Purification and Properties of a Pectin trans-Eliminase in Erwin aroideae Formed in the Presence of Nalidixic Acid

A strain of Erwinia aroideae produces a remarkable amount of pectolytic enzyme when the organism was induced by nalidixic acid for the bacteriocin production. This pectolytic enzyme was purified approximately 60-fold from the induced medium by carboxymethyl-cellulose and Sephadex G–75 gel column chromatographies after batchwise treatment with carboxymethyl- and diethylaminoethyl-celluloses. The purified enzyme was almost homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a molecular weight of about 28,000 to 32,000 was determined for this enzyme. The optimum pH of the enzyme activity was about 8.0 to 8.2. The purified enzyme produced reaction products from pectin and methoxylated pectic acid which had a strong absorption at 235 nm indicating a trans-eliminase reaction. Pectin or pectic acid with higher methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectic acid was a substrate. The limit of degradation of pectin and pectic acid with higher methoxyl content (90% esterified) by the enzyme were 6.5% and 43%, respectively. It was concluded that the enzyme is a new endo-pectin trans-eliminase from bacterial origin.     

Exopectic Acid Transeliminase of an Erwinia

A species of Erwinia was found to produce no other pectolytic enzyme than the two transeliminases of exo-types, namely, an oligogalacturonide transeliminase and an exopectic acid transeliminase. Of the two enzymes, the exopectic acid transeliminase was isolated and its properties were investigated. The results are as follows: (1) Pectic acids having an unsaturated galacturonic acid residue at the non-reducing end of the molecule are susceptible but oxidized or reduced pectic acids resistant to the enzyme action. (2) The enzyme has no activity toward pectinic acid and polymethylpolygalacturonate methyl glycoside. The limit of the enzymatic degradation for citrus pectic acid is 43.8%. (3) The rate of the enzyme activity was maximal with tetragalacturonic acid and followed by acid-soluble pectic acid, acid-insoluble pectic acid, pectic acid and trigalacturonic acid. Unlike the oligogalacturonide transeliminases of Pseudomonas sp. (strain S2) and Erwinia aroideae, the present enzyme shows a considerably high activity toward pectic acids of high molecular weight. (4) The pH-activity curves vary with the buffer employed. (5) The enzyme is activated by Co²⁺ and Mn²⁺ but powerfully inhibited by Cu²⁺ and Hg²⁺. Ca²⁺ has no significant effect on the enzyme activity.     

The CDTA-soluble pectic substances from soybean meal are composed of rhamnogalacturonan and xylogalacturonan but not homogalacturonan

Structural characteristics of pectic substances extracted from soybean meal cell walls (water unextractable solids) with a chelating agent-containing buffer (0.05M 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) and 0.05M NH(4)-oxalate in 0.05M NaOAc buffer) were studied. The arabinogalactans present as side chains to the rhamnogalacturonan backbone were largely removed by enzymatic hydrolysis using endo-galactanase, exo-galactanase, endo-arabinanase, and arabinofuranosidase B. The remaining pectic backbone appeared to be resistant to enzymatic degradation by pectolytic enzymes. After partial acid hydrolysis of the isolated pectic backbone, one oligomeric and two polymeric populations were obtained by size-exclusion chromatography. Monosaccharide and linkage analyses, enzymatic degradation, and NMR spectroscopy of these populations showed that the pectic substances in the original extract contain both rhamnogalacturonan and xylogalacturonan regions, while homogalacturonan is absent.     

Hydrolysis of plant polysaccharides and GLC analysis of their constituent neutral sugars

The release and degradation of sugars from onion cell walls and potato cell wall polysaccharides were followed during hydrolysis with trifluoroacetic acid so that the optimum hydrolysis conditions could be determined. After 6 hr hydrolysis in 2 M acid at 100°, calculated recovery factors of different monosaccharides from potato pectic fractions ranged from 61 to 96%. Lower yields of monosaccharides were obtained from intact onion cell walls, while the yield from cellulose was less than 0.2%. A new GLC column for the separation of alditol acetates derived from cell wall sugars is described.     

