Carrot pectin methylesterase and its inhibitor from kiwi fruit: Study of activity, stability and inhibition

Carrot pectin methylesterase (PME) and its inhibitor (PMEI) from kiwi fruit were successfully purified by affinity chromatography. Enzyme and inhibitor activity and stability and PME–PMEI complex formation, as influenced by intrinsic product factors (pH and NaCl) and extrinsic process factors (temperature and pressure), were studied. The effect of temperature- or pressure-induced denaturation of PME and PMEI on their respective activities was assessed by estimating inactivation kinetic parameters. PME inactivation obeyed first-order kinetics. The enzyme was rather heat-labile but pressure-stable. PMEI inactivation was best described by a model taking into account a processing-stable PMEI intermediate. The behavior of PME and the PME–PMEI complex at elevated temperature or pressure in the presence of pectin was explored by following methanol formation as a function of treatment time. PME catalytic activity was stimulated up to a certain temperature or pressure level before declining. No conclusive evidence was obtained for a temperature-induced dissociation of the PME–PMEI complex, whereas high pressure exposure caused the complex to separate.

Industrial relevance

PME activity control is a major point of interest in the quest of obtaining high quality plant-derived food products. The current study demonstrates that both traditional thermal processing and novel high hydrostatic pressure processing allow stimulation as well as inactivation of PME and, hence, directing the PME-catalyzed pectin hydrolysis. An alternative or additional approach to control endogenous PME activity (e.g. to obtain cloud-stable juices) is through enzyme inhibition using kiwi PMEI. In this context, pH and NaCl boundaries for application were established, the existence of a temperature- and pressure-stable PMEI intermediate was shown and the PME–PMEI complex was proven not to be dissociated at mild temperature and pressure levels. These observations endorse the possibility of inhibiting undesirable PME activity remaining after mild processing.     

The detection of residual pectin methylesterase activity in pasteurized tomato juices

The enzyme pectin methylesterase (PME) is believed to be involved in the destabilization and cloud loss of vegetable juices through the de-esterification of pectin followed by the successive coprecipitation of the pectate with insoluble materials present in the juices. Cloud destabilization is often observed even when the vegetable products have been subjected to thermal treatment to produce sterile products and also to stabilize the cloud. Therefore it is possible that loss of cloud may be because of residual enzymatic activities surviving the thermal treatments. However, so far no evidence demonstrating the existence of residual PME activity in pasteurized juices has been published. In this paper it is reported that in industrial tomato products showing cloud loss residual PME activity is present. It has been possible to detect this very low activity by an affinity chromatography procedure. The method is based on a cyanogen bromide-activated resin which is coupled with a pectin methylesterase inhibitor protein purified from kiwi fruit. This resin binds native PME with high selectivity and the enzyme can be concentrated from the product in a single step. Thus, the very low PME residual activity present in pasteurized juice and generally not detectable with common techniques can be detected and easily determined with the method described in this paper.     

重组猕猴桃果胶甲酯酶抑制剂的抑制活性研究

猕猴桃的果胶甲酯酶抑制剂可有效抑制不同种类植物的果胶甲酯酶活性。主要研究在巴斯德毕赤酵母中成功表达的重组猕猴桃果胶甲酯酶抑制剂(被命名为kWPMEI)的抑制活性。通过研究发现,kWPMEI对桔子果胶甲酯酶的抑制活性最强,其次是胡萝卜,最弱的是番茄。同时kWPMEI对上述三种植物果胶甲酯酶的抑制活性受环境温度和pH的影响比较显著。     

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation

We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6–9) and moderate thermostability (optimum ca. 70 °C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure.     

Purification,properties and heat inactivation of pectin methylesterase from apple (cv Golden Delicious)

Pectin methylesterase from apple (cv Golden Delicious) was extracted and purified by affinity chromatography on a CNBr‐Sepharose®‐PMEI column. A single pectin methylesterase peak was observed. Isoelectric points were higher than 9. Kinetic parameters of the enzyme were determined as K_m = 0.098 mg ml⁻¹ and V_max = 3.86 µmol min⁻¹ ml⁻¹ of enzyme. The optimum pH of the enzyme was above 7.5 and its optimum temperature was 63 °C. The purified PME required the presence of NaCl for optimum activity, and the sodium chloride optimum concentration increased with decreasing pH (from 0.13 M at pH 7 to 0.75 M at pH 4). The heat stability of purified PME was investigated without and with glycerol (50%), and thermal resistance parameters (D and Z values) were calculated showing that glycerol improved the heat resistance of apple PME. © 2000 Society of Chemical Industry     

