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PubMed2001

Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module.

Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5 kDa. Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules. Sequence similarity was seen between the 9.2 kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs). This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose. A high sequence identity of 66% was seen between the 14.2 kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P. cellulosa. The 35.8 kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp. strain KSM-P15, respectively. This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm). Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH 10 and in the presence of 2 mM Ca2+. Highest enzyme activity was detected at 62 degrees C. Pel10Acm was shown to be most active against pectate (i.e. polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins. These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues. Significantly, Pel10A and the P. cellulosa rhamnogalacturonan lyase 11A, in the accompanying article [McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem. J. 355, 167-177], are the first CBM-containing pectinases described to date.

Amino Acid Sequence↗

PubMed2003

Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima.

The ability of the hyperthermophilic bacterium Thermotoga maritima to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium. The pel A gene of T. maritima was functionally expressed in Escherichia coli as the first heterologously produced thermophilic pectinase, and purified to homogeneity. Gel filtration indicated that the native form of PelA is tetrameric. Highest activity (422 units/mg, with a K(m) of 0.06 mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate. In the tradition of pectate lyases, PelA demonstrated full dependency on Ca(2+) for stability and activity. The enzyme is highly thermoactive and thermostable, operating optimally at 90 degrees C and pH 9.0, with a half-life for thermal inactivation of almost 2 h at 95 degrees C, and an apparent melting temperature of 102.5 degrees C. Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known. The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3-8) and the examination of the bond cleavage frequencies.

Amino Acid Sequence↗

PubMed1998

Occupational asthma in fruit salad processing.

BACKGROUND: Three subjects employed in the preparation of fruit for fruit salads reported work-related respiratory symptoms. Their work entailed removing the peel from citrus fruits, primarily oranges, following soaking of the fruits in a bath of enzymes including fungal derived pectinase and glucanase. Objectives To investigate the respiratory symptoms reported by these workers and determine their causes. METHODS: The three workers were investigated by a respiratory physician, including spirometry and serial peak flow measurements. Blood was taken for the measurement of IgE and IgG antibody responses against the enzyme solution. RESULTS: Predominant symptoms in these workers were shortness of breath, chest tightness and wheezing which were all alleviated at weekends and holidays only to occur when returning to work. Serial peak flow measurements showed a clear work-related pattern. All three had strong IgE responses to the enzyme solution used at the workplace and showed distinct patterns of binding in immunoblots. All three improved immensely following withdrawal from the workplace environment. CONCLUSION: Enzymes appear to be widely used in the preparation of fruit and although they are used in liquid form, exposure can occur to induce immunological sensitization and asthma.

Adult↗

PubMed1998

Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium.

The drought-tolerant legume Hedysarum coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions. The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils. With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of H. coronarium. Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature. Enzyme activities, including those of cellulase, pectinase, urease, beta-galactosidase, nitrate and nitrite reductase, were evaluated. The DNA G + C percentage content was determined. Species-specific bacteriophages were isolated and a strain-typing grid established. In order to characterize further and fingerprint the different Rhizobium 'hedysari' isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed-field gel separation) were compared. Adansonian taxonomy yielded similarity clusters of the different isolates.

Bacteriophage Typing↗

PubMed2001

Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts.

AIMS: The objective of this study was to investigate what types of enzymes are being produced by non-Saccharomyces yeasts isolated from grapes in South Africa vineyards and clarified grape juice. These enzyme profiles could pave the way for attributing specific effects in wine to some of these enzymes produced by so-called wild yeasts associated with grape must. METHODS AND RESULTS: In this study 245 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces were screened for the production of extracellular pectinases, proteases beta-glucanases, lichenases, beta-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts, representing 21 species, were previously isolated from grapes and clarified grape juice. The production of all extracellular hydrolytic enzymes screened for was observed except beta-glucosidase activity. The amount and range of enzymes produced varied with different isolates of the same species. CONCLUSION: This study clearly revealed the potential of non-Saccharomyces wine yeasts to produce a wide range of useful extracellular enzymes during the initial phase of wine fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Enzymes produced by indigenous yeasts associated with grapes and juice might be harnessed to catalyse desired biotransformations during wine fermentation.

