How the rice weevil breaks down the pectin network: Enzymatic synergism and sub-functionalization

Pectin is the most complex polysaccharide in nature and highly abundant in plant cell walls and middle lamellae, where it functions in plant growth and development. Phytopathogens utilize plant pectin as an energy source through enzyme-mediated degradation. These pectolytic enzymes include polygalacturonases (PGs) of the GH28 family and pectin methylesterases (PMEs) of the CE8 family. Recently, PGs were also identified in herbivorous insects of the distantly related plant bug, stick insect and Phytophaga beetle lineages. Unlike all other insects, weevils possess PMEs in addition to PGs. To investigate pectin digestion in insects and the role of PMEs in weevils, all PME and PG family members of the rice weevil Sitophilus oryzae were heterologously expressed and functionally characterized. Enzymatically active and inactive PG and PME family members were identified. The loss of activity can be explained by a lack of substrate binding correlating with substitutions of functionally important amino acid residues. We found subfunctionalization in both enzyme families, supported by expression pattern and substrate specificities as well as evidence for synergistic pectin breakdown. Our data suggest that the rice weevil might be able to use pectin as an energy source, and illustrates the potential of both PG and PME enzyme families to functionally diversify after horizontal gene transfer.     

Pectin methyl esterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry

Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point >9, a pH optimum at 7 and temperature optimum at 50 °C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with K _m values of 0.7 mg ml^-1 for pectin with a DE of 70% and 17 mg ml^-1 for pectin with a DE of 25%. The sequences of the NH₂-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands. Received: 15 November 1997 / Accepted: 7 April 1998     

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER*

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.     

Inhibition of pectin methyl esterase activity by green tea catechins

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.     

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.     

Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening

The decrease of strawberry (Fragariaxananassa Duch.) fruit firmness observed during ripening is partly attributed to pectolytic enzymes: polygalacturonases, pectate lyases and pectin methylesterases (PMEs). In this study, PME activity and pectin content and esterification degree were measured in cell walls from ripening fruits. Small green, large green, white, turning, red and over-ripe fruits from the Elsanta cultivar were analyzed. Using the 2F4 antibody directed against the calcium-induced egg box conformation of pectin, we show that calcium-bound acidic pectin was nearly absent from green and white fruits, but increased abruptly at the turning stage, while the total pectin content decreased only slightly as maturation proceeded. Isoelectrofocalisation performed on wall protein extracts revealed the expression of at least six different basic PME isoforms. Maximum PME activity was detected in green fruits and steadily decreased to reach a minimum in senescent fruits. The preliminary role of PMEs and subsequent pectin degradation by pectolytic enzymes is discussed.     

Pectolytic Activity by the Haustorium of the Parasitic PlantOrobancheL. (Orobanchaceae) in Host Roots

The possible involvement of enzymes in the penetration of intrusivecells of the parasitic angiospermOrobancheinto host root tissueswas studied using cytochemical and immunocytochemical methods.Pectin methyl esterase (PME) was detected, with specific antibodies,in the cytoplasm and cell walls ofOrobancheintrusive cells andin adjacent host apoplast. Depletion and chemical changes ofpectins in host cell walls were shown by histochemical stainingwith PATAg, which detects carbohydrates that are sensitive toperiodic acid, especially pectins, and with the monoclonal antibodiesJIM 5 and JIM 7 that label pectins with low and high rates ofesterification, respectively. Galacturonic sequences with lowrates of esterification were more abundant in host cell wallsadjacent to the parasite, which is consistent with pectin de-methylationby PME release from the parasite. Pectins were absent in middlelamellae and in host cell walls neighbouring mature intrusivecells of the parasite, consistent with further degradation ofpectins by other enzymes. These results provide the first directevidence for the presence and activity of a pectolytic enzymein the infection zone of the haustorium of a parasitic angiosperminsitu.Copyright 1998 Annals of Botany Company Broomrape;Orobanche; parasitic weed; haustorium; pectin methyl esterase; pectin; cell wall.     

Immunogold localization of pectin methylesterases in the cortical tissues of flax hypocotyl

Summary Pectin methylesterases (PMEs, EC 3.1.1.11) catalyse the deesterification of pectins. Up to now, most information concerning their location was obtained from biochemical analyses. Taking advantage of specific anti-PME antibodies, we report the precise localization of PMEs at the electron microscopy level within the different cortical tissues of flax hypocotyl. Quantitative data on the densities of immunolabelling have been collected, using anti-PME antibodies as well as JIM5 and JIM7 monoclonal antibodies. Our findings show a co-localization of PMEs and acidic pectins (as revealed by JIM5 antibodies) within specific cell wall microdomains. Moreover, PME epitopes are associated with the cellular membranes, particularly with the plasmalemma.Abbreviations Cdta diamino-1,2 cyclohexane tetra-acetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PME pectin methylesterase - TEM transmission electron microscopy     

Arabidopsis PME17 Activity can be Controlled by Pectin Methylesterase Inhibitor4

The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.     