An exo-poly-alpha-D-galacturonosidase implicated in the regulation of extracellular pectate lyase production in Erwinia chrysanthemi.

Pectic enzymes in the supernatants of Erwinia chrysanthemi cultures in late-logarithmic-phase growth on D-galacturonan were resolved into three components: two pectate lyase isozymes and an exo-poly-alpha-D-galacturonosidase previously unreported in this organism. The hydrolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, preparative electrofocusing in Ultrodex gel, and gel filtration through Ultrogel AcA54. The enzyme had a specific activity of 591 mumol/min per mg of protein, a pI of 8.3, a molecular weight of 67,000, a pH optimum of 6.0, and a Km of 0.05 mM for D-galacturonan. Analyses of reaction mixtures by paper chromatography revealed that the enzyme released only digalacturonic acid from D-galacturonan. The action of the hydrolytic enzyme on D-galacturonan labeled at the nonreducing end by partial digestion with pectate lyase revealed that it rapidly released 4,5-unsaturated digalacturonic acid from 4,5-unsaturated pectic polymers. The production of extracellular exo-poly-alpha-D-galacturonosidase was coordinately regulated with pectate lyase production. The action patterns of the two enzymes appeared complementary in the degradation of pectic polymers to disaccharides that stimulated pectic enzyme production and supported bacterial growth.     

Molecular cloning and cytochemical analysis of <Emphasis Type="Italic">exo</Emphasis>polygalacturonase from carrot

Exopolygalacturonase (exo-PGase, EC 3.2.1.67) attacks the non-reducing terminus of the polygalacturonic acid in pectic molecules, releasing galacturonic acid. We cloned the cDNA of exo-PGase purified from cell homogenates of suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells. The nucleotide sequence of the cDNA (1.4 kb) contains an open reading frame that encodes a 391-amino-acid polypeptide. Sequence homology research showed 97.9% identity to the glycoprotein EP4 obtained from cultured carrot cells and 49.3% identity to the ENOD8 gene product of alfalfa ( Medicago sativa). However, no significant similarity was found to known PGases. The Southern hybridization pattern indicated that this exo-PGase protein is a member of a small-sized gene family. Predominant expression of the exo-PGase gene was detected by in situ hybridization and immunohistochemistry in the root apical meristem and in the elongation region, but not in the root cap. A cross-immunoresponse with anti-exo-PGase also occurred in the root nodule meristem of alfalfa. These results suggest that this exo-PGase plays a role in the degradation of pectic molecules during root development.     

Inhibitory effects of pectic substances on activated hyaluronidase and histamine release from mast cells.

In this paper, we report the effect of pectic substances and D-galacturonic acid, the main constituent of pectic substances, on activated hyaluronidase and histamine release from mast cells. Although D-galacturonic acid itself showed no inhibition, IC50 values of hyaluronidase inhibition were correlated with the D-galacturonic-acid content of pectic substances. It was thought that the polymerization of D-galacturonic acid was necessary for inhibition of activated hyaluronidase. This type of inhibition was suggested to be non-competitive by the Lineweaver-Burk plot. Furthermore, pectic substances, including those purified from Gymnema sylvestre, inhibited histamine release from isolated rat peritoneal mast cells, which had been induced by the antigen. These results suggest that pectic substances may have anti-allergic activities.     

Cell Wall Dissolution in Ripening Kiwifruit (Actinidia deliciosa) : Solubilization of the Pectic Polymers

