Polygalacturonic Acid Trans-eliminase Research Articles

Sucrose, cell wall, and polyamine metabolisms involve in preserving postharvest quality of ‘Zaosu’ pear fruit by L-glutamate treatment

‘Zaosu’ pear fruit is prone to yellowing of the surface and softening of the flesh after harvest. This work was performed to assess the influences of L-glutamate treatment on the quality of ‘Zaosu’ pears and elucidate the underlying mechanisms involved. Results demonstrated that L-glutamate immersion reduced ethylene release, respiratory intensity, weight loss, brightness (L*), redness (a*), yellowness (b*), and total coloration difference (ΔE); enhanced ascorbic acid, soluble solids, and soluble sugar contents; maintained chlorophyll content and flesh firmness of pears. L-glutamate also restrained the activities of neutral invertase and acid invertase, while enhancing sucrose phosphate synthetase and sucrose synthase activities to facilitate sucrose accumulation. The transcriptions of PbSGR1, PbSGR2, PbCHL, PbPPH, PbRCCR, and PbNYC were suppressed by L-glutamate, resulting in a deceleration of chlorophyll degradation. L-glutamate concurrently suppressed the transcription levels and enzymatic activities of polygalacturonases, pectin methylesterases, cellulase, and β-glucosidase. It restrained polygalacturonic acid trans-eliminase and pectin methyl-trans-eliminase activities as well as inhibited the transcription levels of PbPL and Pbβ-gal. Moreover, the gene transcriptions and enzymatic activities of arginine decarboxylase, ornithine decarboxylase, S-adenosine methionine decarboxylase, glutamate decarboxylase, γ-aminobutyric acid transaminase, glutamine synthetase along with the PbSPDS transcription was promoted by L-glutamate. L-glutamate also resulted in the down-regulation of PbPAO, PbDAO, PbSSADH, PbGDH, and PbGOGAT transcription levels, while enhancing γ-aminobutyric acid, glutamate, and pyruvate acid contents in pears. These findings suggest that L-glutamate immersion can effectively maintain the storage quality of ‘Zaosu’ pears via modulating key enzyme activities and gene transcriptions involved in sucrose, chlorophyll, cell wall, and polyamine metabolism.

Repression of cell wall metabolism by calcium lactate enhances the postharvest quality maintenance of Jinfeng pear fruit

Quality deterioration of pear fruit is a significant factor contributing to postharvest losses. Calcium lactate, as a food additive, was investigated in this study to estimate its impacts on quality indexes and cell wall metabolism of Jinfeng pear fruit during storage at room temperature. Results demonstrated that calcium lactate effectively maintained higher levels of titratable acidity, flesh firmness, and soluble solids content in pears. Additionally, it delayed the decline of chlorophyll content and inhibited the changes in L*, a*, b* values, and ΔE of pear fruit. The activities and gene transcriptions of pectin methyltranseliminase, pectate lyase, polygalacturonase, pectinmethylesterase, β-glucosidase, cellulase, and β-galactosidase of pears were also inhibited by calcium lactate. Moreover, calcium lactate delayed the decrease of water-insoluble pectin and cellulose contents, and inhibited the increase of water-soluble pectin content and polygalacturonic acid transeliminase activity. These results imply that postharvest application of calcium lactate can effectively retard the softening process of Jinfeng pears and maintain their postharvest quality by regulating cell wall metabolism.

Effects of iron on the asexual reproduction and major virulence factors of Curvularia lunata

Curvularia lunata (Wakker) Boed, the causal agent of Curvularia leaf spot, is an important fungal pathogen in maize. Iron deficiency or excess plays a crucial role in mycelial growth and virulence of plant pathogenic fungi. In this study, the following pathogenic characteristics of C. lunata under different Fe3+ concentrations were investigated: mycelial growth, conidiation, conidium germination, virulence in maize, toxin and cell wall degrading enzymes (CWDEs) activity. The results showed that iron had a limited impact on the mycelial growth, while it had an obvious impact on mycelial biomass, conidiation, and the conidial germination at a concentration of 40–50 μM Fe3+. Exogenous supplementary of Fe3+ also significantly enhanced the pathogenicity of C. lunata CX-3 strain on maize inbred line Huangzaosi. However, toxin activity was independent of Fe3+ concentration. Exogenous iron application improved polygalacturonase (PG) activity in the early stages of infection, and the activity of cellulase (Cx), pectin polymethyl galacturonase (PMG), polygalacturonic acid transeliminase (PGTE) and pectin methyl transeliminase (PMTE) after 72 h of infection in maize.