Pectinmethylesterase isoforms fromVigna radiata hypocotyl cell walls: kinetic properties and molecular cloning of a cDNA encoding the most alkaline isoform

Peptide maps and partial amino acid sequences of the 3 main pectinmethylesterases (PMEs) solubilized from mung bean hypocotyl cell walls demonstrated that these proteins were different isozymes originating from a small multigene family. A cDNA clone encoding the most alkaline PME (PE) have been obtained by PCR using degenerate oligonucleotide primers. Combining the protein and nucleotide sequencing data, the complete amino acid sequence of PE was determined. The nature protein is composed of 318 amino acids with a calculatedM _r of 34 677 and an estimated pI of 9.84 consistent with the values previously obtained by SDS-PAGE and IEF. It shares most of the conserved regions of previously known PMEs. Enzymatic activities of the three isoforms were differently affected by the presence of cations in the incubation medium but, in all cases, infra-optimal cation concentrations induced two opposite effects: a decrease in theV _max and an increase in the affinity of the enzymes for their substrate. The presence of cations in the assay modulates both the number of enzyme molecules available to the demethylation reaction and the conformation of the pectin and, in turn, the affinity of the PMEs for their substrate.     

Differential cell wall degradation byErwinia chrysanthemi in petiole ofSaintpaulia ionantha

Summary Erwinia chrysanthemi is a soft-rot pathogenic enterobacterium that provokes maceration of host plant tissues by producing extracellular cell-wall-degrading enzymes, among which are pectate lyases, pectin methyl esterases, and cellulases. Cell wall degradation in leaves and petiole tissue of infectedSaintpaulia ionantha plants has been investigated in order to define the structural and temporal framework of wall deconstruction. The degradation of major cell wall components, pectins and cellulose, was studied by both classical histochemical techniques (Calcofluor and periodic acid-thiocarbohydrazide-silver proteinate staining) and immunocytochemistry (tissue printing for detection of pectate lyases; monoclonal antibodies JIM5 and JIM7 for detection of pectic substrates). The results show that the mode of progression of the bacteria within the host plant is via the intercellular spaces of the parenchyma leaf and the petiole cortex. Maceration symptoms and secretion of pectate lyases PelA, -D, and -E can be directly correlated to the spread of the bacteria. Wall degradation is very heterogeneous. Loss of reactivity with JIM5 and JIM7 was progressive and/or clearcut. The primary and middle lamella appear to be the most susceptible regions of the wall. The innermost layer of the cell wall frequently resists complete deconstruction. At the wall intersects and around intercellular spaces resistant domains and highly degraded domains occurred simultaneously. All results lead to the hypothesis that both spatial organisation of the wall and accessibility to enzymes are very highly variable according to regions. The use of mutants lacking pectate lyases PelA, -D, -E or -B, -C confirm the important role that PelA, PelD, and PelE play in the rapid degradation of pectins from the host cell walls. In contrast, PelB and PelC seem not essential for degradation of the wall, though they can be detected in leaves infected with wild-type bacteria. With Calcofluor staining, regularly localised cellulose-rich and cellulose-poor domains were observed in pectic-deprived walls.Abbreviations MAb monoclonal antibody - PATAg periodic acid-thiocarbohydrazide-silver proteinate     

Kiwi fruit PMEI inhibits PME activity,modulates root elongation and induces pollen tube burst in Arabidopsis thaliana

Pectins are major components of primary cell wall that play a crucial role in plant development. After biosynthesis, pectins are secreted in the cell wall by Golgi-derived vesicles under a highly methylesterified form and are de-methylesterified by pectin methylesterases (PME). It is hypothesized that PME might be regulated by pectin methylesterase inhibitor (PMEI). In this paper, we show by isoelectric focalisation and subsequent zymogram that kiwi PMEI was able to inhibit Arabidopsis PME activity by forming a complex. The complexes were stable under a wide range of ionic strength and pH. Moreover, PMEI might be able to form a complex with basic PMEs including three PMEs strongly expressed in root and four PMEs expressed in pollen grains. Finally, exogenous treatment with kiwi PMEI was able to reduce the activity of cell wall resident PMEs with persistent effects such as an increase of the root growth and a dramatic effect on pollen tube stability.     

