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PubMed1971

Purification and properties of an polygalacturonic acid trans-eliminase produced by Bacillus pumilus.

A strain of Bacillus pumilus produced an extracellular pectic enzyme with polygalacturonic acid as the substrate. This enzyme, with optimal activity at pH 8.0 to 8.5, produced reaction products that strongly absorbed light at 232 nm, indicating the presence of a pectic acid trans-eliminase (PATE). Neither pectin esterase nor polygalacturonase was detected in the cell-free culture fluid. Chromatographic examination of the end products revealed the presence of large quantities of unsaturated oligouronides unlike those found with B. polymyxa. It was found that the PATE was produced extracellularly during the negative logarithmic death phase of the organism. The filtrate from sonically treated cells did not show any activity for PATE or hydrolases for lower oligogalacturonides at any time during the growth cycle. The enzyme was inducible. Pectin, National Formulary (NF) was the best inducer, followed by polygalacturonic acid and galacturonic acid. Enzyme activity was markedly stimulated by calcium and other divalent ions. Copper and cobalt ions were inhibitory. The partially purified enzyme showed no significant activity on pectin containing a high methoxyl content (96% esterified). However, pectin NF with a lower methoxyl content (68% esterified) was attacked to a degree by the partially purified and crude enzyme preparations. The initial rate of PATE activity increased up to 60 C, about 16-fold higher than that observed at room temperature. The activation energy was calculated as 12,183 cal/mole. A protective action of calcium chloride against heat inactivation of the PATE was observed. Degradation of polygalacturonic acid by this enzyme produced several unsaturated oligouronides soon after its addition to the substrate. The major endproduct was thought to be different from that of other known PATE enzymes. Paper chromatographic studies and viscosity measurements disclosed the random cleaving nature of the enzyme an endo-PATE.

Ammonium Sulfate↗
PubMed2002

PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases.

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-alpha-D-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.

Amino Acid Sequence↗
PubMed1997

Control of polygalacturonase synthesis in Fusarium oxysporum f.sp. radicis lycopersici.

Genetic control of polygalacturonase (PG) activity from Fusarium oxysporum f.sp. radicis lycopersici was analyzed on pectin and glucose cultures. One exopolygalacturonase from F. oxysporum f.sp. radicis lycopersici was strongly induced, in stationary culture, when the fungus was grown on apple pectin, while on glucose no extracellular PG activity could be detected. Although SDS-PAGE detected the presence of a putative PG band (66 kDa) in both conditions, specific antibodies obtained against the purified PG only detected it in PG-inducing conditions, that is to say, when apple pectin was used as the carbon source. Northern blot analysis of RNA of two isolates of F. oxysporum f.sp. radicis lycopersici (r6 and r2) confirmed that this regulation of PG synthesis was exerted at the transcriptional level. Only one single mRNA species of around 1400 nucleotides was detected on the cultures containing pectin and was absent in glucose-grown cultures. Southern blot analysis of genomic DNA indicated that pg gene seems to be present in a single copy in the genomes of F. oxysporum f.sp. radicis lycopersici r6 and r2 and Fusarium oxysporum f.sp. lycopersici, showing similar hybridization patterns in all species. The partial sequence of this pg gene from F. oxysporum f.sp. radicis lycopersici r6, which is also reported, showed high similarity to diverse PGs already reported. Exopolygalacturonase of F. oxysporum f.sp. radicis lycopersici r6 is heavily glycosylated; its deglycosylated form had a molecular mass of 50 kDa.

Amino Acid Sequence↗
PubMed2006

Wall-associated kinase 1 (WAK1) is crosslinked in endomembranes, and transport to the cell surface requires correct cell-wall synthesis.

The Arabidopsis thaliana wall-associated kinases (WAKs) bind to pectin with an extracellular domain and also contain a cytoplasmic protein kinase domain. WAKs are required for cell elongation and modulate sugar metabolism. This work shows that in leaf protoplasts a WAK1-GFP fusion protein accumulates in a cytoplasmic compartment that contains pectin. The WAK compartment contains markers for the Golgi, the site of pectin synthesis. The migration of WAK1-GFP to the cell surface is far slower than that of a cell surface receptor not associated with the cell wall, is influenced by the presence of fucose side chains on one or more unidentified molecules that might include pectin, and is dependent upon cellulose synthesis on the plasma membrane. WAK is crosslinked into a detergent-insoluble complex within the cytoplasmic compartment before it appears on the cell surface, and this is independent of fucose modification or cellulose synthesis. Thus, the assembly and crosslinking of WAKs may begin at an early stage within a cytoplasmic compartment rather than in the cell wall itself, and is coordinated with synthesis of surface cellulose.

