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PubMed1989

A reappraisal of the terverticillate penicillia using biochemical, physiological and morphological features. III. An evaluation of pectinase and amylase isoenzymes for species characterization.

Polyacrylamide gel electrophoresis of extracellular pectinase and amylase isozymes of 170 mainly terverticillate Penicillium strains was undertaken. The data were coded and subjected to numerical analysis. Variation in intensity of isozymes was observed in repeat analyses of some strains, although most were consistent. Variation was also observed between some representative strains of species. P. viridicatum was more variable than P. brevicompactum and P. hordei for intensity of pectinase activity. There was a correlation between the grouping of the strains on the basis of the isozymes and the species concepts only in some cases. The method proved useful for the identification of strains producing intense activity which provided clear patterns, for example, P. brevicompactum and P. chrysogenum and to a lesser extent P. solitum var. crustosum and P. hordei. The method was also exclusionary in that some species were restricted to a particular cluster or subcluster. Amylase patterns confirmed that strains referred to as single species are not all homogeneous genetically, and that some strains are not simply haploid homokaryons. The genetic heterogeneity of the strains explains some of the problems in the systematics of the terverticillate penicillia.

Amylases↗
PubMed1991

Pectinase production by Neurospora crassa: purification and biochemical characterization of extracellular polygalacturonase activity.

The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1 and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50% with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].

Chromatography↗
PubMed1979

Feulgen cytophotometry of pine nuclei. II. Effect of pectinase used in cell separation.

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.

Cell Separation↗
PubMed1982

Use of pectinase to dissociate plant nuclei for squash preparations: effect of hydration procedures.

Scale tissues taken from cones of pendent Douglas-fir Pseudotsuga menziesii (Mirb.) Franco were treated with pectinase to dissociate nuclei for Feulgen staining. Hydration of fixed tissues with water or a graded series of ethyl alcohol before pectinase treatment resulted in distention and faint staining of nuclei and inadequate disintegration of cell walls. These problems were overcome by adopting citrate buffer hydration. Analysis of digitized scans of nuclei provided information about the distribution of the stained material in density classes and nuclear size which is usually unavailable through the conventional double wavelength DNA measuring method.

Buffers↗
PubMed2002

[Effect of pectinase and cellulase addition on in vitro digestibility of common bean (Phaseolus vulgaris)].

Effect of commercial pectinase and cellulase addition on in vitro digestibility of two common bean (Phaseolus vulgaris) varieties, black (Brunca) and red (Chirripó), was measured. Constant conditions of pH (6.3-6.4), time (60 min), and temperature (40 degrees C) were held, and three enzyme concentrations were tested: 0.25%, 0.5% and 1.0% m/m. In vitro digestibility increases for black beans were 6.9%; 9.5% and 12.3% (p < 0.05), and for red beans were 8.9%; 10.2% and 12.9% (p < 0.05), with increasing pectinase concentration. Effects of cellulase were dependent upon both, enzyme concentration and bean variety. In vitro digestibility of red beans increased by 5.7%; 9.2% and 12.0% with increasing cellulase concentration; digestibility of black beans increased by less than 5%. No significant difference (p > 0.05) in general acceptance of enzyme treated beans (black or red) or nontreated beans was noted.

Cellulase↗
PubMed1993

Ferulic acid esterase from Aspergillus niger: purification and partial characterization of two forms from a commercial source of pectinase.

Two forms of ferulic acid esterase from Aspergillus niger have been isolated from a commercial source of pectinase. One, designated I, has a M(r) of 132,000, is probably dimeric, and has a pI of 3.0. The second, designated II, was partially purified and is monomeric (M(r) 29,000), with a pI of 3.6. Both enzymes were free of pectinase and xylanase activity and released ferulic acid from methyl ferulate. In association with a xylanase, they also released ferulic acid from destarched wheat bran. Ferulic acid esterase II released a small amount of ferulic acid (0.09 unit/mg of protein) in the absence of xylanase. The enzymes had different specificities for a range of methyl ester derivatives of cinnamoyl and benzoyl acids, acetylated xylan and p-nitrophenyl acetate.

Aspergillus niger↗
PubMed1996

Characterization of pectinases and pectin methylesterase cDNAs in pods of green beans (Phaseolus vulgaris L.).

