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PubMedDate not supplied

[Pectolytic enzymes formed by Penicillium and Fusarium micromycetes].

The ability of the two cultures Penicillium sp. and Fusarium sp. to synthesize extracellular pectolytic enzymes was investigated. The cultivation conditions providing a high level of the biosynthesis of these enzymes were identified. The methods of isolating the enzymes by means of organic solvents were developed. The pectinase from Penicillium sp. showed a higher thermostability whereas that from Fusarium sp. displayed a greater acid resistance. Using glutaraldehyde and titanium salts, active immobilized forms of pectinases on silica carriers were prepared.

Drug Stability↗
PubMed2025

Symbionts with eroded genomes adjust gene expression according to host life-stage and environment.

Symbiotic bacteria in long-term host associations frequently undergo extreme genome reduction. While they retain genes beneficial to the host, their repertoire of transcription factors is severely reduced. Here, we assessed whether genome-eroded symbionts can still regulate gene expression by characterizing the transcriptional responses of obligate symbionts in reed beetles to different temperatures and host life stages. These symbionts feature a small genome (~0.5 Mb), encoding for 9-10 essential amino acid biosynthesis pathways, 0-2 pectinases, and 4-5 transcription factors. We found that the symbionts respond to winter conditions by upregulating a heat-shock sigma factor and downregulating translation machinery. Across life stages, symbionts adjusted gene expression to meet the hosts' nutritional demands, upregulating amino acid biosynthesis in larvae, while expression and activity of host and symbiont enzymes involved in plant cell wall breakdown increased in the folivorous adults. In addition, the regulation of symbiont cell morphology genes corresponded to cell shape differences across life stages. Thus, reed beetle symbionts may use their few transcription factors to respond to the host's environment, highlighting the regulatory potential of long-term coevolved symbionts despite severely reduced genomes.

Symbiosis↗
PubMed1999

Cloning, sequence analysis and overexpression of a Saccharomyces cerevisiae endopolygalacturonase-encoding gene (PGL1).

Only a few yeast strains produce pectin-degrading enzymes such as pectin esterases and depolymerases (hydrolases and lyases). Strain SCPP is the only known Saccharomyces strain to produce these pectinases. One of these pectolytic enzymes. PGL1-encoded endopolygalacturonase (EC 3.2.1.15), hydrolyses the alpha-1,4-glycosidic bonds within the rhamnogalacturonan chains in pectic substances. This paper presents the cloning and sequencing of the first S. cerevisiae gene involved in pectin degradation. Few differences were found between the two deduced amino acid sequences encoded by PGL1-1 from a pectolytic (PG+) strain (SCPP) and PGL1-2 from a non-pectolytic (PG-) strain (X2180-1B). Similarities were found with other polygalacturonases from plants and other microorganisms. Of the two S. cerevisiae genes, only the one isolated from strain SCPP was able, by overexpression, to confer endopolygalacturonase activity to a laboratory strain of S. cerevisiae. Overexpression of PGL1-1 gene in a non-pectolytic strain resulted in halo formation on polygalacturonic acid-containing agar plates stained with ruthenium red.

Amino Acid Sequence↗
PubMedDate not supplied

Affinity precipitation of proteins.

Affinity precipitation is being studied as a technique to be introduced at an early stage of downstream processing for the selective isolation of proteins. The technique utilizes a heterobifunctional ligand, which, in addition to having affinity for the target protein(s), possesses another function for controlling precipitation. The latter component is comprised of a polymer which can be made reversibly soluble and insoluble by altering a specific parameter such as pH or temperature. Different polymers of natural and synthetic origin have been used for this purpose. The soluble form of the ligand is used for the affinity binding step and precipitation is induced for obtaining separation of the affinity complex. Some of the polymers used in this laboratory include chitosan, alginate, Eudragit S-100 (copolymer of methacrylic acid and methyl methacrylate) and polyethyleneimine. Chitosan and alginate served as natural ligands for wheat germ agglutinin and pectinase, respectively. The aromatic dye Cibacron Blue 3GA coupled to Eudragit S 100 and polyethyleneimine way used for the affinity precipitation of some model enzymes such as lactate dehydrogenase and alcohol dehydrogenase. As prior removal of cell debris, etc., is essential for affinity precipitation, the possibility of integration of the technique with extraction in aqueous two-phase systems was also demonstrated.

Chemical Precipitation↗
PubMed2004

Preparation of single cells from aggregated Taxus suspension cultures for population analysis.

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism.

Cell Aggregation↗
PubMed2006

A study on the inactivation of micro-organisms and enzymes by high pressure CO2.