Arabidopsis↗
PubMed2000

Study of biocatalyst to produce ethanol from starch. Coimmobilization of glucoamylase and yeast in gel.

This article presents a detailed study on the conditions for achieving a stable biocatalyst to be used in the production of ethanol from starch. Different pellets were used depending on which characteristic of the biocatalyst was being studied: (a) Saccharomyces cerevisiae entrapped in pectin or calcium alginate gel particles; (b) silica containing immobilized glucoamylase entrapped in pectin gel particles; or (c) pectin gel particles, with the silica-enzyme derivative and yeast coimmobilized. The influence of several variables on the mechanical resistance of the particle, on the viability of the microorganism, and on the rate of substrate hydrolysis was studied with biocatalyst. The best conditions found were 6% pectin gel, 2-mm particle diameter, and cure in 0.2M CaCl2.2H2O/60 mM acetate buffer, pH 4.2, for gel preparation; and 6.0 g/L of CaCl2.2H2O in the fermentation medium. Biocatalyst (c) was successfully tested for the production of ethanol from liquefied manioc flour syrup.

Alginates↗
PubMed2001

WAKs: cell wall-associated kinases linking the cytoplasm to the extracellular matrix.

There are only a few proteins identified at the cell surface that could directly regulate plant cell wall functions. The cell wall-associated kinases (WAKs) of angiosperms physically link the plasma membrane to the carbohydrate matrix and are unique in that they have the potential to directly signal cellular events through their cytoplasmic kinase domain. In Arabidopsis there are five WAKs and each has a cytoplasmic serine/threonine protein kinase domain, spans the plasma membrane, and extends a domain into the cell wall. The WAK extracellular domain is variable among the five isoforms, and collectively the family is expressed in most vegetative tissues. WAK1 and WAK2 are the most ubiquitously and abundantly expressed of the five tandemly arrayed genes, and their messages are present in vegetative meristems, junctions of organ types, and areas of cell expansion. They are also induced by pathogen infection and wounding. Recent experiments demonstrate that antisense WAK expression leads to a reduction in WAK protein levels and the loss of cell expansion. A large amount of WAK is covalently linked to pectin, and most WAK that is bound to pectin is also phosphorylated. In addition, one WAK isoform binds to a secreted glycine-rich protein (GRP). The data support a model where WAK is bound to GRP as a phosphorylated kinase, and also binds to pectin. How WAKs are involved in signaling from the pectin extracellular matrix in coordination with GRPs will be key to our understanding of the cell wall's role in cell growth.

Arabidopsis↗
PubMed2003

Effect of natural anionic polymers on the release rate and local concentration of metronidazole at pH near the natural gastric environment.

The aim of the study was to evaluate initially the applicability of certain natural polymers, as metronidazole binding agents in the treatment of ulcerative lesions in the gastric mucous membrane. The investigation involved systems containing metronidazole in combination with sodium alginate, citrus pectin and citrus-apple pectin. The rate of metronidazole release from the investigated preparations was evaluated after acidification to pH in gastric environment. Constant rates were determined, which were from 0.25 h-1 to 0.58 h-1 as well as half-release time, which were from 1.20 h to 2.82 h. Also, dynamic viscosity of the investigated systems was assessed--the dynamic viscosity of the gelatinous form with 2% content of sodium alginate increased by three orders of magnitude under the influence of acid pH, i.e. from about 160 cPa.s to about 30,000 cPa.s. Similarly, the viscosity of the preparation containing 4% of citrus-apple pectin increased after acidification from about 140 cPa.s to almost 40,0000 cPa.s. The use of sodium alginate and citrus-apple pectin as metronidazole carriers may prolong the time of action of the drug on pathologically changed gastric mucosa. The fraction of metronidazole which is in immediate contact with the pathologically changed surface is in concentration exceeding significantly MIC and MBC at usual oral doses.