Tomato fruit maturation is accompanied by a depolymerization of cell wall pectins which is due to the action of endopolygalacturonase (endoPG) preceded by pectin methylesterase (PE) activity. To investigate the role of endoPG and PE in determining the structure of green bean (Phaseolus vulgaris L.) pectins, these pectinases were studied during pod development. Early developmental stages displayed low endoPG or exoPG activities while PE activities were measurable during all stages of pod and seed development. These results do not favour a possible synergistic action of PE and PG. For seeds, the relatively high PE activities concurred with relatively low levels of pectin methyl esterification. At a molecular level, one partial chromosomal clone of 210 bp (PE1V), two partial PE cDNA clones of 660 bp (PE2V and PE3V) from cv. verona and one full-length PE cDNA clone of 1990 bp (PE3M), from cv. Masai were isolated. The identity of the CDNA clones was confirmed by expression in Escherichia coli and immunodetection with antibodies directed towards a tomato fruit PE. Transcripts corresponding with the genomic clone PE1V were not detected but both PE2 and PE3 cDNAs corresponded with mRNAs 1.8 kb in length. In contrast to PE2, PE3 gene expression levels varied significantly in pods from different cultivars suggesting an involvement in determining pod morphology.

Amino Acid Sequence↗
PubMed1993

Pectinase Aspergillus sp. polygalacturonase: multiplicity, divergence, and structural patterns linking fungal, bacterial, and plant polygalacturonases.

Nine forms of Aspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with free alpha-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from other Aspergillus species (72-75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66-71% residue differences toward the Erwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties.

Amino Acid Sequence↗
PubMed1985

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores.

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.

Cellulase↗
PubMed1993

Adsorption of Kluyveromyces marxianus pectinase on CM-Sephadex gels.

New results are presented on the equilibria and kinetics of the ion exchange onto CM-Sephadex of polygalacturonase (pectinase) produced by the fermentation of Kluyveromyces marxianus. It is found that the equilibrium behavior follows the form of the Langmuir isotherm; the equilibrium is strongly affected by pH. High partitioning onto the ion-exchange matrix, with good retention of enzyme activity, is achieved in the pH range 3.5-5.0, and this can be qualitatively explained in terms of simple models for protein adsorption by ion exchange. The kinetics of ion exchange is modeled by assuming that the transfer resistances can be lumped into a single coefficient, and the results show that this gives a reasonable description of the adsorption kinetics. Under optimum conditions protein adsorption is enhanced by electrostatic effects and is extremely fast, and it is suggested that in these circumstances external mass transfer resistance is significant. At pH values close to the isoelectric point, electrostatic interactions are weak and intraparticle diffusion is rate-limiting: pore-blocking by adsorbed proteins appears to be important under these conditions. The results also provide the basis for an efficient single-step purification scheme.

Absorption↗
PubMed2005

Chitooligosaccharides--preparation with the aid of pectinase isozyme from Aspergillus niger and their antibacterial activity.

An isozyme of pectinase from Aspergillus niger with polygalacturonase activity caused chitosanolysis at pH 3.5, resulting in low-molecular weight chitosan (86%), chitooligosaccharides (COs, 4.8%) and monomers (2.2%). HPLC showed the presence of COs with DP ranging from 2 to 6. Charcoal-Celite chromatography and re-N-acetylation of the COs followed by CD, IR, MALDI-TOF-MS and FAB-MS analyses revealed an abundance of chitobiose, chitotriose and chitotetraose. The COs-monomeric mixture showed a bactericidal effect towards Bacillus cereus and Escherichia coli more efficiently than native chitosan. Among the chitooligomers, the hexamer showed maximum antibacterial effect followed by the penta-, tetra-, tri- and dimers. Of the two monomers, only GlcN showed slight bacterial growth inhibition. SEM revealed bactericidal action patterns of COs-monomeric mixture towards B. cereus and E. coli.

Anti-Bacterial Agents↗
PubMed2005

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

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

Amino Acid Sequence↗
PubMed2004

Affinity precipitation of Aspergillus niger pectinase by microwave-treated alginate.