The MAP Kinase Kinase Gene AbSte7 Regulates Multiple Aspects of Alternaria brassicicola Pathogenesis.

Mitogen-activated protein kinase (MAPK) cascades in fungi are ubiquitously conserved signaling pathways that regulate stress responses, vegetative growth, pathogenicity, and many other developmental processes. Previously, we reported that the AbSte7 gene, which encodes a mitogen-activated protein kinase kinase (MAPKK) in Alternaria brassicicola, plays a central role in pathogenicity against host cabbage plants. In this research, we further characterized the role of AbSte7 in the pathogenicity of this fungus using ΔAbSte7 mutants. Disruption of the AbSte7 gene of A. brassicicola reduced accumulation of metabolites toxic to the host plant in liquid culture media. The ΔAbSte7 mutants could not efficiently detoxify cruciferous phytoalexin brassinin, possibly due to reduced expression of the brassinin hydrolase gene involved in detoxifying brassinin. Disruption of the AbSte7 gene also severely impaired fungal detoxification of reactive oxygen species. AbSte7 gene disruption reduced the enzymatic activity of cell wall-degrading enzymes, including cellulase, β-glucosidase, pectin methylesterase, polymethyl-galacturonase, and polygalacturonic acid transeliminase, during host plant infection. Altogether, the data strongly suggest the MAPKK gene AbSte7 plays a pivotal role in A. brassicicola during host infection by regulating multiple steps, and thus increasing pathogenicity and inhibiting host defenses.

Application of sodium silicate retards apple softening by suppressing the activity of enzymes related to cell wall degradation.

During the storage of apples, apple softening is one of the main problems. Sodium silicate has been used to enhance disease resistance and maintain quality of fruits. In the present study, apple fruit (cv. Golden delicious) were treated with 100 mmol L-1 sodium silicate for 10 min and stored at 20 °C to investigate its effects on weight loss, flesh firmness, and the activity of cell wall-degrading enzymes. The results indicated that 100 mmol L-1 of sodium silicate treatment delayed the increase of weight loss and decrease of the flesh firmness in apples. Sodium silicate treatment also suppressed the activity of polygalacturonic acid transeliminase and pectin methyltranseliminase, pectin methylgalacturonase, polygalacturonase, cellulase and β-galactosidase in the fruit. Delaying apple softening by sodium silicate treatment is closely related to the inhibition of the activity of cell wall-degrading enzymes and weight loss. © 2018 Society of Chemical Industry.

Inhibitory effects of sodium silicate on the fungal growth and secretion of cell wall‐degrading enzymes by Trichothecium roseum

AbstractTrichothecium roseum causes decay in muskmelons, apples, tomatoes and mangoes, which leads to economic losses. In this study, we investigated the effect of sodium silicate on the growth of T. roseum and the cell wall‐degrading enzymes (CWDEs) secreted by the hyphae. The results indicated that sodium silicate significantly inhibited mycelial growth and spore germination of T. roseum. The sodium silicate treatment also retarded the secretion of several CWDEs, including pectate lyase (PL), polygalacturonic acid transeliminase (PGTE), pectin methyltranseliminase (PMTE), pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β‐glucosidase. These results suggest that sodium silicate exerts its effects on T. roseum through direct inhibition of its growth and secretion of CWDEs.

Optimization of Fermentation Reaction Conditions for Preparation of PATE Enzyme by Erwinia carotovora IFO3830 by TFCCRD Method

The fermentation conditions for preparation of polygalacturonic acid transeliminase (PATE) enzyme by Erwinia carotovora IFO3830 were optimized for seed ratio, vibration rate, and temperature by the TFCCRD method. The results indicated that the optimum fermentation conditions for E. carotovora IFO 3830 were that seed ratio, vibration rate, and temperature were 5% v/v, 113 min(-1), and 29 degrees C, respectively.