Alginates↗
PubMed1985

Dietary fiber constituents of selected fruits and vegetables.

This study compared the dietary fiber (DF), neutral detergent fiber (NDF), cellulose, hemicellulose, lignin, and pectin content of selected fruits and vegetables. Apples and peaches (fresh and canned), oranges (fresh), strawberries (fresh, canned, and frozen), carrots, green beans, and potatoes (fresh, fresh cooked, canned, and frozen), and tomatoes (fresh, fresh cooked, and canned) were studied. When possible, two varieties, two stores, and name and store brands were chosen. Samples were analyzed for NDF, acid detergent fiber, 72% sulfuric acid, lignin, and pectin. From those values, DF, cellulose, and hemicellulose were calculated. Fresh fruits in gm/100gm wet weight had decreasing DF, NDF, and hemicellulose values as follows: apples, peaches, strawberries, and oranges. Apples were highest in cellulose; strawberries, highest in lignin; and oranges, highest in pectin. Fresh-cooked vegetables in gm/100gm wet weight have decreasing DF and NDF values as follows: green beans, carrots, potatoes, and tomatoes. Green beans were highest in cellulose and hemicellulose; potatoes highest in lignin; and carrots highest in pectin. On a wet-weight basis, fresh apples and peaches, fresh-cooked green beans, canned carrots, and canned and frozen potatoes were higher in DF and NDF than other forms of the fruit or vegetable. There were few differences according to stores, brands, or varieties of fruits and vegetables. On a dry-weight basis, fresh apples, peaches, strawberries, green beans, and tomatoes appear to have higher DF and NDF contents than their processed counterparts. Fresh-cooked carrots and fresh potatoes appear to have less DF and NDF than their canned and frozen counterparts.

Cellulose↗
PubMed1986

Relationship between colonic luminal pH, cell proliferation, and colon carcinogenesis in 1,2-dimethylhydrazine treated rats fed high fiber diets.

The comparative effects of different fibers on colonic luminal pH, crypt cell proliferation, and colon carcinogenesis were studied in 120 male Sprague-Dawley rats. The animals were divided into five equal groups and fed either a basal fiber free diet or the basal diet supplemented with 10% pectin, cellulose or guar, or 20% oat bran for up to 30 weeks. 1,2-Dimethylhydrazine was given at 20 mg/kg body weight as a weekly s.c. injection for 12 weeks. Food intake and weight gain were similar in all diet groups. At sacrifice, in vivo pH measurements showed that compared to fiber free rats, all fibers significantly acidified large bowel luminal contents (P less than 0.05). In the guar group 62.5% of rats developed colonic tumors compared to 33.4% of the fiber free rats (P less than 0.05). The yield of proximal colonic adenocarcinomas in the oat bran, pectin, and guar groups was increased by 4.5 to 5 times over the fiber free level (P less than 0.05-0.025). Pectin and guar provided the greatest stimulus to cell proliferation. A lower luminal pH was associated with a higher tumor yield and increased epithelial cell proliferation. Thus, acidification of colonic contents by high fiber diets failed to inhibit rat colon carcinogenesis, while the consumption of soluble fibers, such as oat bran, pectin, and guar, was associated with enhancement of proximal colon carcinogenesis.

1,2-Dimethylhydrazine↗
PubMed1979

[Model trials for isolation of soft rot bacteria by media containing pectic substances (author's transl)].

1. Under aerobic conditions bacterial soft rots are caused in the most cases by pectolytic Erwinia, Pseudomonas, and Bacillus spp. 2. Using 7 soft rotting strains several selective media were tested for their ability to sure a quick and exact isolation and differentiation of these pathogens. By bile salt-lactose-medium all Erwinia spp. under test could be isolated. For isolation of pectinolytic Pseudomonas strains the D4 medium of Kado and Heskett was suitable as the best one. 3. By the use of substrats containing pectic substances the results of isolation could be essentially improved: So, a sure differentiation of soft rotting bacteria and saprophytic organisms already upon the substrate and in addition, the isolation of pectinolytic Bacillus strains also became possible. The pectinolytic activity of the test strains on pectin-double layer-media was dependent upon the composition of the basal medium. 4. On the base of the results we obtained an isolation scheme is proposed, that allowed to indicate the pectolytic active bacilli (after heating to 80 degrees C for 10 min) on thioglycollate medium covered by a pectin layer, the Erwinia spp. on Stewart-Medium covered by pectin layer and the pectolytic pseudomonads on FPA-medium described by Sands, Hankin and Zucker (containing citrus pectin) to the equal time. 5. The trials also demonstrated, that the preparing of double layer-media instead of sodium polypectate mostly being recommended in literature lower estered pectic compounds can be used successfully.