Plant pectin methylesterase and its inhibitor from kiwi fruit: Interaction analysis by surface plasmon resonance

Two surface plasmon resonance (SPR)-based interaction analysis methods were successfully implemented to explore the binding between plant PME and kiwi PMEI. In a first method, plant PMEs were immobilised on a chip surface via amine coupling. This experimental setup allowed studying the effect of pH and ionic strength on the PME–PMEI interaction kinetics. Strong binding was obtained at pH < 7 and at low salt concentrations, whereas both pH ? 8 and [NaCl] of ca. 1.0 M effectively caused dissociation. In a second method, kiwi PMEI was immobilised on a chip surface to which streptavidin had been covalently attached. Hereto, PMEI was biotinylated by means of a NHS-biotin reagent. With this immobilisation strategy, the effect of (partial) thermal or high pressure-induced denaturation of PME on its affinity towards PMEI was investigated. A notable degree of enzyme inactivation was required before interaction characteristics were significantly altered. Any incomplete inactivation of PME resulted in binding to the PMEI surface.     

Inhibition of tomato (Licopersicon esculentum Mill.) pectin methylesterase by pulsed electric fields

High intensity pulsed electric fields were utilized for inhibition of pectin methylesterase (PME) from tomato and a 93.8% reduction of the initial activity was achieved with 400 pulses of 0.02-ms pulse-width at 24 kV/cm. The classical exponential decay model as well as Hülsheger’s and Fermi’s empirical models adequately described the enzymatic inhibition. Hülsheger’s equation yielded parameter values of 0.7 kV/cm, 0.48 ms, and 39 kV/cm, for critical electric field intensity, critical treatment time, and the independent constant factor, respectively. For Fermi’s equation, the critical electric field and steepness parameters ranged from 10.8 to 28 kV/cm and 5.7 to 11 kV/cm, respectively. The residual PME activity reduction as a function of the energy supplied per volume unit was described by an exponential decay model with the constant equal to 0.068 (m³/GJ).     

Process stability of Capsicum annuum pectin methylesterase in model systems, pepper puree and intact pepper tissue

Process stability studies towards temperature and/or pressure on pepper pectin methylesterase (PME) were carried out in different systems (purified form, crude extract, pepper pieces and puree) at pH 5.6. Within the temperature range studied (22–80 °C, 5 min), pepper PME in pure form and crude extract was gradually inactivated showing a biphasic inactivation behaviour, indicating the presence of isoenzymes of different thermostability. Pepper samples heated for 15 min showed a maximum of residual PME activity around 55 °C. Isothermal inactivation of pepper PME in purified form and crude extract at pH 5.6 could be described by a biphasic inactivation model for the temperature range studied (62–76 °C). A stable behaviour towards high-pressure/temperature treatments (400–800 MPa/25–60 °C) was observed for crude extract and purified pepper PME. PME in pepper puree samples revealed to be very pressure stable. Mild temperatures combined with pressure treatments seem to increase the extractability from PME in pepper tissue, probably due to the effect on the cell structure.     

Production and optimization of a kiwi pectin methylesterase inhibitor in Pichia pastoris GS115

Gene sequence coding of a kiwi pectin methylesterase inhibitor was optimized, synthesized, and expressed in Pichia pastoris GS115 based on P. pastoris preferred codon usage. The expression level of the recombinant protein (kwPMEI) increased by 89.74% after codon optimization. Expression conditions of recombinant strains were optimized. The highest production of kwPMEI was achieved using 0.8% sorbitol (added every 24 h), 0.05% oleic acid (added at the beginning of induction), and 0.5% methanol (added every 12 h). kwPMEI was purified using Ni²⁺ chelating affinity chromatography and 17 mg of the protein was harvested from 60 mL of a culture supernatant. Activity analysis showed that kwPMEI efficiently inhibited the activity of different plant PMEs. High expression levels and purification of kwPMEI will promote applications in fruit and vegetable juices.     

Development of an immunological toolbox to detect endogenous and exogenous pectin methylesterase in plant-based food products

An immunological toolbox consisting of five monoclonal antibodies (MA) was developed for the detection of endogenous as well as exogenous pectin methylesterase (PME) in plant-based food products. Based on cross-reactivity with plant PME, the MA were subdivided into two groups. MA of group one recognise exclusively PME from Aspergillus aculeatus (fPME), which could be visualised upon infusion. Three different infusion techniques were compared for three different types of plant tissue, revealing a homogenous distribution of exogenous PME upon pressure-and vacuum-assisted infusion. The MA of group two recognise fPME as well as PME from different plant sources like tomato, carrot, strawberry, broccoli and apple. The use of these antibodies allowed detection of endogenous PME in tomato which revealed an overall presence of PME in the pericarp of tomato fruit. Moreover, the PME detected by the MA of group two in the plant cell wall of tomato coincided with the localisation of two isoenzymes of tomato PME, as detected by isoenzyme-specific MA. The obtained results highlight the versatility of the developed antibodies as probes to detect PME in the context of food processing.     