This study addresses some microbial inactivation phenomena induced by high pressure CO2 over micro-organisms and enzymes. The activity of four selected enzymes was measured before and after treatment with CO2 under pressure in both buffer solutions and natural cellular environment (E. coli cells and tomato paste). Results are reported for acid phosphatase, alkaline phosphatase, ATPase, and pectinase at different conditions of temperature, CO2 pressure, and treatment time (32-40 degrees C, 85-150 bar, 30-70 min). The results obtained show that the high pressure CO2 treatment induces an inactivation of cellular enzymatic activity higher than the one caused on the same enzymes in solution. However, the measured activity difference is not caused by a damage at the enzymes molecular level but is a consequence of the permeabilization of the cellular envelopes which leads to a release of unmodified enzymes from the cells with simultaneous drop of enzymatic cellular activity. The reported data suggest that the bacterial cell death is probably due not to a selective effect of high pressure CO2 treatment but to simultaneous detrimental action of CO2 on cellular membrane and cell wall.

Carbon Dioxide↗
PubMed1989

Enzyme activity electrophoresis: development and applications.

The development of rocket enzyme activity electrophoresis for the detection and quantification of various proteinases, lipases and pectinases is presented. Rocket enzyme activity electrophoresis is more sensitive than the radial diffusion assay and often enables distinction between qualitatively different enzymes present in the same samples, whereas the radial diffusion assay only provides information on the overall enzyme activity. However, calibration and optimization of the enzyme activity electrophoretic assay have to be performed for each new enzyme-substrate system to be analyzed. Some of the common pitfalls in the development of new enzyme activity electrophoretic assays are presented. Enzyme activity electrophoresis can be applied in combination with other electrophoretic assays. Particularly the combination of enzyme activity electrophoresis with various immunoelectrophoretic methods can provide detailed information on the enzymes studies.

Electrochemistry↗
PubMed1981

[Characterization of polygalacturonase covalently bonded to Sepharose].

For the soluble endo-polygalacturonase (EC 3.2.1.15.) from Aspergillus spec., investigated in the present work, the defined substrate turnover U at 50% loss of viscosity if 0.2% and is independent on the reaction temperature. In the case of the covalently-bonded enzyme, the following linear equation applies to U, depending on the specific activity A and in the limits from A = O [U] and Amax: U = [U] + S square root of A. U is influenced by the kind of linkage, the conditions of immobilization and the properties of the carrier: it is a measure of the postulated conformational change of the polygalacturonase. The characteristic limiting value for the substrate turnover at A = O [U] is also temperature-independent and proves to be a true increment of binding, whereas Amax depends essentially on the porosity of the carrier. Polygalacturonase-sepharose complexes with a real substrate turnover U of 3--20% were prepared by varying systematically the kind of linkage and the specific activity A. It was found that with increasing U these complexes were, as a rule, inhibited to a lesser extent by a non-competitive pectinase inhibitor than the soluble polygalacturonase. Furthermore, their ability to liberate or enrich oligomeric galacturonic acids with a degree of polymerization greater than 3 was markedly reduced.

Aspergillus↗
PubMed1984

[Occurrence and analysis of the mycotoxin emodin].

Besides other mould species, Aspergillus wentii Wehmer is assumed to form the hydroxyanthraquinone derivative emodin which has toxic and gene mutagenic properties. As a pectinase producer this fungus is used for the industrial isolation of pectinolytic enzymes for the food industry so that a contamination of such preparations by emodin cannot be excluded. However, preliminary thin-layer chromatographic studies of extracts of the corresponding pectinolytic enzyme preparations and Aspergillus wentii Wehmer did not point to a detectable toxin formation or contamination (the limit of detection lay at 5 mg emodin/kg substrate or 0.3 mg emodin/kg enzyme).

Animals↗
PubMed1993

The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa.

Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes.

4-Butyrolactone↗
PubMed2003

The effect of carbohydrate carbon sources on the production of constitutive and inducible laccases by Botryosphaeria sp.

The influence of carbohydrates: glucose, fructose, galactose, galacturonic acid, xylose, lactose, sucrose, pectin and inulin, were evaluated as sole carbon source for the production of laccases by the ascomycete, Botryosphaeria sp. Veratryl alcohol, a laccase inducer, was added to culture media to study inducible laccase production on the same carbon sources. Inulinase and pectinase were also produced when Botryosphaeria sp. was grown on inulin, and galacturonic acid and pectin, respectively, and their levels were less in the presence of veratryl alcohol. Botryosphaeria sp. produced constitutive laccases on all carbon sources examined, and veratryl alcohol increased the laccase production on most of carbon sources studied except for inulin and galacturonic acid. Evidence is presented that Botryosphaeria sp. is also pectinolytic.