Bacillus↗
PubMed1976

Antidiarrheal agents in the treatment of acute diarrhea in children.

To evaluate the efficacy of antidiarrheal agents in the treatment of diarrheal illnesses, a study was conducted with children in Guatemala who had an acute diarrheal illness. Eighty patients, aged 3 to 11 years, were hospitalized and treated for two days with one of five agents: kaolin-pectin suspension concentrate (Kao-Con), kaolin suspension, pectin suspension, diphenoxylate-atropine liquid (Lomotil), or placebo. Although the patients receiving kaolin-pectin produced stools that tended to be more formed than those of the placebo-treated group patients, the study did not demonstrate any effect by any of the agents tested in influencing the frequency of bowel movement, the water content of the stools, or the weight of stools. Kaolin-pectin suspension and diphenoxylate-atropine liquid do not appear to be useful in the relief of acute nonspecific diarrhea in children.

Acute Disease↗
PubMed1995

Influence of psyllium preparations on plasma and liver lipids of cholesterol-fed rats.

Rats were fed a semi purified diet containing 0.5% cholesterol and 10% fiber (cellulose, pectin, psyllium seed and defatted psyllium husk). One additional group of rats was fed cholesterol (0.5%) as part of a fiber-free diet and another was fed the fiber free diet without cholesterol. Cellulose had virtually no effect on serum or liver lipids. Pectin had a lipid lowering effect. Psyllium seed exerted an effect on total serum cholesterol equal to that of pectin but gave higher levels of HDL-cholesterol. The effects of psyllium seed on liver lipids were more pronounced than those of pectin. Defatted psyllium husk feeding virtually normalized liver size and serum triglyceride levels and produced lower serum total cholesterol levels and higher HDL-cholesterol than observed in normal controls. Defatted psyllium husk feeding also yielded liver lipid values which were in the normal range. Fecal wet and dry weights were significantly higher in rats fed either psyllium preparation.

Analysis of Variance↗
PubMed1996

[Comparison of the quality of frozen raspberries depending on the use of various additives].

The aim of the study was to determine the effect of sugar, pectin preparation, and L-ascorbic acid addition on selected physicochemical traits and organoleptic properties of frozen raspberries. Dry sugar or in the form of 60% syrup at a dose of 10 g/100 g of fruit was used with pectin or 0.5% L-ascorbic acid. A 1.5%. solution of the pectin preparation was applied at a dose of 10 g/100 g of fruit alone or with a 0.5% addition of L-ascorbic acid. Sorted fruits in 500 g samples treated with additives were frozen in plastic containers at -40 C, the storage temperature of -28(-)-30 C being reached after about 90 min. The evaluation was conducted at a stage of raw material, directly after freezing, and after 3-, 6-, 9-, and 12-month storage. Fresh fruit contained 13.4% of dry matter, 4.1% of total sugars, 2.12% of acids as citric acid, 48.3 mg/100 g of vitamin C, and 60.8 mg/100 g of anthocyanins. The additives contributed to the differentiation of frozen fruit samples in the level of dry matter, sugars, and vitamin C, and brought about some dilution of the remaining components. Freezing and 1-year storage of fruit not treated with any additives decreased the content of vitamin C in 100 g to 21.6 mg and of anthocyanins to 45.5 mg. In the samples with the additives these values were 23.4-29.2 mg /the samples of an enriched vitamin C content not being taken into consideration/and 47.1-56.3 mg, respectively. The organoleptic quality of frozen raspberries without additives evaluated in a 5-score scale was 3.78 and that of fruit with the additives 4.21-4.74. Of the analyzed variants of the additives used in freezing raspberries for direct consumption the 60% syrup of sucrose may be recommended on account of high organoleptic quality and simple application. The 1.5% solution of pectin preparation may be used in freezing raspberries for diabetics and the other groups of people who do not tolerate sugar in their diet.