Inactivation of pectin methylesterase and stabilization of opalescence in orange juice by dynamic high pressure

The effect of dynamic high pressure homogenization (DHP) alone or in combination with pre-warming on pectin methylesterase (PME) activity and opalescence stability of orange juice was studied. DHP without heating reduced PME activity by 20%. Warming the juice (50 °C, 10 min) prior to homogenization significantly increased the effectiveness of DHP. PME inactivation was further increased by adjusting the pH downward prior to treatment. Accelerated shelf-life study at 30 °C revealed that opalescence stability can be increased by several days by DHP treatment, even in the presence of active PME. These results suggest that the opalescence stability of orange juice treated by DHP does not depend entirely on PME activity but also on particle size reduction and structural changes to pectin resulting from the treatment. The freshness attributes of orange juice treated by warming was improved by DHP treatment.     

不同发酵条件对重组猕猴桃果胶甲酯酶抑制剂表达量的影响

将已在毕赤酵母中成功表达的重组猕猴桃果胶甲酯酶抑制剂为研究对象,通过改变发酵培养时间、培养基装填量及培养基配方,对该重组蛋白的表达量进行研究。最终确定使用BMMY培养基、表达时间为96h、培养基装填量为10%时为最佳表达条件,在最佳表达条件下,该重组蛋白的表达量最高可达0.2g/L。     

Orange Juices and Concentrates Stabilization by a Proteic Inhibitor of Pectin Methylesterase

A proteic inhibitor of pectin methylesterase (PME), recently discovered in kiwi, was used to stabilize cloud of orange juice concentrate with “Cut-back,” 42° Brix. To concentrated and pasteurized orange juice were added increasing amounts of fresh juice (12° Brix) with PME 0.8 U/mL at the fresh to pasteurized ratios 6.6 to 38%. The PME inhibitor (65% pure), from kiwi, was added at 50 mg/L. After 8 mo at 5°C samples were compared with controls containing the same amount of fresh juice with PME inhibited by pasteurization. Pasteurized samples were not different from those treated with PME inhibitor. Conversely, where PME was not inhibited, cloud stability decreased with increasing amounts of fresh juice. Use of kiwi PME inhibitor in fruit juice production has potential advantages.     

The kinetics of inactivation of pectin methylesterase and polygalacturonase in tomato juice by thermosonication

The ultrasonic inactivation kinetics of polygalacturonase (PG) and pectin methylesterase (PME) in tomato juice were studied at a frequency of 20 kHz, amplitude of 65 μm and temperatures between 50 and 75 °C. Thermal treatments at the same temperatures were conducted to separate the effects of heat and ultrasound. The thermal inactivation of PG was described by a fractional conversion model with PG 1 remaining stable, whereas the inactivation of PG by combined ultrasonic and heat treatment (thermosonication) was best described by first order biphasic kinetics, with both PG1 and PG2 inactivated at different rates. The thermal and thermosonication inactivation of PME was described well by first order kinetics. The inactivating effect of combined ultrasound and heat was synergistic. Thermosonication enhanced the inactivation rates of both PME and PG. The inactivation rate of PME was increased by 1.5–6 times and the inactivation rate of PG2 by 2.3–4 times in the temperature range 60–75 °C, with the highest increase corresponding to the lowest temperature.     

Pectin methylesterase activity in juices from mandarins, oranges and hybrids

The pectin methylesterase (PME) activity of fresh and processed juices obtained from Valencia-late oranges, Clementine mandarins (cultivar Nules) and citrus hybrids (Ortanique) was studied. The PME activity was measured using a modification of the traditional method of Rouse and Atkins [1], based on the titration of carboxylic groups generated by PME during the hydrolysis of a commercial pectin solution. Instead of maintaining the pH at a given value by addition of NaOH, the maximum rate of pH decrease is used as measurement of the PME activity. An exponential decay function accurately represents the evolution of pH with time from a starting value of pH = 7.8 (optimum pH of PME activity for all assayed cultivars). This function allows an easy calculation of the PME activity. The total PME activity measured was similar in Clementine and Valencia-late juices, and approximately twice that of Ortanique juices. Ratios between thermostable and total PME activity were 4.0–8.4% for Clementine, 12.6–12.7% for Valencia-late and 24.2–34.2% for Ortanique juices. Soft heat treatments (70 °C, 30 s or 74 °C, 10 s) were enough to reduce the total PME activity of Clementine juices under the maximum residual level (10%) for commercial life of refrigerated juices, according to Irwe and Olson [2]. An erratum to this article can be found at     