Ascomycota↗
PubMed2005

Functional subgenomics of Clostridium thermocellum cellulosomal genes: identification of the major catalytic components in the extracellular complex and detection of three new enzymes.

Clostridium thermocellum produces the most efficient enzyme-complex for the degradation of polysaccharides in biomass, the large extracellular cellulosome. The draft complete genomic sequence of Clostridium thermocellum was screened for open reading frames (ORF) containing cellulosomal dockerin sequences. Seventy-one putative cellulosomal genes were detected. One third of these ORFs may be involved in cellulose hydrolysis. Most of the others showed homology to hemicellulases, pectinases, chitinases, glycosidases or esterases potentially involved in the unwrapping of cellulose fibers. To identify the predominant catalytic components, cellulosomes were purified and the components were separated by an adapted two-dimensional gel electrophoresis technique. The apparent major spots were identified by MALDI-TOF/TOF. Ten of the components were previously known: the structural protein CipA, the endo-glucanases Cel8A, Cel5G, Cel9N, the cellobiohydrolases Cbh9A, Cel9K, Cel48S, the xylanases Xyn10C, Xyn10Z, and the chitinase Chi18A. In addition, three hitherto unknown major components were detected, Cel9R, Xyn10D and Xgh74A. These major components in the cellulosomal particles most probably constitute the essential enzymes for crystalline cellulose hydrolysis.

Catalysis↗
PubMed2006

Effect of lemnan, pectin from Lemna minor L., and its fragments on inflammatory reaction.

An effect of apiogalacturonanic pectin of duckweed Lemna minor L. (lemnan LM) was studied on the inflammatory response to ovalbumin injected intradermally into the footpad of control and ovalbumin-fed mice. Lemnan LM (1-2 mg per mouse) was found to enhance by as much as 50-60% the footpad swelling in control mice. Oral administration of ovalbumin was shown to result in sensitization that increased inflammation. Ovalbumin admixed with lemnan was found to increase by two-fold footpad edema in comparison with the mice receiving ovalbumin alone. Apple pectin used as a reference compound failed to influence the inflammatory reaction. Degradation of lemnan was performed to elucidate the active region of the polysaccharide macromolecule. The apiogalacturonanic fragment (LMP) obtained using a digestion of lemnan LM with pectinase was shown to increase the footpad response in both control and ovalbumin-fed mice. Fragment LMPH deprived of some terminal apiose residues as a result of partial acidic hydrolysis failed to have an effect on the inflammatory response.Thus, the data obtained reveal an enhancement by lemnan of the inflammatory response. The ramified apiogalacturonan seemed to be the active region of the lemnan macromolecule.

Administration, Oral↗
PubMed1994

Water-soluble carbodiimide for the fluorescent measurement of the carboxyl group produced by enzyme reactions.

Fluorescent measurement of the carboxyl group was achieved using water-soluble carbodiimide, EDC (1-ethyl-3-(3- dimethylaminopropyl)carbodiimide). Detection of the active intermediate from the glucuronic acid-EDC reaction mixture using the OPA (o-phthalaldehyde) reagent described in a previous paper was greatly improved to about 4 ng/tube (17 pmol) glucuronic acid owing to a modification of the OPA cocktail. Moreover, fluorescent EDAN (N-1-ethylenediamino-naphthalene) labeling of the carboxyl group was also enabled by EDC catalysis. Quenching of the excess fluorescence of EDAN by OPA increased the sensitivity of EDC-EDAN to a level above the EDC-OPA method. In addition to the assay of enzymes concerning the uronic acid metabolism such as pectinase and pectinesterase, lipase and protease actions could be measured by the EDC-OPA and EDC-EDAN quenching methods, respectively. Thus, these two fluorescent means of detecting carboxyl groups seemed to have extensive application not only to acidic sugars but also to various carboxylic compounds regardless of their solubility.

Carboxylic Acids↗
PubMed1996

The rumen: a unique source of enzymes for enhancing livestock production.