Pectic polysaccharides solubilized in vivo during ripening, were isolated using phenol, acetic acid, and water (PAW) from the outer pericarp of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang and A.R. Ferguson var deliciosa `Hayward') before and after postharvest ethylene treatment. Insoluble polysaccharides of the cell wall materials (CWMs) were solubilized in vitro by chemical extraction with 0.05 molar cyclohexane-trans-1,2-diamine tetraacetate (CDTA), 0.05 molar Na₂CO₃, 6 molar guanidinium thiocyanate, and 4 molar KOH. The Na₂CO₃-soluble fraction decreased by 26%, and the CDTA-soluble fraction increased by 54% 1 day after ethylene treatment. Concomitantly, an increase in the pectic polymer content of the PAW-soluble fraction occurred without loss of galactose from the cell wall. The molecular weight of the PAW-soluble pectic fraction 1 day after ethylene treatment was similar to that of the Na₂CO₃-soluble fraction before ethylene treatment. Four days after ethylene treatment, 60% of cell wall polyuronide was solubilized, and 50% of the galactose was lost from the CWM, but the degree of galactosylation and molecular weight of pectic polymers remaining in the CWMs did not decrease. The exception was the CDTA-soluble fraction which showed an apparent decrease in molecular weight during ripening. Concurrently, the PAW-soluble pectic fraction showed a 20-fold reduction in molecular weight. The results suggest that considerable solubilization of the pectic polymers occurred during ripening without changes to their primary structure or degree of polymerization. Following solubilization, the polymers then became susceptible to depolymerization and degalactosidation. Pectolytic enzymes such as endopolygalacturonase and β-galactosidase were therefore implicated in the degradation of solubilized cell wall pectic polymers but not the initial solubilization of the bulk of the pectic polymers in vivo.     

Structure of the d-galactan isolated from aloe barbadensis miller

Hot-water extraction of the pulp obtained by dehydrating the jelly of the fleshy leaves of Aloe barbadensis furnished a mixture of polysaccharides containing mainly pectic acid, along with a d-galactan, a glucomannan, and an arabinan. The pectic acid was partly removed by treatment with calcium chloride, and the resulting, hexose-enriched, polysaccharide mixture was fractionated through a column of DEAE-cellulose to yield a d-galactan containing d-galactose (92.9% and d-galacturonic acid (3.8%). Hydrolysis of the permethylated d-galactan furnished 2,3,4,6-tetra-, 2,3,6-tri-, and 2,3-di-O-methylgalactose in the molar ratios of 1:26:1. On periodate oxidation, the d-galactan reduced 0.95 molar equivalent of the oxidant per hexosyl residue, and liberated one molar equivalent of formic acid per 26 galactosyl residues. Smith degradation of the d-galactan afforded mainly threitol. From these results, a structure has been assigned to the repeating unit of the d-galactan. The number-average, molecular weight of the peracetylated galactan has been found to be 3.74 x 10⁴.     

Heterogenous Degradation of Myofibrillar Proteins

Pectic substances were extracted from the vegetables with oxalate buffer of pH 4.25 and, after saponification, fractionated into two components, weakly acidic pectic polysaccharide (WAP) and pectic acid, by DEAE-cellulose and Sephadex G-100 chromatographies. The galacturonic acid content (17.3~25.8%) of WAPs was much lower than that of pectic acids, though the neutral sugar compositions of both pectic substances were almost the same. The arabinose-galactose side chains were found to be very long or highly branched in WAPs compared with those in pectic acids.

All the WAPs were appreciably hydrolyzed by exo- and endopolygalacturonases. The limited-degradation products (the residual polysaccharides; i.e., the rhamnogalacturonan segments) obtained by endopolygalacturonase from both WAPs and pectic acids showed a similar behavior on Sephadex G-100 and Sepharose CL-4B gel filtrations; each of the rhamnogalacturonan segments was eluted in the void volume of the Sephadex G-100 column. From these results, we concluded that WAPs are probably an inherent pectic component of the cell walls of the vegetables.     

A simple procedure for the synthesis of alpha-32P-nucleoside triphosphates.

Chemical analysis of grapefruit (Citrus paradisi) pectic polysaccharides demonstrated that galacturonic acid constitutes 78% by weight of the total carbohydrates found. The remaining 22% was accounted for by a number of sugars which include galactose, glucose, arabinose, xylose, and mannose and, by weight, galactose accounted for almost 50% of the total neutral sugar components found in these pectic polysaccharides. Treatment of pectic polysaccharides with galactose oxidase followed by reduction of oxidized galactose residues with tritiated potassium borohydride resulted in the labeling of pectic polysaccharides. Analysis of the labeled polysaccharides demonstrated that of the total radioactivity incorporated more than 90% was recovered in the galactose residues. These results clearly demonstrate the successful utilization of the galactose oxidase/tritiated potassium borohydride method in labeling plant pectic polysaccharide.     

