0.1 m NaCl, pH 8.5 and 30°C was low, between 4 and 8 units g−1 dry matter, but activities up to 60 units g−1 dry matter were obtained at higher temperatures. The optimum temperature was 60°C with the enzyme stable up to 40°C with 5 min heating. The mean activation energy for PE in the three samples was calculated at 39.2 kJ mol−1 K−1. The optimum pH was high at 10.0 probably due to the PE assay measuring the extraction/solubilization and stability of the enzyme in addition to its activity. Optimum activity was obtained in 0.15 m NaCl with optimum stability at 0.5 m.

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.

The Tomato Fruit Cell Wall

A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat-inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue.

Detection of pectinesterase and polygalacturonase from salivary secretions of living greenbugs, Schizaphis graminum (Homoptera: Aphididae)

Pectinesterase (EC 3.1.1.11) and polygalacturonase (EC 3.1.1.15) were successfully detected in induced salivary secretions of living greenbugs, Schizaphis graminum (Rondani). The method involved the construction of agarose-pectin gel plates on which the aphids were allowed to feed through Parafilm, followed by staining of the gels with ruthenium red. The specific activities of pectinesterase and polygalacturonase were detected by formation of dark red circular and clear halos, respectively, around the site of stylet sheaths left by the aphids. The assay indicates the saliva of biotypes E and C contains both enzymes. The method combines a living, feeding organism with an enzyme assay system in a unique and simple way and provides conclusive evidence for the nature of secreted materials. It also provides an opportunity to investigate the formation of stylet sheaths by this and other aphid species. The toxicity of the enzymes to host plants and the importance of the enzymes to greenbug feeding are discussed.

Extracellular Enzymes of Colletotrichum nicotianae Causing Tobacco Leaf Anthracnose in Nigeria

AbstractBioassays for extracellular enzyme production were carried out in cultures of Colletotrichum nicotianae and in tobacco leaves infected with the same organism. Pectinolytic and cellulolytic enzymes were produced abundantly in culture and in diseased leaves. The activity of pectinesterase was highest at 40 °C and about pH 6. The greatest activity of carboxylcellulase was between 40° and 50 °C and at pH 6. These findings are discussed in relation to the probable roles of these enzymes in pathogenicity.

INFLUENCE of GLUCONIC ACID ON THERMAL PROCESSING REQUIREMENTS FOR CANNED WHOLE PEELED TOMATOES

Methods of detecting the activities of endogenous tomato pectin esterase and polygalacturonase were developed and used to measure heat resistance parameters for enzymes in tomato homogenates. Addition of 0.44% gluconic acid reduced heat resistance of pectin esterase which is responsible for gel formation in canned whole peeled tomatoes. However, heat inactivation of tomato polygalacturonase, which is responsible for general degradation of texture/consistency in tomato products, was not significantly affected by addition of up to 1.65% by wt gluconic acid. Pectic enzymes in canned whole peeled tomatoes packed in juice in 303 × 406 cans were inactivated in 35 min at 98.9 and 101.7°C, 26 min at 110.0°C or 25 min at 118.3°C.

Pectin Lyase Production by a Penicillium italicum Strain

Growth and concomitant production of an extracellular pectin lyase (PL) [poly(methoxylgalactosiduronate) endolyase; EC 4.2.2.10] were investigated in a group of 16 fungi grown in liquid medium containing pectin as a supplementary carbon source. Culture filtrates of both Penicillium italicum (CECT 2294) and P. expansum (CECT 2275) showed the highest PL activity and contained polygalacturonase but not pectinesterase activity. The effect of the inoculum size, the carbon source (sucrose and glucose syrup), and the presence of pectin on the production of PL by P. italicum was studied. The presence of 2.6 mM glycerophosphate in the culture medium enhanced the appearance of PL but was not inhibitory for the in vitro activity. However, glycerol inhibited the enzyme nearly 50% at such a concentration.