Candida↗

PubMed2002

Extracellular enzymatic activity profiles in yeast and yeast-like strains isolated from tropical environments.

AIMS: The objective of this study was to investigate the extracellular enzymatic activity (EEA) profile of yeasts isolated from tropical environments of the Brazilian rain forest. This screening survey could constitute the first approach in selecting yeast strains of environmental origin potentially exploitable as enzyme producers. METHODS AND RESULTS: In this study, 348 yeast (193 ascomycetes and 155 basidiomycetes) and 46 yeast-like strains (Aureobasidium pullulans) were screened for their EEA profile. The spread occurrence of extracellular amylases, esterases, lipases, proteases, pectinases and chitinases appeared to be a strain-related character. CONCLUSIONS: Yeasts isolated from tropical environments could represent a promising source of EEA. Selected strains showed maximum levels of EEA under acidic or neutral conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the potential for yeasts isolated from extreme environments as sources of industrially relevant enzymes for biotechnological purposes.

Ascomycota↗

PubMed2000

Polarization confocal microscopy and congo red fluorescence: a simple and rapid method to determine the mean cellulose fibril orientation in plants.

The mean or net preferential orientation of cellulose fibrils in plant cell walls is detected with polarization confocal laser scanning microscopy using the fluorescence dichroism of Congo Red. Single cells, arrays of cells in a tissue, or the epidermis of whole organs can be assayed in vivo. Aerial parts require an extra pectinase treatment because of the cuticle, which is impermeable to aqueous solutions. Peeling off the epidermis can be an elegant alternative, especially for leaves. With this method the net preferential fibril orientation can be related to the symmetry axis of the cell in quantitative terms. Data issuing from this approach are useful in current research on plant biomechanics.

Cell Wall↗

PubMed1996

Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937.

The secretion of extracellular pectinases, among which there are least six isoenzymes of pectate lyase and one pectin methylesterase, allows the phytopathogenic bacterium Erwinia chrysanthemi to degrade pectin. A gene coding for a novel pectin methylesterase has been cloned from an E. chrysanthemi strain 3937 gene library. This gene, pemB, codes for a 433-amino-acid protein. The PemB N-terminal region has the characteristics of lipoprotein signal sequences. We have shown that the PemB precursor is processed and that palmitate is incorporated into the mature protein. The PemB lipoprotein is not released into the extracellular medium and is localized in the outer membrane. The PemB sequence presents homology with other pectin methylesterases from bacterial and plant origin. pemB-like proteins were detected in four other E. chrysanthemi strains but not in Erwinia carotovora strains. PemB was overproduced in Escherichia coli and purified to homogeneity. PemB activity is strongly increased by non-ionic detergents. The enzyme is more active on methylated oligogalacturonides than on pectin, and it is necessary for the growth of the bacteria on oligomeric substrates. PemB is more probably involved in the degradation of methylated oligogalacturonides present in the periplasm of the bacteria, rather than in a direct action on extracellular pectin. pemB expression is inducible in the presence of pectin and is controlled by the negative regulator KdgR.

Amino Acid Sequence↗

PubMed1997

Mutual control of the PecS/PecM couple, two proteins regulating virulence-factor synthesis in Erwinia chrysanthemi.

The Erwinia chrysanthemi pecS mutant displays constitutive production of virulence factors, such as pectinases or cellulases. Complementation of the pecS mutation can be obtained in the presence of the pecS wild-type gene on a low-copy-number plasmid. Moreover, the resulting plasmid decreases the expression of a pecS::uidA chromosomal fusion, indicating the existence of an autoregulation mechanism. This negative autoregulation was confirmed and quantified by analysis of the pecS transcripts using primer-extension experiments. Band-shift assays and DNase I footprinting experiments demonstrated that the PecS protein could bind to the intergenic regulatory region, located between the pecS and pecM genes, with a relatively high affinity (apparent dissociation constant (K'[d]) close to 4nM). These PecS-binding sites overlap the pecS and pecM promoters. The comparison of these new PecS-binding sites with those previously characterized on the target genes confirms the absence of a consensus. This observation was in accordance with the results of the missing-contact experiments performed on the pecS-pecM intergenic regulatory region and the celZ operator. Concurrently, we demonstrated that the PecS protein negatively controls the expression of the divergently transcribed pecM gene located 400bp upstream from the pecS gene. By following the efficiency of pecS autoregulation in a double E. chrysanthemi pecM-pecS mutant, we established that the PecM protein potentiates PecS activity in vivo.

