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PubMed1991

Synthesis of different pectinases by filamentous growing A. niger mutants.

Mutants of A. niger K 69/26, prepared by multistep mutagenesis (UV, MNNG, heating) have been screened for pectinase activities. Mutants with altered levels of certain pectinases, such as endo- and exopolygalacturonase (PG vis, red), pectinesterase (PE) and pectinlyase (PL), were isolated. The enzyme activities of the best mutants M 1348/126 were increased 2-3-fold compared to the parent strain after a 6-d cultivation of filamentous mycelium on a shaker. Further mutagenesis of mutants with decreased pectinase activities (e.g. Se3) produced revertants. PG (vis) synthesis of revertant Se5 was increased 1.7 times compared to the control strain K 69/26. Independent of these increased rates, the general level of pectinase activities synthesized by the filamentous mycelium of A. niger mutants amounts to about 10-20% compared with those produced by aggregated mycelium. It appears that the enzyme synthesis related to mycelium structure is independent of the mechanism which regulates the level of pectinase synthesis within a specific morphological structure.

Aspergillus niger↗
PubMed2001

Isolation and characterization of rabbit caecal pectinolytic bacteria.

Two hundred and thirty colonies from the caecal contents of six rabbits were picked up and, after a 2-d incubation, were microscopically characterized using Gram staining. Large Gram-negative (34%) and small Gram-negative (30%) irregular rods, Gram-negative (27%) and Gram-positive (8%) cocci were found. Eleven isolates (Bacteroides ovatus (6 strains), B. thetaiotamicron, B. caccae, B. stercoris, B. capillosus and Capnocytophaga ochracea) were identified using commercial tests for measuring their catalase activity, metabolite production, etc., and testing their growth in 20% bile. Bacteria belonging to the genus Bacteroides were demonstrated to be the principal pectinolytic organisms in the rabbit caecum.

Animals↗
PubMed1998

Batch foam fractionation of kudzu (Pueraria lobata) vine retting solution.

The aqueous protein solution from kudzu (Pueraria lobata) vine retting broth, without the addition of other surfactants, was foam-fractionated in a vertical tubular column with multiple sampling ports. Time-varying trajectories of the total protein levels were determined to describe the protein behavior at six positions along the 1-m column. The lowest two trajectories of this batch process represented a loss of proteins from the bulk liquid and tended to merge and decay together in time; the other trajectories displayed a gain in proteins in the foam phase. These upper column port protein concentration trajectories generally increased in time up to 45 mm, followed by a decrease, reflecting the removal of proteins from the column ports. The foam became dryer as it passed up the column to the top port. The protein concentration was about 5-8x higher in the top port foam than in the initial bulk solution, mainly as a result of liquid drainage from the foam along the column axis. This concentration increase in the collected foam was dependent on the initial pH of the bulk solution. The mol-wt profile of the proteins in the concentrated foam effluent was determined by one-dimensional gel electrophoresis. An analysis of the gel electropherograms indicated that the most abundant proteins could be cellulases and pectinases.

Aspergillus niger↗
PubMed1994

Production of ethanol from enzymatically hydrolyzed orange peel by the yeast Saccharomyces cerevisiae.

We extended our previous investigations of enzymatic hydrolysis of polysaccharides in orange peel by commercial cellulase and pectinase enzymes to higher, more practical concentrations of orange peel solids. High yields of saccharification could be maintained even at substrate concentrations as high as 22-23%, but the rates of solubilization and saccharification decreased 2-3-fold. We also tested the fermentability of these hydrolysates by the yeast Saccharomyces cerevisiae, which revealed the presence of inhibitory compounds. These compounds could be removed by the filtration of hydrolyzed peel. Successful fermentations of filtered hydrolysates were achieved after pH adjustment with calcium carbonate.

Cellulase↗
PubMed2003

Quantitative determination of saturated oligogalacturonic acids in enzymatic digests of polygalacturonic acid, pectin and carrot pomace by on-line LC-ESI-MS.