Increasing competition in the livestock industry has forced producers to cut costs by adopting new technologies aimed at increasing production efficiency. One particularly promising technology is feeding enzymes as supplements for animal diets. Supplementation of diets for non-ruminants (e.g., swine and poultry) with fibrolytic enzymes, such as cellulases, xylanases and beta-glucanases, increases the feed conversion efficiency and growth rate of the animals. Enzymatic hydrolysis of plant cell wall polymers (e.g., cellulose, xylan, beta-glucans) releases glucose and xylose and eliminates the antinutritional effects of beta-glucans and arabinoxylans. Enzyme supplementation of diets for ruminants has also been shown to improve growth performance, even though the rumen itself represents the most potent fibrolytic fermentation system known. Implementation of this technology in the livestock industry has been limited largely because of the cost of development and production of enzymes. Over the last decade, however, developments in recombinant DNA technology have increased the efficiency of existing microbial production systems and facilitated exploitation of alternative sources of industrial enzymes. The ruminal ecosystem is among the novel enzyme sources currently being explored. Understanding the role of enzymes in feed digestion through characterization of the enzymology and genetics involved in digestion of feedstuffs by ruminants will provide insight required to improve the products currently available to producers. Characterization of genes encoding a variety of hydrolytic enzymes, such as cellulases, xylanases, beta-glucanases, amylases, pectinases, proteases, phytases and tannases, will foster the development of more efficacious enzyme supplements and enzyme expression systems for enhancing nutrient utilization by domestic animals. Characteristics of the original source organism need no longer restrict the production of a useful enzyme. Recent reports of transgenic plants expressing fibrolytic or phytase activity and of transgenic mice able to produce endoglucanase in the pancreas speak to the feasibility of improving feed digestion through genetic modification of the feedstuffs and the animals.

Journal Article↗
PubMed1998

Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems.

A decade ago, Pugsley and colleagues reported the existence of a large region of Klebsiella DNA, distinct from the Klebsiella gene encoding pullulanase, which was necessary for secretion of this enzyme to the cell surface in Escherichia coli (d'Enfert et al., 1987a,b). The pul genes it contained proved to be the tip of an iceberg. The sequences reported before 1992 (d'Enfert et al., 1987a,b; d'Enfert & Pugsley, 1989; Pugsley & Reyss, 1990; Reyss & Pugsley, 1990) included only one gene (pulD) that matched any sequence in the data base; a 220 amino acid residue segment of PulD was 32% identical with a portion of the filamentous phage-encoded protein, pIV. But by the time the sequence of the 18.8 kb DNA fragment that contained the pul genes had been completed (Possot et al., 1992), reports of sets of homologous genes in several species of Gram-negative plant and animal pathogens had appeared. For the most part, these gene clusters were cloned by their ability to complement mutants that produced, but failed to secrete, proteins normally found in the extracellular milieu; when tested, the mutants showed reduced pathogenicity or were totally avirulent. The secreted proteins included hydrolytic enzymes such as cellulase and pectinase from plant pathogens, and proteases and toxins from animal pathogens. The multi-gene family necessary for secretion of these enzymes is now known as the type II system or the main terminal branch (MTB) of the general secretion pathway (GSP). As summarized by Pugsley et al. (1997), the current tally includes type II systems from Klebsiella oxytoca (pul), Erwinia chrysanthemi and carotovora (out), Xanthomonas campestris (xps), Pseudomonas aeruginosa (xcp), Aeromonas hydrophila (exe), and Vibrio cholerae (eps). A second type II system (sps) necessary for deposition of the S-layer on the cell surface in A. hydrophila is more similar to the X. campestris than A. hydrophila genes (Thomas & Trust, 1995). The biggest surprise has been the discovery of a complete set of type II secretion genes in E. coli K12. The E. coli genes are not expressed under normal growth conditions, and a search is underway to find inducing conditions and secretion substrates (Francetic & Pugsley, 1996). Impressive progress has already been made in defining components of the pathway. What remains to be understood in mechanistic detail is how this protein secretion system functions.

Bacterial Proteins↗
PubMed1999

Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL.

Erwinia chrysanthemi secretes, by the type II secretory pathway, a large number of enzymes, including cellulases and pectinases. This process requires the products of the out genes, which are widely conserved in Gram-negative bacteria. The Out proteins are thought to form a membrane-associated multiprotein complex. Here, we investigated interaction between OutE, the putative ATP binding component, and OutL, an inner membrane protein. We showed, by limited proteolysis, genetic suppression and the yeast two-hybrid system, that OutE and OutL interact directly. Analysis of truncated forms of OutE demonstrated that the N terminus of OutE (residues 1-97) is important for the OutE/OutL interaction. Moreover, results from the yeast two-hybrid system suggested that OutE and OutL are each able to form homomultimers. The region required for homomultimerisation of OutE is located in its C terminus. Limited proteolysis assay indicated that OutE induces a conformational change in OutL, in both its cytoplasmic and periplasmic domains. Moreover, the secretion process requires a conformational change in OutE which depends on both the interaction with OutL and on the presence of an intact Walker A motif in OutE. Our results support the view that interaction occurring on the cytoplasmic side influences the events occurring in the outer membrane. We discuss a model in which OutE uses ATP to control the assembly of the type II secretion machinery.

Bacterial Outer Membrane Proteins↗