Cell-wall polysaccharides and glycoproteins of parenchymatous tissues of runner bean (Phaseolus coccineus).

1. Polymers were solubilized from the cell walls of parenchyma from mature runner-bean pods with minimum degradation by successive extractions with cyclohexane-trans-1,2-diamine-NNN'N'-tetra-acetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue, which contained cross-linked pectic polysaccharides and Hyp-rich glycoproteins. These were solubilized with chlorite/acetic acid and cellulase. The polymers were fractionated by anion-exchange chromatography, and fractions were subjected to methylation analysis. 2. The pectic polysaccharides differed in their ease of extraction, and a small proportion were highly cross-linked. The bulk of the pectic polysaccharides solubilized by CDTA and Na2CO3 were less branched than those solubilized by KOH. There was good evidence that most of the pectic polysaccharides were not degraded during extraction. 3. The protein-containing fractions included Hyp-rich and Hyp-poor glycoproteins associated with easily extractable pectic polysaccharides, Hyp-rich glycoproteins solubilized with 4M-KOH+borate, the bulk of which were not associated with pectic polysaccharides, and highly cross-linked Hyp-rich glycoproteins. 4. Isodityrosine was not detected, suggesting that it does not have a (major) cross-linking role in these walls. Instead, it is suggested that phenolics, presumably linked to C-5 of 3,5-linked Araf residues of Hyp-rich glycoproteins, serve to cross-link some of the polymers. 5. There were two main types of xyloglucan, with different degrees of branching. The bulk of the less branched xyloglucans were solubilized by more-concentrated alkali. The anomeric configurations of the sugars in one of the highly branched xyloglucans were determined by 13C-n.m.r. spectroscopy. 6. The structural features of the cell-wall polymers and complexes are discussed in relation to the structure of the cell walls of parenchyma tissues.     

Pectic enzymes associated with Penicillium digitatum decay of citrus fruit

Pectin methylesterase was the only pectic enzyme detectablein extracts from rind of sound or Penicillium digitatum-infectedoranges. No pectic enzymes were detected in juice from soundor infected fruit. Extracts from infected rind, and juice frominfected fruit, had macerating activity. Chromatographic analysesof rind extracts, and juice from infected fruit, showed galacturonicacid as a possible product of the degradation of pectic substances.Orange juice contained a thermo labile inhibitor of pectic ‘chain-splitting’,and macerating enzymes.     

Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged

The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.     

Pectic and cellulolytic enzymes in soybeans

The dominant cell wall degrading enzymes detected in extracts from 6-day-old, dark grown, Lee soybean seedlings carried out random (endo) and hydrolytic cleavages of pectic acid, pectin and methylcellulose. The pH optima of these activities were 6·0, 6·5 and 5·7 respectively. Monogalacturonic acid and another unidentified product accumulated during the degradation of polygalacturonic acid.     

5,6-Anhydro-l-ascorbic acid. A reactive intermediate for the formation of 6-substituted derivatives of l-ascorbic acid

Cell-wall material was isolated from the alcohol-insoluble residue of carrot by treatment with Pronase, phenol—acetic acid—water, and aqueous 90% methyl sulphoxide. Some pectic material was solubilised, but the major component was a highly esterified, acidic arabinogalactan. The purified cell-wall material, which contained ～1% of protein, was sequentially extracted with water at 80°, ammonium oxalate at 80°, and m and 4m KOH at 20°, to leave a residue of α-cellulose, which contained some pectic material. From the hot-water-soluble fraction, a major pectic polymer was isolated by anion-exchange chromatography. Methylation analysis showed that it was a rhamnogalacturonan, probably having highly branched arabinan and slightly branched galactan side-chains linked to O-4 of rhamnopyranosyl residues. An unusual feature of this pectic polymer is that it contained a small but significant proportion of 1,4-linked xylopyranosyl residues. From the alkali-soluble fractions, a range of pectic polymers associated with various amounts of xylans and possibly xyloglucans was isolated. The main linkages present in these complexes were 1,4-linked galactopyranosyluronic acid, 1,4-linked galactopyranosyl, and 1,5-linked arabinofuranosyl residues, terminal arabinofuranosyl and galactopyranosyl groups, and, in some fractions, 1,4-linked xylopyranosyl residues. The possible association of some of these polymers with proteins and phenolics is discussed.     