A method for the quantitative determination of oligogalacturonic acids (OGAs) by on-line high-performance liquid chromatographic (HPLC) separation and mass spectrometric detection via an electrospray interface (ESI-MS) without additional desalting steps was developed. Saturated OGAs up to a degree of polymerization (dp) of 3 were quantified by comparison with reference compounds. The calibration plots showed high linearity (R(2)>0.99), and the detection limits for dp 1, 2, and 3 were 11, 28, and 6 ng per injection, respectively. Non- and partially methyl-esterified OGAs with a dp of 3 and 4 were calculated semi-quantitatively as dp 3. The analytical system was used for the quantification of OGAs of digests obtained by incubation of polygalacturonic acid, pectin, and carrot pomace with commercial enzyme preparations. Furthermore, methyl-esterified OGAs up to a dp of 12 containing up to 4 methyl esters were detected in a pectin digest.

Cellulases↗
PubMed2006

Interactions between yeasts and grapevines: filamentous growth, endopolygalacturonase and phytopathogenicity of colonizing yeasts.

It has been clearly established that phytopathogenic fungi, bacteria, and viruses exert biotic stresses on plants. Much less is known, however, about the interactions between enological species of yeast and their host plants. In a previous study, we described how Saccharomyces cerevisiae, the most common enological yeast, can act as a grapevine (Vitis vinifera L.) pathogen, causing growth retardation or plant death. In the present in vitro study on 11 strains of yeast belonging to different genera, which often occur on the surfaces of vineyard grapes and V. vinifera, a link was found to exist between strain phytopathogenecity and pseudohyphal growth habits and/or endopolygalacturonase activity. The results obtained here are consistent with earlier findings showing that the phytopathogenicity of yeast strains depends on the filamentous growth process, and show that endopolygalacturonase alone is not responsible for the invasion of plants tissues. The mechanisms observed here may be of significant ecological importance and may help to explain the long periods of yeast survival found to occur in vineyards.

Ecosystem↗
PubMed2006

Enhanced ethanol fermentation of brewery wastewater using the genetically modified strain E. coli KO11.

We have used liquid waste obtained from a beer brewery process to produce ethanol. To increase the productivity, genetically modified organism, Escherichia coli KO11, was used for ethanol fermentation. Yeast was also used to produce ethanol from the same feed stock, and the ethanol production rates and resulting concentrations of sugars and ethanol were compared with those of KO11. In the experiments, first the raw wastewater was directly fermented using two strains with no saccharification enzymes added. Then, commercial enzymes, alpha-amylase, pectinase, or a combination of both, were used for simultaneous saccharification and fermentation, and the results were compared with those of the no-enzyme experiments for KO11 and yeast. Under the given conditions with or without the enzymes, yeast produced ethanol more rapidly than E. coli KO11, but the final ethanol concentrations were almost the same. For both yeast and KO11, the enzymes were observed to enhance the ethanol yields by 61-84% as compared to the fermentation without enzymes. The combination of the two enzymes increased ethanol production the most for the both strains. The advantages of using KO11 were not demonstrated clearly as compared to the yeast fermentation results.

Beer↗
PubMed2004

Molecular characterization and in planta detection of Sclerotinia sclerotiorum endopolygalacturonase genes.

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, secretes multiple pectinolytic enzymes that facilitate penetration, colonization, and maceration of the plant tissues. Molecular analysis has previously revealed that the pectinolytic system of the fungus is organized as a multigene family, among which a subfamily of three members encoding for neutral endopolygalacturonase (endoPG) isoforms has been characterized. Here we describe the isolation and characterization of three additional endoPG-encoding genes ( pg5, pg6, and pg7) that belong to distinct phylogenetic groups. Pairwise sequence comparison between the known endoPGs from S. sclerotiorum revealed 43% to 97% identity, and the genomic organization of the pectinolytic system showed a great similarity to that of the related necrotroph Botrytis cinerea. During plant pathogenesis, a sequential expression of the endoPG-encoding genes was shown.

Ascomycota↗
PubMed2006

Production of polysaccharidases in different carbon sources by Leucoagaricus gongylophorus Möller (Singer), the symbiotic fungus of the leaf-cutting ant Atta sexdens Linnaeus.

Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens, produces polysaccharidases that degrade leaf components by generating nutrients believed to be essential for ant nutrition. We evaluated pectinase, amylase, xylanase, and cellulase production by L. gongylophorus in laboratory cultures and found that polysaccharidases are produced during fungal growth on pectin, starch, cellulose, xylan, or glucose but not cellulase, whose production is inhibited during fungal growth on xylan. Pectin was the carbon source that best stimulated the production of enzymes, which showed that pectinase had the highest production activity of all of the carbon sources tested, indicating that the presence of pectin and the production of pectinase are key features for symbiotic nutrition on plant material. During growth on starch and cellulose, polysaccharidase production level was intermediate, although during growth on xylan and glucose, enzyme production was very low. We propose a possible profile of polysaccharide degradation inside the nest, where the fungus is cultured on the foliar substrate.

Agaricales↗
PubMed2000

Phenotypic characterization of polysaccharidases produced by four Prevotella type strains.

Four ruminal Prevotella type strains, P. ruminicola JCM8958T, P. bryantii B 4T, P. albensis M384T, and P. brevis ATCC19188T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100-170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species.

Animals↗
PubMed2000

Effect of agglutinins from Rhizobium leguminosarum strain 252 on the activity of hydrolytic enzymes.

Cells of the nitrogen-fixing soil bacterium Rhizobium leguminosarum 252 and its hemagglutination-deficient mutant strain 252/7 were found to possess the activities of a variety of hydrolytic enzymes. The agglutinin proteins of rhizobia diminished beta-glucosidase activity, pectinolytic activity, and acid and alkaline phosphatase activities while completely inhibiting proteolytic enzyme activity in the bacterial cell. The results here show that rhizobial agglutinins are capable of affecting enzyme functioning in Rhizobium.

Acid Phosphatase↗
PubMed2001

Cloning and sequence analysis of the endopolygalacturonase gene from the pitch canker fungus, Fusarium circinatum.

The fungus Fusarium circinatum causes pitch canker disease on mature pine trees and root rot and damping-off of pine seedlings. Endopolygalacturonases (endoPGs) play a major role during penetration of plants by fungi. Digestion of the pectic polysaccharides in the plant primary cell walls is one of the earliest functions of endoPGs during infection. The research objective was to clone and characterize the gene encoding endopolygalacturonase in F. circinatum. A 970-bp DNA fragment was cloned by using degenerate PCR amplification from F. circinatum DNA. Sequence data for this fragment were used to design specific primers for use in genome walking to amplify and sequence the remaining portion of the F. circinatum endoPG gene (Fcpg). The amino acid sequence predicted from this gene showed 90% and 87% similarity to Fusarium oxysporum and Fusarium moniliforme endoPGs, respectively.

Chromosome Walking↗
PubMed2002

The pipg1 gene of the oomycete Phytophthora infestans encodes a fungal-like endopolygalacturonase.

Endopolygalacturonases (endoPGs) are plant cell wall-degrading enzymes that have been implicated in the invasion of plant tissue by pathogenic microbes. EndoPGs have been described from bacteria, plants, insects and numerous species of phytopathogenic fungi. In this study, we describe the first endoPG sequence from oomycetes, a unique group of eukaryotic plant pathogens that exhibit fungal-like filamentous growth but share little taxonomic affinity to fungi. The characterized gene, pipg1, was identified from the potato late-blight pathogen, Phytophthora infestans, and was predicted to encode a secreted glycoprotein with all the signature sequences of endoPGs. Pipg1 was expressed during preinfection and infection stages. Phylogenetic analysis of endoPGs indicated that pipg1 forms a unique class that is significantly more similar to fungal endoPGs than to plant or bacterial ones. This unexpected affinity between PIPG1 and fungal endoPGs contrasts with phylogenies obtained using ribosomal sequences or compiled protein sequences from mitochondrial and chromosomal genes, raising interesting questions about the evolution of these enzymes in oomycetes.

Amino Acid Sequence↗
PubMed2000

Endopolygalacturonase genes and enzymes from Saccharomyces cerevisiae and Kluyveromyces marxianus.