Pectin methylesterase in Citrus bergamia R.: purification, biochemical characterisation and sequence of the exon related to the enzyme active site

Three forms of pectin methylesterase (PME) were purified, from bergamot fruit (Citrus bergamia R.), to homogeneity by ion-exchange and affinity chromatography. The isoforms, named PME I, PME II and PME III, according their elution order on a heparin–sepharose column, were characterized for their relative molecular mass, activity kinetic parameters and thermostability. The molecular mass was estimated to be 42 kDa for the three forms, and the apparent K_m values for citrus pectin were 0.9 mg/ml for PME I and 0.5 mg/ml for PME II and PME III. The optimum pH values lie within the range 6.5–9.0, depending on salt concentration. Thermal behaviours of the three PME isoforms were studied in a temperature range from 65 °C to 80 °C with the less abundant PME I isoform showing a higher heat resistance. Moreover, the complete exon 2 sequence of PME gene was acquired (GenBank accession no. DQ458770) using a PCR-based approach on well-known Citrus genomic DNA present in the NCBI database.     

Firming of fruit tissues by vacuum-infusion of pectin methylesterase: Visualisation of enzyme action

Apple pieces were vacuum-impregnated with either a pectin methylesterase (PME) and calcium solution or with water prior to pasteurization. Pasteurized apple pieces impregnated with PME and calcium showed a significantly higher firmness. Moreover, solid state ¹³C NMR spectroscopy of apple cell wall residues revealed an increase of their molecular rigidity. Exogenous PME addition involved a decrease from 82% to 45% of apple pectin degree of methyl-esterification. Microscopic observations of apple slices immunolabelled with antibodies specific for pectins showed that (i) demethyl-esterification was more intense in the cell wall region lining intercellular spaces (demonstrating a key role for these intercellular channels in the enzyme penetration in the tissue during vacuum-infusion) and that (ii) the number of calcium-dimerized deesterified homogalacturonan chains increased. The results corroborate the hypothesis that vacuum-impregnated PME action liberates free carboxyl groups along pectin chains that could interact with calcium, increasing the rigidity of pectins and finally the mechanical rigidity of apple tissue.     

Relationship between pectin methylesterase activity and the formation of methanol in Concord grape juice and wine

Pectin methylesterase (PME) activity in grapes was studied. Concord grape PME has optimum activity at pH 7·5 and the activity was the highest among several grape varieties tested. PME activity in Concord grapes increased continuously throughout the maturation period and reached a maximum at harvest. Consequently, this reflected the large amount of methanol formation during wine fermentation.     

A review of pectin methylesterase inactivation in citrus juice during pasteurization

BackgroundPectin, naturally found in citrus, plays a key role in the quality of the obtained juices. Pectin methylesterase enzyme (PME) influences the cloud stability, viscosity, color, mouth feeling and flavor of the juices by de-esterification of pectin. Iinactivation of PME is introduced as a pasteurization index in citrus juices, due to its higher thermal resistance than the spoilage microorganisms.Scope and approachInactivation of PME using different thermal (conventional, microwave and ohmic heating) and non-thermal (pulsed electric field, high pressure processing and high pressure carbon dioxide) processes is important in juice production. The aim of this study was to review the effect of these processing methods on the PME inactivation in different citrus juices.Key finding and conclusionUsing non-thermal methods in combination with moderate thermal methods can be more effective in PME inactivation with minimum loss in citrus juice quality.     

Extraction and characterisation of pectin methylesterase from black carrot (Daucus carota L.)

This study was carried out to determine some of the biochemical properties of pectin methylesterase (PME) from black carrot. The enzyme showed very high activity in a broad pH range of 6.5–8.5, with the optimum pH occurring at 7.5. The optimum temperature for maximal PME activity was found to be 55 °C. NaCl enhanced PME activity, particularly at 0.2 M. K_m and V_max values for black carrot PME using apple pectin as substrate were found to be 2.14 mg/ml (r² = 0.988) and 3.75 units/ml, respectively. The enzyme was stable between the temperatures of 30–50 °C/5 min whereas it lost nearly all of its activity at 70 °C/5 min. E_a and Z values were found to be 196.8 kJmol⁻¹ (r² = 0.996) and 2.16 °C (r² = 0.995), respectively.