Ascorbic Acid↗
PubMed1997

Endogenous production of methanol after the consumption of fruit.

After the consumption of fruit, the concentration of methanol in the human body increases by as much as an order of magnitude. This is due to the degradation of natural pectin (which is esterified with methyl alcohol) in the human colon. In vivo tests performed by means of proton-transfer-reaction mass spectrometry show that consumed pectin in either a pure form (10 to 15 g) or a natural form (in 1 kg of apples) induces a significant increase of methanol in the breath (and by inference in the blood) of humans. The amount generated from pectin (0.4 to 1.4 g) is approximately equivalent to the total daily endogenous production (measured to be 0.3 to 0.6 g/day) or that obtained from 0.3 liters of 80-proof brandy (calculated to be 0.5 g). This dietary pectin may contribute to the development of nonalcoholic cirrhosis of the liver.

Acetone↗
PubMed2001

Development of segments and appendages in embryos of the desert scorpion Paruroctonus mesaensis (Scorpiones: Vaejovidae).

The scanning electron microscope was used to study the changing features of scorpion embryos from the blastula through early stages in the development of appendages. The earliest scorpion fossils (Silurian period) have structures more advanced than the embryos herein, so the possibility is considered that these embryos still retain and display some features indicative of evolutionary patterns in adult pre-Silurian ancestors. The blastodisc stage is followed by a knob-like germinal center that gives rise to most of the embryo body. The germinal center elongates on the ventral surface of the spherical yolk mass. The broad cephalic lobe is first delineated from the following pedipalpal segment. The limbbuds for the pedipalps and anterior walking legs appear, as additional segments are added at a growth zone at the rear of the embryo body. Initially, in the cephalic lobe there are no limbbuds; then the cheliceral buds emerge from the posterior part of the lobe. The stomodeum appears first in the anterior half of the cephalic lobe, but an oral groove forms and the mouth is displaced posteriorly within the groove. This repositioning allows space anteriorly for invagination (semilunar grooves) of epithelium for the brain and medial eyes. The mouth is directed ventrally in all stages of this study. The widespread chelicerae are initially posterior to the mouth, but later move anterior and dorsal to it. Small limbbud bulges on mesosomal segments disappear later and never become protruding appendages. Metasomal segments are produced free from the yolk surface in a ventral flexure beneath the embryo body. The telson starts as two spherical lobes, but later elongates and tapers distally, not yet developing the sharp sting (aculeus) seen in Silurian and all subsequent scorpions. The walking legs are digitigrade, as in most fossil aquatic scorpions. Segments are delineated in the appendages; the chelicerae and pedipalps are divided distally for chela (claw) formation. Bilateral swellings (limbbuds) on the third abdominal segment become larger than the others, indicating the site of pectine formation. The early fin-like pectines are somewhat posterior in the mesosoma, suggesting ancestral swimming, maneuvering, and balancing for the elongate abdomen. The pectinal surface is initially smooth but later transverse striations increase the surface area as a possible respiratory adaptation. Pectinal teeth (present in Silurian and all subsequent scorpions) and forward movement and merging of anterior abdominal segments are not yet evident in embryos of this study.

Animals↗
PubMed1986

Influence of dietary fiber on lipids and aortic composition of vervet monkeys.

