Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes.
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ABSTRACT:
SUBMITTER: Werb Z
PROVIDER: S-EPMC1185864 | biostudies-other | 1978 Sep
REPOSITORIES: biostudies-other
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Project description:1. Polyacrylamide beads containing entrapped 35S-labelled proteoglycan molecules have been prepared. 2. The measurement of release of radioactivity provides an extremely sensitive assay for proteoglycan-degrading enzymes, including proteinases and hyaluronidase. 3. The amount of label released is a logarithmic function of enzyme concentration or time of incubation. Experiments were made in an attempt to explain this. 4. Assays were made by the new method at several pH values, and with the inclusion of inhibitors to identify the proteoglycan-degrading enzymes of rabbit ear cartilage. 5. A previously undescribed proteinase active against proteoglycan at pH4.5 but unaffected by pepstatin, was discovered. The enzyme was named cathepsin F, and was partially purified and characterized; it was detected in human articular cartilage.
| S-EPMC1183726 | biostudies-other
Project description:SUMMARY:Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation.
| S-EPMC4248655 | biostudies-literature
Project description:The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and beta-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian beta-mannosidases.
| S-EPMC91384 | biostudies-literature
Project description:Biofilm-degrading enzymes could be used for the gentle cleaning of industrial and medical devices and the manufacture of biofilm-resistant materials. We therefore investigated 20 species and strains of the bacterial genus Lysobacter for their ability to degrade experimental biofilms formed by Staphylococcus epidermidis, a common nosocomial pathogen typically associated with device-related infections. The highest biofilm-degradation activity was achieved by L. gummosus. The corresponding enzymes were identified by sequencing the L. gummosus genome. Partial purification of the biofilm-degrading activity from an extract of extracellular material followed by peptide mass fingerprinting resulted in the identification of two peptidases (α-lytic protease and β-lytic metalloendopeptidase) that were predicted to degrade bacterial cell walls. In addition, we identified two isoforms of a lysyl endopeptidase and an enzyme similar to metalloproteases from Vibrio spp. Potential peptidoglycan-binding C-terminal fragments of two OmpA-like proteins also co-purified with the biofilm-degrading activity. The L. gummosus genome was found to encode five isoenzymes of α-lytic protease and three isoenzymes of lysyl endopeptidase. These results indicated that the extracellular digestion of biofilms by L. gummosus depends on multiple bacteriolytic and proteolytic enzymes, which could now be exploited for biofilm control.
| S-EPMC3979864 | biostudies-literature
Project description:The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degradation, an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period associated with the appearance of these enzymes. These first peroxidases were not able to degrade lignin directly and used diffusible metal cations to attack its phenolic moiety. The phylogenetic analysis of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are included, suggests that later in evolution these enzymes would have acquired the ability to degrade nonphenolic lignin using a tryptophanyl radical interacting with the bulky polymer at the surface of the enzyme. Here, we track this powerful strategy for lignin degradation as a phenotypic trait in fungi and show that it is not an isolated event in the evolution of Polyporales. Using ancestral enzyme resurrection, we study the molecular changes that led to the appearance of the same surface oxidation site in two distant peroxidase lineages. By characterization of the resurrected enzymes, we demonstrate convergent evolution at the amino acid level during the evolution of these fungi and track the different changes leading to phylogenetically distant ligninolytic peroxidases from ancestors lacking the ability to degrade nonphenolic lignin.
| S-EPMC6016776 | biostudies-literature
Project description:Microarrays have become established tools for describing microbial systems, however the assessment of expression profiles for environmental microbial communities still presents unique challenges. Notably, the concentration of particular transcripts are likely very dilute relative to the pool of total RNA, and PCR-based amplification strategies are vulnerable to amplification biases and the appropriate primer selection. Thus, we apply a signal amplification approach, rather than template amplification, to analyze the expression of selected lignin-degrading enzymes in soil. Controls in the form of known amplicons and cDNA from Phanerochaete chrysosporium were included and mixed with the soil cDNA both before and after the signal amplification in order to assess the dynamic range of the microarray. We demonstrate that restored prairie soil expresses a diverse range of lignin-degrading enzymes following incubation with lignin substrate, while farmed agricultural soil does not. The mixed additions of control cDNA with soil cDNA indicate that the mixed biomass in the soil does interfere with low abundance transcript changes, nevertheless our microarray approach consistently reports the most robust signals. Keywords: comparative analysis, microbial ecology, soil microbial communities
2009-06-01 | GSE13977 | GEO
Project description:Transcriptional profiling with next-generation sequencing methods demonstrated that a Neurospora crassa mutant with the three most highly expressed beta-glucosidase genes deleted had a transcriptional response to cellobiose similair to that of wild type N. crassa exposed to cellulose.