Affinity precipitation is a simple, single plate separation process in which the complex of a smart macroaffinity ligand with the target protein (from a crude broth) can be selectively precipitated by application of a suitable stimulus. Alginate is a copolymer of guluronic acid and mannuronic acid residues and precipitates with Ca(2+) ions. It was found to bind to pectinase present in a commercial preparation of Aspergillus niger, Pectinex Ultra-SPL. Microwave pretreatment of alginate at 75 degrees C was found to enhance the selectivity of the affinity precipitation. Using microwave-treated alginate, 83% of the enzyme activity with 20-fold purification could be recovered. SDS-PAGE upon silver staining confirmed the enhanced selectivity of affinity precipitation when microwave-treated alginate was used.

Alginates↗
PubMed2003

Low molecular weight chitosans--preparation by depolymerization with Aspergillus niger pectinase, and characterization.

The viscosity of a chitosan solution was rapidly lowered in the presence of pectinase from Aspergillus niger at pH 3.0 and 37 degrees C. The low molecular weight chitosans (LMWC) had a molecular weight in the range 20,000-5000 Da. Circular dichroism spectra showed a decrease in the segment of acetylated glucosamine units, whereas X-ray diffraction and CP-MAS 13C NMR indicated higher crystallinity and polymorphism in LMWC. The latter on thermal drying resulted in structural alterations, and yielded an insoluble product. FT-IR and X-ray diffraction showed no evidence of either Schiff's base linkage or any annealed polymorph. CP-MAS 13C NMR showed marked changes in the chain conformations of LMWC, which are believed to be responsible for its loss of solubility and functionality.

Acetylation↗
PubMed2003

Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation.

Bacillus sp. DT7 produced very high levels of alkaline and thermotolerant pectinase by solid state fermentation. Production of this enzyme was affected by nature of solid substrate, level of moisture content, presence or absence of carbon, nitrogen, mineral and vitamin supplements. Maximum enzyme production of 8050 U/g dry substrate was obtained in wheat bran supplemented with polygalacturonic acid (PGA; 1%, w/v) and neurobion (a multivitamin additive; 27 micro l/g dry substrate) with distilled water at 75% moisture level, after 36 h of incubation at 37 degrees C.

Bacillus↗
PubMedDate not supplied

A smart bioconjugate of alginate and pectinase with unusual biological activity toward chitosan.

The commercial preparation of pectinase (Pectinex Ultra SP-L) was conjugated to alginate by noncovalent interactions by employing 1% alginate during the conjugation protocol. The optimum "immobilization efficiency" was 0.76. The pH optimum and the thermal stability of the enzyme remained unchanged upon conjugation with alginate. The soluble bioconjugate showed a 3-fold increase in V(max)/K(m) as compared to the free enzyme when the smart biocatalyst was used for chitosan hydrolysis. Time course hydrolysis of chitosan thus showed higher conversion of chitosan into reducing oligosaccharides/sugars. The smart bioconjugate could be reused five times without any detectable loss of chitosanase activity.

Alginates↗
PubMedDate not supplied

Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed.

Alginates↗
PubMedDate not supplied

Effect of pectinase on rumen fermentation in sheep and lambs.

In trial with adult wethers and weaned lambs the effect of enzymatic preparation Pektofoetidin G3x (mostly pectinase and cellulase) on rumen fermentation was studied. After 4 weeks of Pektofoetidin G3x application (0.54 g per day and animal) to adult wethers no statistically significant differences in total volatile fatty acids (VFA), acetic acid, propionic acid, butyric acid, ammonia in the rumen contents and urea in blood were determined between control and enzyme treated group. In comparison of fermentation parameters in wethers (mean of 1-3 hours after feeding) and lambs (2-3 hours after feeding) the significant differences in mol % of acetic acid (63.3 in control, 54.6 in experimental group, P less than 0.01), propionic acid (24.6, vs. 31.3, P less than 0.001), acetate: proprionate ratio (2.54, vs. 1.77, P less than 0.01) and in energy efficiency of VFA production (76.0%, vs. 79.1%, P less than 0.001) were determined. These differences between wethers and lambs suggest more intensive fermentation in lambs than in adult sheep. On the basis of these results it is possible to suggest, that in adult animals the efficiency of application of enzymatic preparations is low. In utilization of enzymatic preparations more important role, probably, is that of ruminal ecosystem itself, that, if once fully developed, is perfectly resistant to biotechnological interferences.

Acetates↗