A semipurified, cholesterol-free diet containing 40% carbohydrate can produce aortic sudanophilia or aortic atherosclerosis in vervet monkeys (Cercopithecus aethiops pygerethrus) depending on the particular carbohydrate fed. Four groups of vervet monkeys (three males and three females per group) were fed semipurified diets containing lactose. Two of the groups were also fed 15% cellulose (C) or 15% cellulose plus 0.1% cholesterol (CC); the two other groups were fed 15% pectin (P) or 15% pectin plus 0.1% cholesterol (PC). The average serum total cholesterol and low density lipoprotein cholesterol levels over the entire feeding period (mg/dl +/- SEM) were, for C, 156 +/- 14 and 95 +/- 5; for P, 173 +/- 15 and 112 +/- 8; for CC, 187 +/- 27 and 122 +/- 21; and for PC, 155 +/- 11 and 108 +/- 7. Cholesterol levels at autopsy (mg/dl +/- SEM) were, for C, 103 +/- 6; for P, 108 +/- 16; for CC, 92 +/- 9; and for PC, 106 +/- 7. Aortic sudanophilia (percentage of area) was, for C, 5.9 +/- 2.7; for P, 13.5 +/- 9.4; for CC, 5.3 +/- 2.1; and for PC, 21.6 +/- 10.3. Dietary pectin led to more severe sudanophilia (increased by 129% in the absence of cholesterol and by 308% in its presence) than did cellulose. Analysis of aortic glycosaminoglycans (GAG) revealed that dermatan sulfate levels fell in both cholesterol-fed groups, and chondroitin sulfate fell in aortas of group CC. Heparan sulfate levels were unaffected by cholesterol feeding. Hexuronic acid, galactosamine and hexosamine levels were elevated in the pectin-fed monkeys, but levels were unaffected by dietary cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Cell-wall antigens in mesophyll cells and mesophyll-derived protoplasts of sugar beet: possible implication in protoplast recalcitrance?

We have investigated the possible relation between plant cell-wall constituents and the recalcitrance of the cell to regenerate organs and whole plants in vitro. A temporal and spatial expression of several carbohydrate epitopes was observed both within leaf tissue used for protoplast isolation and within new walls reformed by recalcitrant mesophyll protoplasts of sugar beet ( Beta vulgaris L.); these include four pectic epitopes, one xyloglucan (rhamnogalacturonan I) epitope, two carbohydrate motifs of arabinogalactan proteins (AGPs) and callose. The walls of mesophyll cells and newly formed walls of protoplasts were similar with respect to the presence of large amounts of pectins recognized by JIM7 antibodies, the scarcity of JIM5-pectins and the complete absence of LM5-responding pectin molecules. Their main differences were the significantly higher accumulation of LM6-recognizing pectins and the very conspicuous greater accumulation of AGPs and callose in walls deposited by protoplasts than in those synthesized by donor cells.

Antigens↗
PubMed2005

Purification and characterization of a novel pectinase from Acrophialophora nainiana with emphasis on its physicochemical properties.

An extracellular pectinase (PECI) was purified to apparent homogeneity from liquid state cultures of the thermophilic fungus Acrophialophora nainiana by ultrafiltration and a combination of gel filtration and ion-exchange chromatographic procedures. The molecular masses of PECI were 35,500 and 30,749 Da, as determined by SDS-PAGE and mass spectrometry, respectively. It was more active at 60 degrees C and pH 8.0 and showed high stability at 50 degrees C with half-life of 7 days. However at 60 and 70 degrees C, PECI was much less stable with half lives of approximately 20 and 3 min, respectively. The thermostability of purified PECI was also investigated by fluorescence and circular dichroism spectroscopy. Fluorescence revealed that the unfolding transition region was observed between 45 and 70 degrees C. A major decrease in the stability was found at 70 degrees C. Circular dichroism measurements at pH between 5.0 and 9.0 showed a transition temperature (T(m)) range of 50-55 degrees . The thermodynamic analysis of these results showed that EPGI is thermal stable protein exhibiting maximum stability (DeltaG(25)) of 22.65 and 19.19 kcal/mol at pH 8.0 and 9.0, respectively. The apparent K(m) value on pectin from citrus fruits was 4.22 mgml(-1). PECI exhibited no detectable activity of pectin methylesterase, endo-polygalacturonase, mannanase, xylanase and cellulase. However, it showed exo-polygalacturonase and pectin lyase activities. The presence of carbohydrate was detected in the pure PECI. It was activated by l-tryptophan, DEPC, DTT, DTNB, DTP, l-cystein and beta-mercaptoethanol and inhibited by NBS, Fe(2+), Cu(2+), Zn(2+), Mn(2+), Al(3+) and Ca(2+). The enzyme showed homology with a pectin lyases from Xanthomonas campestris and Bacillus licheniformis.

Amino Acid Sequence↗