In‐situ pectin de‐esterification of alkali‐treated fruit waste materials

AbstractWaste peel and pulp materials from orange, lime and mango fruits were treated with sodium carbonate which increased the pH and resulted in the de‐esterification of pectin in situ. Chemical and enzymic (pectinesterase) de‐esterification occurred in each of the treated fruit waste materials. A specific carbonate enhancement of pectinesterase activity which was clearly demonstrated in orange peel was found to a lesser extent in lime pulp but not at all in mango peel. During treatment of the dispersions with Na2CO3, each of the three fruit waste materials gelled. The pectin degree of esterification of the orange and lime dispersions was below 50% (low methoxyl pectin) on gelling but the mango peel dispersions gelled at pectin degree of esterification levels above 50% although the gelation mechanism was that of a low DE pectin. The degree of esterification of the soluble pectin in the mango dispersion was, however, found to be very much lower than the overall pectin degree of esterification determined. It is suggested that in‐situ pectinesterase activity is restricted to the soluble pectin in mango peel whereas in the citrus residues both soluble and bound pectin are de‐esterified.

Amidated pectins—characterization and enzymatic degradation

Partially amidated pectins with degrees of amidation between 10 and 40% are prepared by de-esterification with ammonia in the heterogeneous phase. The mol. wt distribution monitored by gel permeation chromatography (GPC) has been hardly changed by this procedure. From measurements of intrinsic viscosities of GPC fractions it is concluded that amidated groups have no effect on molecular shape in solution (ionic strength 0.1). The Mark-Houwink equation for pectins obtained formerly [ η ] = 0.0955 M w 0.73 [η] = 0.0955 M w 0.73 can also be applied to partially amidated pectins. In ion exchange chromatography the fractionation depends only on the share of free carboxyl groups in the pectin molecules. Both amidated and esterified carboxyl groups in the units of the pectin chain are detected in a similar manner, they are not discriminated. At the beginning of enzymatic hydrolysis the activity of polygalacturonase is not essentially reduced by amidated groups if pectinesterase activity is sufficient. Hydrolysis of amidated pectins with an excess of pectolytic enzymes and long reaction times is restricted to the amount of non-amidated groups. No long-range effects of amidated units are observed.

Solubilization of Cell Wall Bound, Thermostable Pectinesterase from Valencia Orange

ABSTRACTComplete solubilization of citrus pectinesterase is not effected by alkaline salt buffers but is partially enhanced after degradation of the cell wall by pectinase and cellulase. After heating at 70°C for 2 min, the pectinesterase fractions solubilized after enzymatic cell wall degradation possessed three times the residual activity of the pectinesterase fractions solubilized by alkaline salt buffer. Isoelectric focusing prints of pectinesterase activity and SDS polyacrylamide gel electrophoresis suggest increased solubilization of a heat stable pectinesterase isozyme after cell wall degradation.

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

AbstractDe‐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.

Role of pectolytic enzymes in the development of woolliness in peaches

SummaryThe role of pectolytic enzymes in the development of woolliness in peaches (Prunus persica, cv. Peregrine), stored at different temperatures, was studied. All peaches became woolly during a ripening period of 12 days at 10°C following storage at 2°C, whereas 75% of those stored at –0.6°C developed this disorder. Only 6% of fruit showed symptoms of woolliness after 48 h at 23°C prior to storage. The sugar content, as well as the viscosity of soluble pectin, of peaches stored after the delay period reached a maximum during the 15th day of cold storage, after which it decreased until the end of the ripening period. In peaches stored immediately, however, the maximum sugar content and viscosity was reached during the ripening period. Pectinesterase (PE) activity fluctuated between the different treatments and its possible role in the development of woolliness is not clear. The polygalacturonase (PG) activity of those peaches which became woolly after being stored directly at either temperature was markedly lower over the first 21 days’ storage than peaches which were initially subjected to delay before storage. A sharp increase in PG-activity of the peaches which developed woolliness was observed during the ripening period.

Effect of Assay Temperature on Activity of Citrus Pectinesterase In Fresh Orange Juice

ABSTRACTThe effect of temperature on pectinesterase activity in crude enzyme extract from Valencia pulp was determined. The effect is best described by the regression equation: LN PEU = 14.3446 −3415 (1/T) with a correlation coefficient of 0.991. The activation energy was determined to be 28,392 joules/mol. This regression equation was used to predict the enzyme activities of different juice samples and verified by assay of Valencia juice at various temperatures. Duncan analysis indicated no significant differences in the predicted pectinesterase activities of the concentration‐corrected crude extract and collective means of the Valencia juices at the respective temperature.