The gene encoding endopolygalacturonase (EC 3.2.1.15) has been cloned, sequenced and expressed from three strains of Saccharomyces cerevisiae (including nonsecretors) and three strains of Kluyveromyces marxianus. Both control and coding regions showed small differences within each species, one including loss of a potential glycosylation site. Two non-secreting S. cerevisiae strains (FY1679 and var. uvarum) had non-transcribed copies of functional genes. Maximum enzyme activity was achieved with the S. cerevisiae FY1679 gene in an expressing vector, with an enzyme activity of 51 micromol of reducing sugar released from polygalacturonic acid microg protein(-1) min(-1), the highest so far reported for a yeast.

Amino Acid Sequence↗
PubMed2000

Regulation of endopolygalacturonase gene expression in Botrytis cinerea by galacturonic acid, ambient pH and carbon catabolite repression.

The phytopathogenic fungus Botrytis cinerea produces a set of endopolygalacturonases (endoPGs) which are involved in the enzymatic degradation of pectin in plant cell walls. The endoPG-encoding genes of B. cinerea are differentially expressed when the fungus is grown in liquid culture on different carbon sources. A basic constitutive expression level was observed for two genes, Bcpg1 and Bcpg2, which encode basic isozymes. Galacturonic acid was shown to induce the expression of Bcpg4 and Bcpg6. Low pH of the culture medium resulted in induced expression of the Bcpg3 gene. Expression of the Bcpg5 gene was inducible; however the inducing factors could not be identified. Finally, galacturonic acid-induced expression of the Bcpg4 gene was repressed by the presence of more-favourable carbon sources, such as glucose.

Arabinose↗
PubMed2002

Improved Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.): assessment of macerating enzymes and sonication.

Agrobacterium -mediated transformation of shoot apices of sunflower (Helianthus annuus L.) was evaluated following wounding by cell-wall-digesting enzymes and sonication. The frequency of explants with regenerated shoots expressing GUS (beta-glucuronidase) or GFP (green fluorescent protein) increased following treatment with the macerating enzymes cellulase Onozuka R-10 and pectinase Boerozym M5, whereas treatment with macerozyme R-10 had a negative effect. When a combination of cellulase (0.1%) and pectinase (0.05%) was used, the rate of explants with uniformly GUS-positive shoots increased at least twofold. The transient expression of reporter genes was also enhanced using sonication (50 MHz; 2, 4 and 6 s), but stable expression in regenerated shoots following 4 weeks of selection did not increase with this treatment. Enzyme treatment alone (0.1% cellulase and 0.05% pectinase) was superior to a combined treatment of sonication and enzymes with respect to stable transformation. Polymerase chain reaction analyses of shoots recovered by grafting from transformation experiments using GFP as the reporter gene demonstrated the stable integration of the transgene. Regenerated plants were fertile and seeds could be harvested.

Cellulase↗
PubMed2004

Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense.

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.

Cell Wall↗
PubMed2005

Biosynthesis and cell-wall deposition of a pectin-xyloglucan complex in pea.

Golgi-enriched enzyme preparations prepared from etiolated pea epicotyls incorporated [U-(14)C]galactose from UDP-[U-(14)C]galactose into the 1,4-beta-galactan sidechains of a pectin-xyloglucan complex. This complex could bind to paper and was degraded both by pectin-degrading enzymes and by a xyloglucan-specific endoglucanase. Gel permeation chromatography was used to assess the molecular size of the complex and of enzymically-degraded, galactan-containing fragments of it. Etiolated pea stems were labelled with [U-(14)C]sucrose for 1 h, and the newly-synthesised cell wall polysaccharides were extracted with EDTA or NaOH and fractionated by ion-exchange chromatography. The NaOH-extracted, acidic radioactive polysaccharides obtained in this way were also degraded both by pectin-degrading enzymes and by xyloglucan-specific endoglucanase. Analysis of the radioactive sugar composition indicated that neutral sugars characteristic of both pectin and xyloglucan were present. Analysis of the total non-radioactive, neutral sugar composition of the NaOH-extracted, acidic cell-wall polysaccharides indicated that pectin-xyloglucan complexes were a general feature of the cell wall in this tissue.

Cell Wall↗