Optimization of fermentation conditions for preparation of polygalacturonic acid transeliminase by Erwinia carotovora IFO3830.

This paper is concerned with optimization of the fermentation conditions for preparation of alkaline pectin lyase (poly-galacturonic acid trans-eliminase, PATE) by Erwinia carotovora IFO3830 (E.C.IFO3830). The orthogonal matrix method was adopted as the experimental design method for media. The effects of media composition on the growth rate of E.C.IFO3830 are in the order of pectin > KH(2)PO(4)/Na(2)HPO(4) > MgSO(4) > (NH(4))(2)SO(4) > glucose > L-glutamate sodium, and those on PATE activity are in the order of glucose > pectin > L-glutamate sodium > KH(2)PO(4)/Na(2)HPO(4) > MgSO(4) > (NH(4))(2)SO(4). The strain of E.C.IFO3830 grows quickly and easily near an initial pH of 7.0, and especially at the alkaline range of pH 7.0-8.0. The growth rate profiles indicate that E.C.IFO3830 grows very quickly; its generation time t(1/2) is about 0.2 h. The appropriate fermentation period is about 10-20 h for a high level productivity of biomass and 25-35 h for high productivity of PATE enzyme.

Characterization of intracellular polygalacturonic acidtrans-eliminase fromKlebsiella oxytoca, Yersinia enterocolitica, andErwinia chrysanthemi

Intracellular polygalacturonic acidtrans-eliminase (PATE) was purified and characterized fromKlebsiella oxytoca andYersinia enterocolitica, enterobacteria unable to macerate plant tissue. The well-studied PATE from a strain ofErwinia chrysanthemi, a phytopathogen able to macerate plant tissue and cause soft-rot disease, was included for comparison. PATE from all strains displayed endo-splitting activity with pH optima between pH 8.5 and 9.0E. chrysanthemi had three isozymes (pls at pH 9.4, 9.0, and 7.8),K. oxytoca had two isozymes (pIs at pH 5.9 and 5.3), andY. enterocolitica had one isozyme (pI at pH 5.8). Molecular weights for theKlebsiella andYersinia PATEs were 71,000 and 55,000, respectively, compared with 33,000 for theErwinia PATE. Unlike theErwinia enzyme, theKlebsiella andYersinia PATEs did not require divalent cations for activity and could not macerate plant tissue without addition of pectinmethylesterase. The polygalacturonic acid-degrading enzymes found inK. oxytoca andY. enterocolitica appear to represent a separate type of PATE enzyme. It is unlikely that these organisms are phytopathogens; however, their ability to degrade polygalacturonic acid is probably advantageous to their survival in environments containing decomposing plant residues.

Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia, and Klebsiella species

Various enterobacteria exhibit several patterns of synthesis and excretion of polygalacturonic acid trans-eliminase (PATE), one of the enzymes involved in degradation of pectic substances. In a strain of Erwinia chrysanthemi, PATE is an extracellular enzyme and is almost totally excreted. In strains of Yersinia enterocolitica and Yersinia pseudotuberculosis, PATE is a periplasmic and cytoplasmic enzyme. In a strain of Erwinia carotovora, PATE activity is extracellular, periplasmic, and cytoplasmic. In an "oxytocum" strain of Klebsiella pneumoniae, PATE is entirely cytoplasmic; none is found in the periplasm and almost none in culture supernatants. Cells (but not supernatants, because PATE is not excreted) of K. pneumoniae show much higher levels of PATE activity when grown on polygalacturonate than when grown on other carbon sources. A clinical strain of Y. enterocolitica synthesizes high levels of PATE activity (almost all cell-bound) when grown in the presence of gluconate, glycerol, or polygalacturonate, and considerably less activity when grown on glucose. A clinical strain of Y. pseudotuberculosis synthesizes barely detectable quantities of cell-bound PATE regardless of the carbon source. The phytopathogenic Erwinia strains synthesize and excrete high levels of PATE activity when grown on polygalacturonate, galacturonate, gluconate, or glycerol. Growth on glucose represses PATE activity in the Erwinia strains; the extent of repression differs between the two strains. The different patterns of synthesis and excretion are discussed in the light of the possible catabolic and (or) cytolytic (plant tissue-macerating) functions and the related ecological significance of PATE in these enterobacteria.