Biochemistry of papaya (Carica papayaL.) fruit ripening: changes in RNA, DNA, protein and enzymes of mitochondrial, carbohydrate, respiratory and phosphate metabolism

SummaryFruits of papaya cv Coorg Honey Dew were analysed at five ripening stages for RNA, DNA, protein and enzyme activity. Ripening was characterized by an increase in the activities of invertase, cellulase, pectinesterase, catalase, peroxidase, adenosine triphosphatase, acid phosphatase, polyphenol oxidase and mitochondrial glutamate dehydrogenase; and falling levels of amylase, ribonuclease, mitochrondrial adenosine triphosphatase, NADH diphorase and malic dehydrogenase, and of the RNA, DNA, protein and mitochondrial protein contents.

Polyuronide solubilization during ripening of normal and mutant tomato fruit

The amount and molecular size of soluble polyuronide extractable from ripening tomatoes is markedly affected by residual enzyme activity. The efficacy of phenol-acetic acid-water treatment to remove this residual activity is demonstrated. Data obtained using treated wall preparations confirms that there is an increase in soluble polyuronide during normal ripening and that this also occurs in the ‘Never-ripe’ mutant, and to a lesser degree in the ‘ripening-inhibitor’ mutant. However, changes in the molecular size of this polyuronide during normal ripening were not as extensive as previously reported and few changes were apparent in either of the mutants. Measurements were also made of polygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1.1.11) activity during ripening. The level of polygalacturonase activity does not appear to correlate with the amount of soluble polyuronide released, but may be related to the extent of depolymerisation. No relationship was apparent between the level of pectinesterase and either soluble polyuronide released or depolymerization.

Bacteroides pectinophilus sp. nov. and Bacteroides galacturonicus sp. nov.: two pectinolytic bacteria from the human intestinal tract.

Studies on the physiological characteristics of two obligately anaerobic, rod-shaped bacteria from the human intestinal tract indicated that the organisms represented two previously undescribed species of Bacteroides, for which we propose the names Bacteroides pectinophilus (type strain, N3) and Bacteroides galacturonicus (type strain, N6). Both strains were pectinophilic; that is, they utilized as fermentable substrates for growth only pectin and a few related compounds. The two species differed significantly from each other in guanine plus cytosine content of the DNA, in substrate utilization patterns, and in other phenotypic characteristics. Both species deesterified pectin by means of an extracellular pectinesterase (EC 3.1.1.11) activity. Polygalacturonate (the main component of deesterified pectin) was depolymerized extracellularly with formation of unsaturated products by both species. The depolymerizing activity required Ca2+, functioned at a higher rate when polygalacturonate was the substrate as compared with pectin, and had an alkaline pH optimum. These data, as well as viscosity decrease studies and identification of products formed from polygalacturonate, indicated that the extracellular depolymerizing activity of either species was characteristic of an exopectate (exopolygalacturonate) lyase. The exopectate lyase activity had an unusual action pattern that resulted in terminal cleavage of unsaturated trigalacturonic acid units from polygalacturonate. An unsaturated trimer was the major product that accumulated in cell-free reaction mixtures, where it was not cleaved further. Growing cells of both Bacteroides species released the exopectate lyase into the external environment by processes that did not involve cell lysis to any significant extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Pectinolytic activity ofClostridium thermocellum: Its use for anaerobic fermentation of sugar beet pulp

Clostridium thermocellum is well known for its ability to convert cellulose into ethanol and to hydrolyse hemicellulose. The present work shows its ability to hydrolyse model pectins and to use them for growth. The main products on these substrates as well as on sugar beet pulps were as follows: acetate, ethanol and methanol. Galacturonase and lyase activities were measured in the fermentation broths. As shown by the accumulation of methanol in the medium, there is a pectin esterase activity but this one seems to be very low.