Bacterial Proteins↗

PubMed1997

Antagonistic effect of CRP and KdgR in the transcription control of the Erwinia chrysanthemi pectinolysis genes.

The main virulence factors of the phytopathogenic bacteria Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall. The cyclic AMP receptor protein (CRP) was identified as the main activator of the pectinolysis genes. Gel shift and DNase I footprinting experiments showed that the purified E. chrysanthemi CRP protein binds specifically to the promoter regions of seven pectinolysis genes (pelB, pelC, pelD, pelE, ogl, kduI and kdgT) whose expression is positively regulated in vivo by CRP. In contrast, no interaction was observed between CRP and the promoter-operator region of pelA, whose expression is negatively regulated in vivo by CRP. Primer extension experiments demonstrated that each of the pelB, pelC, pelE and kduI genes is expressed from a unique sigma70 promoter, whereas ogl and kdgT possess three and two functional promoters respectively. The position of the CRP binding site relative to the transcription start site suggests that CRP acts as a primary activator at the pelB (via the CRP binding site 1), pelC, pelE, pelD, kdgTP1 and oglP2 promoters. In contrast, transcription at the kduI, oglP1 promoters seems to require another transcriptional activator in synergy with CRP. Investigation of the simultaneous binding of CRP and KdgR, the main repressor of pectinolysis genes, to the regulatory regions of pelB, pelC, pelD, pelE, ogl, kduI and kdgT genes showed that binding of KdgR is preferential and exclusive in the case of ogl and kdgT, whereas the binding of these two regulators is independent in the case of pelB, pelC, pelD, pelE and kduI. Taken together, our data suggest that the antagonistic effects of CRP and KdgR on the expression of the pectinolysis genes occur by different mechanisms, including direct competition between the two regulators or between the repressor and RNA polymerase for the occupation of a common DNA region on the target genes.

Bacterial Proteins↗

PubMed2001

Two transporters, TogT and TogMNAB, are responsible for oligogalacturonide uptake in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft rot of plants by secreting pectinases which cleave pectin, a polysaccharide cementing the plant cell wall constituents. We demonstrated that two transporters mediate the uptake of the extracellularly formed oligomers in E. chrysanthemi. TogMNAB, a multicomponent transporter member of the ATP-binding cassette (ABC) superfamily, is only partially responsible for the uptake of pectic oligomers. Its action is completed by that of the second transporter, TogT, a member of the glycoside-pentoside-hexuronide (GPH) family (TC no. 2.2) which includes transporters involved in the uptake of complex sugars, mostly oligosaccharides and glycosides. Each transport system, TogMNAB and TogT, is able to independently mediate the transport of oligogalacturonides and the simultaneous inactivation of both is necessary to give a total absence of growth with pectin as the carbon source. The togT gene constitutes an independent transcriptional unit. Its expression is induced in the presence of pectic derivatives and it is subject to catabolite repression. In vitro, the repressor KdgR and the activator CRP both interact directly with the togT regulatory region. The decreased pathogenicity of single and double togT, togM mutants indicated that a deficiency in uptake of pectic oligomers leads to reduced bacterial multiplication which, in turn, limits plant maceration.

ATP-Binding Cassette Transporters↗

PubMed2004

The RhaS activator controls the Erwinia chrysanthemi 3937 genes rhiN, rhiT and rhiE involved in rhamnogalacturonan catabolism.