Donor strains of the soft-rot bacterium Erwinia chrysanthemi and conjugational transfer of the pectolytic capacity.

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F'lac(+) plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F'lac(+) heterogenote by repeated platings of single Lac(+) colonies on lactose minimal agar, do not segregate (as does the parent F'lac(+) heterogenote) into Lac(-) or F(-) clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), lac(+), leu(+), lys(+), mcu(+) (multiple carbohydrate utilization), pat(+) (production of polygalacturonic acid trans-eliminase), thr(+), and trp(+) in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade(+), leu(+), and thr(+) transfer were higher than those of the other markers tested to date. This donor strain transfers lac(+) genes during a 6-h mating on membranes; most of the Lac(+) recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr(+) and leu(+) enter the recipient as proximal markers and that lac(+) enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F'lac(+) into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat(+) recombinants and not in Pat(-) recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.

Enzymatic Degradation of Polygalacturonic Acid by Yersinia and Klebsiella Species in Relation to Clinical Laboratory Procedures

As scored by several specified plating procedures, clinical and environmental strains of Yersinia enterocolitica, Yersinia pseudotuberculosis, and Klebsiella pneumoniae "Oxytocum" showed detectable, albeit generally weak, ability to digest polygalacturonic (pectic) acid. None of these bacterial strains had the vigorous and rapid pectolytic activity on these polygalacturonic acid-containing media that is typical of soft-rot Erwinia species, although some of the Oxytocum strains came fairly close. Analyses of the pectolytic enzyme contents of the cells and culture supernatants of the Yersinia and Klebsiella species revealed that readily detectable quantities of cell-bound polygalacturonic acid trans-eliminase and hydrolytic polygalacturonase were formed by the Yersinia and Klebsiella species; however, the total units of enzyme activity produced by these bacteria were, in general, lower than were produced by soft-rot Erwinia species. Furthermore, unlike the situation in soft-rot Erwinia cultures, these pectolytic enzymes of Yersinia and Klebsiella species were not excreted rapidly and massively into the growth medium. Cultures of other enterobacteria (Citrobacter species, Enterobacter species, Erwinia amylovora, Erwinia herbicola, Escherichia coli, Proteus species, Salmonella typhimurium, and Serratia marcescens) showed no pectolytic ability whatsoever by any of the plating procedures used and (to the extent they were so examined) produced no pectolytic enzymes detectable either in their cells or culture supernatants. This slow or weak release of pectolytic enzymes by Yersinia and Klebsiella species has a bearing on clinical laboratory procedures suitable for detecting their pectolytic activity; methods adequate for this purpose are detailed.

Polygalacturonic acid trans-eliminase in the osmotic shock fluid of Erwinia rubrifaciens: characterization of the purified enzyme and its effect on plant cells.

An endopolygalacturonic acid trans-eliminase (EC 4.2.2.2), released by osmotic shock of Erwinia rubrifaciens cells, has been purified to near homogeneity (3, 100-fold) by column chromatography on diethylaminoethyl-cellulose, phosphocellulose, and hydroxyapatite-cellulose followed by isoelectric focusing. It has a molecular weight of 41,000, s20,w of 3.09S, an isoelectric point of pH 6.25, pH optimum of 9.5, and a temperature optimum of 37 C and requires Ca2+ with an optimum concentration of 0.5 to 1.0 mM. Mg2+ could not substitute for Ca2+. Tyrosinyl residues seem essential for enzyme catalysis based on rapid inactivation by tetranitromethane. The enzyme prefers unmethylated polygalacturonic acid as the substrate, cleaving alpha-1,4-glycosidic linkages randomly to form unsaturated galacturonides at a Vmax of 1,166 mumol of product/min per mg of protein and a Km of 5 mg of polygalacturonic acid per ml. Over 90% of the enzyme activity is released from osmotically shocked E. rubrifaciens cells. Unlike E. rubrifaciens, trans-eliminase is not released from Erwinia carotovora cells by osmotic shock treatment, but enzyme activity is detected in the culture medium. The release of the enzyme is reduced fivefold by the addition of dibutyryl cyclic adenosine 5'-monophosphate. The hypersensitive reaction in tobacco leaves was induced within 60 min after injection of less than 1 mug of purified E. rubrifaciens trans-eliminase. Single cells of tobacco in suspension culture are readily killed by the enzyme, whereas tobacco protoplasts remain unaffected when treated in the same manner. These results indicate that endopolygalacturonic acid trans-eliminase is a constitutive enzyme possibly located in the periplasmic space of the E. rubrifaciens cell and releases enzyme into the culture medium in the presence of substrate. The release of the enzyme in tobacco tissue and the trans-eliminative cleavage of plant cell wall components may be steps leading to hypersensitivity of the tobacco tissue.