Structure of the D-fructan isolated from garlic (Allium sativum) bulbs

Extraction of defatted garlic bulbs with hot water yielded a mixture of polysaccharides containing pectic acid, a D-galactan, and a fructan component. The pectic acid was partially removed as calcium pectate, and the galactan-enriched portion was separated by fractional precipitation with alcohol; on concentration and several fractionations, the supernatant liquor furnished the fructan component, which contained fructose (94.4%) and glucose (4.3 %). Methanolysis and hydrolysis of the permethylated fructan gave (a) 1,3,4,6-tetra-O-methyl-D-fructose, (b) 2,3,4,6-tetra-O-methyl-D-glucose, (c) 2,4,6-tri-O-methyl-D-glucose, and (d) 3.4,6-tri-O-methyl-D-fructose in the ratio (a + b): (d) = 1:20.3. On periodate oxidation, the fructan reduced one molar equivalent of the oxidant per hexosyl residue, and liberated one molar equivalent of formic acid per 51 hexosyl residues. On Smith degradation, the major product was glycerol, and ～2 % of the glucose survived. From these results, and from the fact that the fructan is hydrolyzed by β-D-fructofuranosidase, a linear, inulin-type of structure is suggested for it.     

Autolysis-Iike Release of Pectic Polysaccharides from Regions of Cell Walls Other Than the Middle Lamella

Cell walls of a storage organ (potato tubers) showed autolysis-likeactivity. After 20 h of incubation in water at 35°C, thepurified cell walls released approximately 10% of the cell walldry weight as pectic polysaccharides containing about 40% ofthe total galacturonic acid present in the cell walls. Virtuallyno neutral polysaccharides were found in the soluble fraction.The pectic polysaccharides were heterogeneous in galacturonicacid content and had a very large molecular size. The releaseof pectic polymers was caused neither by enzymatic reactionsnor by ß-elimination, but by a chelation of Ca²⁺ and/orother metal ions during the cell wall isolation. Ultrastructuralobservations clearly showed that these pectic polysaccharideswere released not from the middle lamella, but from the primarycell wall adjacent to the plasma membrane. These results indicatethat nearly half of cell wall pectic polysaccharides are heldin the primary wall only by Ca²⁺- and/or other metal-bridgesand that these pectic polymers are not associated with the middlelamella. (Received March 20, 1989; Accepted October 3, 1989)     

Distribution of methoxyl groups in apple pectic substances

The distribution of methoxyl groups in apple pectic substances was investigated by means of fractionation on ion-exchange and gel-filtration columns and by means of degradation of pectin fractions by pectin lyase and pectate lyase. Pectin fragments thus obtained were fractionated by gel-permeation chromatography and high-pressure liquid chromatography. It was concluded that a heterogeneous intermolecular distribution of the methoxyl groups exists with peaks at degrees of esterification of about 50%, 70% and 95%. The intramolecular distribution of the methoxyl groups cannot be distinguished from a random distribution. Since plant pectin esterases cause a blockwise de-esterification, it is unlikely that the biosynthesis of apple pectic substances passes through a stage of 100% esterification after which partial de-esterification by pectin esterase occurs.     

Structure of a pectic polysaccharide fraction from zea shoots

A pectic fraction, accounting for about 0.3% of the total cell wall polysaccharide, was derived from the hot water extract of an insoluble fraction of the buffer-homogenate of Zea shoots. The pectic polysaccharide fraction was characterized by fragmentation analysis after hydrolysis with acid and Erwinia carotovora pectate lyase. The results suggest that the fraction consists of mostly a linear homopolygalacturonan with neutral sugar components or a homogalacturonan and a rhamnogalacturonan with neutral sugar components.