Jan Kopecny | Academy of Sciences of the Czech Republic (original) (raw)

Papers by Jan Kopecny

Research paper thumbnail of Genetically Modified Ruminal Bacteria Protect Sheep from Fluoroacetate Poisoning

Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalog... more Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10 6 to 10 7 cells ml ؊1 in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep.

Research paper thumbnail of The effect of yellow affinity substance on cellulases of Ruminococcus flavefaciens

Letters in Applied Microbiology, 1997

J. KOPEC NÝ AND B. HODROVÁ . 1997. Cellulolytic cultures of Ruminococcus flavefaciens produced a ... more J. KOPEC NÝ AND B. HODROVÁ . 1997. Cellulolytic cultures of Ruminococcus flavefaciens produced a yellow affinity substance (YAS) with a strong affinity to microcrystalline cellulose (MC). YAS was bound to MC in the range of pH from 5 to 8 and at temperatures from 10°C to 60°C. The positive effect of YAS on adsorption of ruminococcal cellulases was demonstrated by comparing the adsorption behaviour of endoglucanases and cellobiohydrolases onto MC and YAS-treated MC. HPLC chromatography proved the presence of two yellow compounds with affinity to cellulose as well as to ruminococcal cellulases. Both YAS compounds were sensitive to oxidation. The observed YAS properties showed a close relation to YS of Clostridium thermocellum.

Research paper thumbnail of Butyrivibrio hungatei sp. nov. and Pseudobutyrivibrio xylanivorans sp. nov., butyrate-producing bacteria from the rumen

International Journal of Systematic and Evolutionary Microbiology, 2003

Research paper thumbnail of Cleavage of di- and tripeptides by Prevotella ruminicola

Anaerobe, 1995

Preaotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a signific... more Preaotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested. Streptococcus boaishydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobncter amylophilus, Ruminococcus spp. and Veillonella paraula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P, ruminicola.Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Alaa and Ala5; anothet was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro--methoxynapthylamide and was similar to dipeptidyl peptidase type IV DPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. A11 of the enzymes, and particularly those active against Ala2'p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures' 1997 Academic press

Research paper thumbnail of Peptidases of the Rumen Bacterium, Prevotella ruminicola

Anaerobe, 1997

Preaotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a signific... more Preaotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested. Streptococcus boaishydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobncter amylophilus, Ruminococcus spp. and Veillonella paraula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P, ruminicola.Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Alaa and Ala5; anothet was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro--methoxynapthylamide and was similar to dipeptidyl peptidase type IV DPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. A11 of the enzymes, and particularly those active against Ala2'p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures' 1997 Academic press

Research paper thumbnail of Molecular biology, genetics and biotechnology Effect of DNA extraction and sample preservation method on rumen bacterial population

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isol... more The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, À80 C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using highthroughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at À80 C was found as the optimal method to study ruminal bacterial profile.

Research paper thumbnail of Genetically Modified Ruminal Bacteria Protect Sheep from Fluoroacetate Poisoning

Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalog... more Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10 6 to 10 7 cells ml ؊1 in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep.

Research paper thumbnail of The effect of yellow affinity substance on cellulases of Ruminococcus flavefaciens

Letters in Applied Microbiology, 1997

J. KOPEC NÝ AND B. HODROVÁ . 1997. Cellulolytic cultures of Ruminococcus flavefaciens produced a ... more J. KOPEC NÝ AND B. HODROVÁ . 1997. Cellulolytic cultures of Ruminococcus flavefaciens produced a yellow affinity substance (YAS) with a strong affinity to microcrystalline cellulose (MC). YAS was bound to MC in the range of pH from 5 to 8 and at temperatures from 10°C to 60°C. The positive effect of YAS on adsorption of ruminococcal cellulases was demonstrated by comparing the adsorption behaviour of endoglucanases and cellobiohydrolases onto MC and YAS-treated MC. HPLC chromatography proved the presence of two yellow compounds with affinity to cellulose as well as to ruminococcal cellulases. Both YAS compounds were sensitive to oxidation. The observed YAS properties showed a close relation to YS of Clostridium thermocellum.

Research paper thumbnail of Butyrivibrio hungatei sp. nov. and Pseudobutyrivibrio xylanivorans sp. nov., butyrate-producing bacteria from the rumen

International Journal of Systematic and Evolutionary Microbiology, 2003

Research paper thumbnail of Cleavage of di- and tripeptides by Prevotella ruminicola

Anaerobe, 1995

Preaotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a signific... more Preaotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested. Streptococcus boaishydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobncter amylophilus, Ruminococcus spp. and Veillonella paraula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P, ruminicola.Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Alaa and Ala5; anothet was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro--methoxynapthylamide and was similar to dipeptidyl peptidase type IV DPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. A11 of the enzymes, and particularly those active against Ala2'p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures' 1997 Academic press

Research paper thumbnail of Peptidases of the Rumen Bacterium, Prevotella ruminicola

Anaerobe, 1997

Preaotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a signific... more Preaotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested. Streptococcus boaishydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobncter amylophilus, Ruminococcus spp. and Veillonella paraula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P, ruminicola.Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Alaa and Ala5; anothet was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro--methoxynapthylamide and was similar to dipeptidyl peptidase type IV DPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. A11 of the enzymes, and particularly those active against Ala2'p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures' 1997 Academic press

Research paper thumbnail of Molecular biology, genetics and biotechnology Effect of DNA extraction and sample preservation method on rumen bacterial population

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isol... more The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, À80 C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using highthroughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at À80 C was found as the optimal method to study ruminal bacterial profile.