Preparation, separation and degradation of oligouronides produced by the polygalacturonic acid transeliminase of bacillus pumilus

Production of the end products of polygalacturonic acid degradation on a large scale was done by reacting free galacturonic acid with Bacillus pumilus polygalacturonic acid transeliminase (PATE, EC 4.2.2.2) to obtain a mixture of the barium salts of several oligouronides. Small amounts of the unsaturated oligouronides were separated by paper chromatography. Large quantities of unsaturated oligouronides were separated on a AG-I-X8 (formate) column by applying a sample of mixed oligouronides and stepwise elution was carried out with sodium formate buffer (pH 4.7). The unsaturated oligouronides were identified on the basis of chromatographic mobilities, Sephadex gel filtration data, COOH/CHO ratio, thiobarbituric acid-reacting material, bromine uptake, and chemical and enzymatic degradation data as unsaturated tri-, tetra-, and hexagalacturonic acids. The chemical degradation of these unsaturated oligouronides, done with 6 N HCl by heating at 100° for 30 min, gave qualitatively identical products of hydrolysis. These products compared with authentic standards, were identified as galacturonic acid, formic acid, 5-formyl-2-furancarboxylic acid, and 2-furancarboxylic acid. Analysis of the enzymatic breakdown products of the higher unsaturated uronides showed that a minimum of four galacturonic acid units was required for the action of purified endo-PATE from B. pumilus. The unsaturated trimer was not attacked, thus accounting for its accumulation as the major end product of polygalacturonate degradation by this enzyme.

Pectic enzymes produced by Pseudomonas fluorescens, an organism associated with “pink eye” disease of potato tubers

No pectin methylesterase (PME) activity was observed in crude or dialyzed extracts from macerated potato tuber tissue inoculated with Pseudomonas fluorescens; however, pectic lyase (syn. polygalacturonic acid transeliminase, PATE) activity was observed. Two PATE enzymes (peaks 1 and 2) were eluted from a pH 9.4 DEAE-cellulose column using a gradient of pH 7.6 Tris-HCl buffer (0.01–0.1 M). Enzyme in peak 1 was about 6 times more active than enzyme in peak 2 based on reducing group assays, and 10 times more active in viscosity reduction of 1% Na-polypectate (NaPP) at pH 8.5. No increase in absorbancy was observed at 515 nm of clarified reaction mixtures, indicating that saturated oligouronides did not accumulate. Other properties of the two peaks: optimum pH range was 8.5–9.5, substrate preference was NaPP vs. pectin, addition of Ca2+ (0.001 M) enhanced activity while EDTA (0.001 M) decreased activity to [Formula: see text], cellulose acetate electrophoresis revealed one band of protein per peak, and heat of inactivation was 51–60C. Thin-layer chromatography of hydrolytic products from NaPP revealed unsaturated uronides and pectic fragments after 2 h hydrolysis; after 96 h hydrolysis only unsaturated uronides were observed. Molecular weight estimations by Sephadex G-200 gel filtration were about 18 000 (peak 1) and 22 500 (peak 2). Enzyme in either peak macerated 400-μ sections of potato tissue.

A New Pectolytic Enzyme in Erwinia aroideae Formed in the Presence of Nalidixic Acid

Pectolytic enzyme formation by whole cells of Erwinia aroideae was markedly stimulated when nalidixic acid was added to a culture medium. The activity of pectolytic enzyme was markedly stimulated by nalidixic acid when the activity was measured by the decrease of viscosity of pectin, while activities of both polygalacturonic acid trans-eliminase and polygalacturonase which were measured respectively by the increase of optical density at 230 nm and the liberation of aldehyde groups, were not stimulated. The analysis of pectolytic enzyme by carboxymethyl cellulose column chromatography indicated that there was a significant difference in the elution profiles between the pectolytic enzyme induced by nalidixic acid and that synthesized under normal conditions. Therefore, we conclude that two enzymes are distinct protein species.