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The linear regions of pectin are composed of an acidic sugar, D-galacturonic acid. The ramified regions of pectin also include neutral sugars, and are rich in L-rhamnose residues. E. chrysanthemi is able to degrade these polysaccharides, polygalacturonate and rhamnogalacturonate. In E. chrysanthemi, the production of pectinases acting on linear regions is induced in the presence of polygalacturonate by a mechanism involving the repressor KdgR. The induction of the two adjacent E. chrysanthemi genes, designated rhiT and rhiN, is maximal after the simultaneous addition of both polygalacturonate and L-rhamnose. The rhiT product is homologous to the oligogalacturonide transporter TogT of E. chrysanthemi. The rhiN product is homologous to various proteins of unknown function, including a protein encoded by the plant-inducible locus picA of Agrobacterium tumefaciens. Both rhiT and rhiN are highly induced during plant infection. Various data suggest that RhiT and RhiN are involved in rhamnogalacturonate catabolism. RhiN is able to degrade the oligomers liberated by the rhamnogalacturonate lyase RhiE. The induction of the rhiTN operon in the presence of polygalacturonate results from control by the repressor KdgR. The additional induction of these genes by rhamnose is directly mediated by RhaS, a protein homologous to the activator of rhamnose catabolism in Escherichia coli. The virulence of an E. chrysanthemi rhaS mutant towards different host plants was clearly reduced. In this phytopathogenic bacterial species, RhaS positively regulates the transcription of the rhaBAD operon, involved in rhamnose catabolism, of the rhiE gene and of the rhiTN operon. The regulator RhaS plays a larger role in E. chrysanthemi than in other enterobacteria. Indeed, the RhaS control is not restricted to the catabolism of rhamnose but is extended to the degradation of plant polysaccharides that contain this sugar.

Bacterial Proteins↗

PubMed2001

Characterization of pectin lyase produced by an endophytic strain isolated from coffee cherries.

AIMS: The effect of endophytic bacterial activity on the quality of coffee beverage was studied. METHODS AND RESULTS: A survey of the micro-organisms in coffee cherries was performed before harvesting, and their growth on the main nutrients available in coffee cherries was determined in vitro. CONCLUSION: Many endophytic bacteria were isolated from surface-sterilized coffee cherries. One of the pectinolytic strains was physiologically and phenotypically characterized, and was tentatively identified by partial 16S rDNA sequencing as Paenibacillus amylolyticus. This endophytic strain produced an extracellular pectinase with maximal activity at 40 degrees C and pH 7.9, and was thermostable up to 45 degrees C. EDTA and metal ions had little effect on pectin lyase activity. Km and Vmax values were 4.6 mg ml(-1) and 94.0 10(-8) mol min(-1) ml(-1), respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Pectin lyases have been found in fungi but rarely in bacteria, and this isolate is a promising tool for regulation studies of these enzymes.

Bacillaceae↗

PubMed2002

Enzymes for animal feeding from Penicillium chrysogenum mycelial wastes from penicillin manufacture.

AIMS: Several enzymes from mycelial wastes of Penicillium chrysogenum fungal cultures from penicillin manufacture were studied as supplements for animal feeding. METHODS AND RESULTS: Proteins were precipitated with tannic acid, acetone, ethanol or polyethyleneglycol 6000. The levels of the enzyme activities found in the different precipitates were similar. The activities of invertase, beta-1,3-glucanase, lipase and tannase were higher and those of amylase, cellulase, pectinase, protease and xylanase were lower. Precipitation with tannic acid was selected as an easy and simple method. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: An enzymatic precipitate from these P. chrysogenum wastes, comparable to a commercial enzymatic additive used in animal feeding, was obtained.

Animal Feed↗

PubMed2000

Inhibition of Helicobacter pylori hemagglutination by polysaccharide fractions from roots of Panax ginseng.

Polysaccharides from the roots of Panax ginseng were extracted by hot water and fractionated by using ethanol precipitation and ion exchange chromatography. Fractions FC (crude extract), F1 (fraction precipitated by ethanol), F1N (fraction unbound to DEAE-Sepharose CL-6B), and F1A (bound fraction) were obtained. Their carbohydrate analyses showed that acidic fraction F1A contains higher amounts of galactose, arabinose and uronic acids, in comparison to FC and F1. Fraction F1N mainly consists of glucose. The inhibition of Helicobacter pylori-induced hemagglutination revealed different inhibitory activities of these fractions. In particular, acidic fraction F1A showed a remarkable inhibitory activity (minimum inhibition concentration was 0.25 mg/ml) among the polysacharide fractions. However, digestion of the fraction F1A with pectinase resulted in a lower molecular weight oligosaccharide fraction F1AP which was non-inhibitory at the concentration of 4 mg/ml. Comparison of inhibitory activities and carbohydrate compositions of isolated fractions indicates that the activity correlated with the contents of galactose, arabinose, and uronic acids. These data suggest that acidic polysaccharides may be responsible for the inhibitory activity.