Demethylesterification of the primary wall by PECTIN METHYLESTERASE35 provides mechanical support to the Arabidopsis stem

Secondary cell walls, which contain lignin, have traditionally been considered essential for the mechanical strength of the shoot of land plants, whereas pectin, which is a characteristic component of the primary wall, is not considered to be involved in the mechanical support of the plant. Contradicting this conventional knowledge, loss-of-function mutant alleles of Arabidopsis thaliana PECTIN METHYLESTERASE35 (PME35), which encodes a pectin methylesterase, showed a pendant stem phenotype and an increased deformation rate of the stem, indicating that the mechanical strength of the stem was impaired by the mutation. PME35 was expressed specifically in the basal part of the inflorescence stem. Biochemical characterization showed that the activity of pectin methylesterase was significantly reduced in the basal part of the mutant stem. Immunofluorescence microscopy and immunogold electron microscopy analyses using JIM5, JIM7, and LM20 monoclonal antibodies revealed that demethylesterification of methylesterified homogalacturonans in the primary cell wall of the cortex and interfascicular fibers was suppressed in the mutant, but lignified cell walls in the interfascicular and xylary fibers were not affected. These phenotypic analyses indicate that PME35-mediated demethylesterification of the primary cell wall directly regulates the mechanical strength of the supporting tissue.     

Phenylephrine,a small molecule,inhibits pectin methylesterases

Pectin methylesterases (PMEs) catalyze pectin demethylation and facilitate the determination of the degree of methyl esterification of cell wall in higher plants. The regulation of PME activity through endogenous proteinaceous PME inhibitors (PMEIs) alters the status of pectin methylation and influences plant growth and development. In this study, we performed a PMEI screening assay using a chemical library and identified a strong inhibitor, phenylephrine (PE). PE, a small molecule, competitively inhibited plant PMEs, including orange PME and Arabidopsis PME. Physiologically, cultivation of Brassica campestris seedlings in the presence of PE showed root growth inhibition. Microscopic observation revealed that PE inhibits elongation and development of root hairs. Molecular studies demonstrated that Root Hair Specific 12 (RHS12) encoding a PME, which plays a role in root hair development, was inhibited by PE with a Ki value of 44.1?μM. The biochemical mechanism of PE-mediated PME inhibition as well as a molecular docking model between PE and RHS12 revealed that PE interacts within the catalytic cleft of RHS12 and interferes with PME catalytic activity. Taken together, these findings suggest that PE is a novel and non-proteinaceous PME inhibitor. Furthermore, PE could be a lead compound for developing a potent plant growth regulator in agriculture.     

Developmental localization and methylesterification of pectin epitopes during somatic embryogenesis of banana (Musa spp. AAA)

Background

The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development.

Methodology/Principal Findings

Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment.

Conclusions/Significance

These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana.     

Structure and functional features of olive pollen pectin methylesterase using homology modeling and molecular docking methods

Pectin methylesterases (PMEs), a multigene family of proteins with multiple differentially regulated isoforms, are key enzymes implicated in the carbohydrates (pectin) metabolism of cell walls. Olive pollen PME has been identified as a new allergen (Ole e 11) of potential relevance in allergy amelioration, since it exhibits high prevalence among atopic patients. In this work, the structural and functional characterization of two olive pollen PME isoforms and their comparison with other PME plants was performed by using different approaches: (1) the physicochemical properties and functional-regulatory motifs characterization, (2) primary sequence analysis, 2D and 3D comparative structural features study, (3) conservation and evolutionary analysis, (4) catalytic activity and regulation based on molecular docking analysis of a homologue PME inhibitor, and (5) B-cell epitopes prediction by sequence and structural based methods and protein-protein interaction tools, while T-cell epitopes by inhibitory concentration and binding score methods. Our results indicate that the structural differences and low conservation of residues, together with differences in physicochemical and posttranslational motifs might be a mechanism for PME isovariants generation, regulation, and differential surface epitopes generation. Olive PMEs perform a processive catalytic mechanism, and a differential molecular interaction with specific PME inhibitor, opening new possibilities for PME activity regulation. Despite the common function of PMEs, differential features found in this study will lead to a better understanding of the structural and functional characterization of plant PMEs and help to improve the component-resolving diagnosis and immunotherapy of olive pollen allergy by epitopes identification.     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

Polygalacturonase, Pectate Lyase and Pectin Methylesterase Activity in Pathogenic Strains of Phytophthora capsici Incubated under Different Conditions

Pectolytic enzymes are found mainly in fungi and bacteria. The most widely occurring enzymes are polygalacturonase (PGs), pectin methylesterase (PMEs) and pectate lyase (PLs) produced during the infection process and during culturing. The secretion of these enzymes results in the disorganization of the plant cell walls, which is responsible for the pathogenicity of the pathogens. These enzymes degrade the pectin of plants causing maceration of plant tissues and the enzyme activity increases under favourable environmental conditions. We have found that Phytophthora capsici , a pathogenic oomycete, produces levels of these three enzymes equal to those produced by soft-rotting Erwinia chrysanthemi . The activity of PGs, PLs and PMEs was investigated at the optimum temperature, pH and ionic strength in highly pathogenic P. capsici strains cultivated in two kinds of liquid medium containing either crude pepper extracts plus pectin or pectin as the carbon source. Virulence tests and enzymes activity showed that there was a high correlation between the enzyme activity and the pathogenicity of P. capsici . The effects of different carbon sources on the enzyme activity showed that pepper extract plus pectin was the best source for the carbon source.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

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 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/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 5 mM 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 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.