Pectic enzymes produced by Erwinia chrysanthemi and their effects on plant tissue

Erwinia chrysanthemi produced extracellular enzymes which degrade pectic acid in a trans-eliminative manner. Isolate 307, from carnation, produced at least two, and isolate EC 23, from Philodendron, produced at least four polygalacturonic acid trans-eliminases. The enzymes produced by each isolate were separated from each other and purified by ammonium sulfate fractionation, diethylaminoethylcellulose column chromatography, and electrofocusing. Molecular weights for the six enzymes ranged between 30,000 and 36,000 as determined by gel filtration in Sephadex G-75 and sucrose density gradient centrifugation. The isoelectric points of two enzyme fractions from isolate 307 were pH 9·1 and 7·9 and those for the four enzyme fractions from isolate EC 23 were pH 9·4, 8·4, 7·9 and 4·6. Random cleavage of pectic acid was greatest with the enzyme fractions having isoelectric points at pH 9·4 and 9·1, intermediate for the fraction with an isoelectric point at pH 4·6, and least with the fractions with isoelectric points at pH 8·4 and 7·9. All of the trans-eliminases exhibited a Ca 2+ requirement and had pH optima between pH 8·2 and 9·8. All but one of the purified polygalacturonic acid trans-eliminase fractions from both E. chrysanthemi isolates induced electrolyte loss, maceration and cell death of potato tissue. The enzyme fraction with an isoelectric point at pH 4·6 from isolate EC 23 did not macerate potato, carrot or cucumber tissue even though it readily degraded pectic acid prepared from onion, carrot and cucumber as well as commercial sources of sodium polypectate, polygalacturonic acid and pectin N.F.

Metabolic regulation of polygalacturonic acid trans-eliminase in Erwinia.

Strains of Erwinia carotovora and Erwinia aroideae, formerly thought only to be inducible, are constitutive for polygalacturonic acid trans‐eliminase. Maximum enzyme formation, however, occurs in the presence of polygalacturonic acid. A release from strong catabolic repression enables glucose‐grown cultures to initiate their attack on polygalacturonic acid and, thereby, eliminates the necessity for the entry of the polymer into the cell. A low constant proportion (less than 25%) of the total polygalacturonic acid trans‐eliminase is present in the culture fluid during the exponential growth‐phase of Erwinia carotovora. The enzyme may be regarded as an exoenzyme. The exact location of polygalacturonic acid trans‐eliminase in the cells was not established; it could be truly intracellular or reside between the cell‐wall and membrane.

Production and catabolite repression of the constitutive polygalacturonic acid trans-eliminase of Aeromonas liquefaciens.

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.

Extracellular and intracellular polygalacturonic acid trans-eliminases of Erwinia carotovora

Polygalacturonic acid trans-eliminase (PATE) was purified from the culture fluid of Erwinia carolovora, completely free from contamination by polygalacturonase and pectinesterase. The enzyme attacks polygalacturonic acid, in preference to pectin; however, pectin is attacked to an extent dependent upon the degree of demethoxylation of the pectin molecule. Polygalacturonic acid and oligouronides are degraded by Erwinia PATE at rates which decrease as the chain length of the substrate decreases; digalacturonic and unsaturated digalacturonic acids are not attacked by this enzyme. Erwinia PATE acts randomly, has an optimum pH of 8.5, and is stimulated by calcium. The major endproduct from polygalacturonic acid is O-(4-deoxy-β- l-5- threo-hexopyranos-4-enyluronic acid)-(1→4)- d-galacturonic acid (“unsaturated digalacturonic acid”); lesser amounts of unsaturated trigalacturonic acid and saturated mono- and digalacturonic acids are formed. From the cells of the same organism, an intracellular enzyme wag isolated which has properties similar to those of the extra-cellular polygalacturonic acid trans-eliminase.