Helicobacter pylori↗

PubMed2004

In vitro anti-adhesive activity of an acidic polysaccharide from Panax ginseng on Porphyromonas gingivalis binding to erythrocytes.

A polysaccharide with high uronic acid content from the roots of Panax ginseng was found to inhibit the ability of Porphyromonas gingivalis to agglutinate erythrocytes. This polysaccharide showed a strong inhibitory activity (minimum inhibitory concentration 0.25 mg/mL), but treatment with pectinase resulted in non-inhibitory hydrolyzed products. In contrast, the inhibition by the acidic polysaccharide from the leaves of Artemisia capillaris was negligible. The carbohydrate composition of the two polysaccharides indicated that the anti-adhesive activity may be correlated with glucuronic acid content, one of the components of glycosaminoglycans. Low molecular weight heparin and sucrose octasulfate revealed stronger inhibitory effects on bacterial binding, than the acidic polysaccharide from P. ginseng.

Anticoagulants↗

PubMed1992

Chemical structure and biological activity of polysaccharides from Hibiscus sabdariffa.

Three water-soluble polysaccharides have been isolated from flower buds of Hibiscus sabdariffa L. (HIB 1,2,3). The neutral polysaccharides (HIB 1 and 2) are composed of arabinans and arabinogalactans of low relative molecular mass. The major fraction was investigated by methylation analysis, pectinase-treatment, mild acid hydrolysis and NMR studies, and it was shown to be a pectin-like molecule (Mr = 10(5)d). The main chain is composed of alpha-1,4-linked GalA (24% methyl-esterified) and alpha-1,2-linked Rha. Side chains are built of Gal and Ara and are connected to the main chain via C-4 of every third Rha. Its structure seems to be different from polysaccharide structures described in other species of the Hibiscus genus and the Malvaceae family. All fractions were assayed for possible immune-modulating effects. All fractions showed some activity, but the main acidic fraction was contaminated with lipopolysaccharide, and therefore its shown activity has to be discussed carefully.

Animals↗

PubMed1988

Cell expansion and single-cell separation induced by colchicine in suspension-cultured soybean cells.

Single plant cells have been obtained without the preparation of protoplasts by culturing pieces of soybean callus tissue with colchicine. Cell expansion and separation were evoked by colchicine (1 mM) within a week of culture. Microscopic observation showed that cells took on a spherical shape in the presence of colchicine and then separated into single cells. Addition of colchicine to the culture medium did not affect the composition of cell wall polysaccharides, but a uronic acid-rich extracellular polysaccharide appeared during cell expansion and separation. Addition of microtubule stabilizers, glycerol (300 mM) or dimethyl sulfoxide [3% (vol/vol)], inhibited the secretion of the polysaccharide as well as cell expansion and separation. The extracellular polysaccharide elicited by colchicine was isolated by ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sepharose CL-6B from the conditioned medium of colchicine-treated soybean cells. The purified 18-kDa polysaccharide immediately enhanced cell expansion and separation when added to soybean callus tissue cultured in medium containing colchicine, even at low concentrations (0.1 mM). The polysaccharide was composed of galacturonic acid and, after digestion with a pectinase preparation, had no effect on the cells. Methylation analysis suggests that the polysaccharide consists of approximately 100 sequential alpha-1,4-galacturonic acids. The galacturonan increased the viability of separated cells cultured in medium containing colchicine, and the single cells obtained did not produce a wound-response callose. (Aminoethoxyvinyl)glycine, a specific inhibitor of ethylene production, extensively decreased the cell expansion and separation but did not inhibit the formation of the extracellular polysaccharide, suggesting that the polysaccharide may exert its effect by stimulating ethylene production.

Journal Article↗