2012-04-05 | GSE36719 | GEO
Project description:Microarrays have become established tools for describing microbial systems, however the assessment of expression profiles for environmental microbial communities still presents unique challenges. Notably, the concentration of particular transcripts are likely very dilute relative to the pool of total RNA, and PCR-based amplification strategies are vulnerable to amplification biases and the appropriate primer selection. Thus, we apply a signal amplification approach, rather than template amplification, to analyze the expression of selected lignin-degrading enzymes in soil. Controls in the form of known amplicons and cDNA from Phanerochaete chrysosporium were included and mixed with the soil cDNA both before and after the signal amplification in order to assess the dynamic range of the microarray. We demonstrate that restored prairie soil expresses a diverse range of lignin-degrading enzymes following incubation with lignin substrate, while farmed agricultural soil does not. The mixed additions of control cDNA with soil cDNA indicate that the mixed biomass in the soil does interfere with low abundance transcript changes, nevertheless our microarray approach consistently reports the most robust signals. Keywords: comparative analysis, microbial ecology, soil microbial communities We used lignin degradation as a model process to demonstrate the use of an oligonucleotide microarray for directly detecting gene expression in soil communities using signal amplification instead of template amplification to avoid the introduction of PCR bias. In the current study, we analyzed mRNA isolated from two distinct soil microbial communities and demonstrate our ability to detect the expression of a small subset of lignin degrading genes following exposure to a lignitic substrate. We also included purified control amplicons and mixed target experiments with pure P. chrysosporium genomic cDNA to determine the level of interference from soil biomass on target hybridization.
2010-05-18 | E-GEOD-13977 | biostudies-arrayexpress
Project description:Transcriptional profiling with next-generation sequencing methods demonstrated that a Neurospora crassa mutant with the three most highly expressed beta-glucosidase genes deleted had a transcriptional response to cellobiose similair to that of wild type N. crassa exposed to cellulose. N. crassa was pregrown in Sucrose and transferred to Avicel (cellulose), Cellobiose, Sucrose or media with no carbon added. Biological triplicates used to identify differentially expressed genes in WT on Avicel. Single libraries for mutant strains identify which genes show similair expression on cellobiose as in the WT on cellulose.
2012-04-04 | E-GEOD-36719 | biostudies-arrayexpress
Project description:For efficient removal of intra- and/or extracellular hydrogen peroxide by dismutation to harmless dioxygen and water (2H(2)O(2) → O(2) + 2H(2)O), nature designed three metalloenzyme families that differ in oligomeric organization, monomer architecture as well as active site geometry and catalytic residues. Here we report on the updated reconstruction of the molecular phylogeny of these three gene families. Ubiquitous typical (monofunctional) heme catalases are found in all domains of life showing a high structural conservation. Their evolution was directed from large subunit towards small subunit proteins and further to fused proteins where the catalase fold was retained but lost its original functionality. Bifunctional catalase-peroxidases were at the origin of one of the two main heme peroxidase superfamilies (i.e. peroxidase-catalase superfamily) and constitute a protein family predominantly present among eubacteria and archaea, but two evolutionary branches are also found in the eukaryotic world. Non-heme manganese catalases are a relatively small protein family with very old roots only present among bacteria and archaea. Phylogenetic analyses of the three protein families reveal features typical (i) for the evolution of whole genomes as well as (ii) for specific evolutionary events including horizontal gene transfer, paralog formation and gene fusion. As catalases have reached a striking diversity among prokaryotic and eukaryotic pathogens, understanding their phylogenetic and molecular relationship and function will contribute to drug design for prevention of diseases of humans, animals and plants.
| S-EPMC3523812 | biostudies-literature