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Enviromental DNA-inexhaustible source of unique bacterial genes
Culka, Martin ; Černá, Věra (advisor) ; Vaněk, Ondřej (referee)
Search for new enzymes or variants of known ones is now a hot issue in enzymological research. The classical culture-based approach, however, often fails when applied on environmental samples, because they contain uncultured microorganisms at most. For this reason, a new approach has been developed - the metagenomics. This approach is based on direct isolation of total DNA (RNA) from specific environment and its subsequent sequence-based (genotype) or function-based (phenotype) analysis. In this work, the metagenomic approach has been used to find new forms of penicillin G acylase, the enzyme that catalyze cleavage or formation of acyl - β-lactam nucleus bond and is used in industry for synthesis of semi-synthetic β-lactam antibiotics, in eleven samples from 4.5 m soil horizon. Sequence analysis of PCR amplicons on metagenomic templates revealed nucleotide sequences of major part of potential structural penicillin acylase genes. After translation it has been found that the sequences are most homologous to penicillin amidase from Conexibacter woesei. Further perspective of this metagenomic study is amplification of at least one complete structural gene of environmental penicillin acylase. However, the cloned regions of the gene can also be used to create hybrid penicillin acylases using gene shuffling...
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Enviromental DNA-inexhaustible source of unique bacterial genes
Culka, Martin ; Černá, Věra (advisor) ; Vaněk, Ondřej (referee)
Search for new enzymes or variants of known ones is now a hot issue in enzymological research. The classical culture-based approach, however, often fails when applied on environmental samples, because they contain uncultured microorganisms at most. For this reason, a new approach has been developed - the metagenomics. This approach is based on direct isolation of total DNA (RNA) from specific environment and its subsequent sequence-based (genotype) or function-based (phenotype) analysis. In this work, the metagenomic approach has been used to find new forms of penicillin G acylase, the enzyme that catalyze cleavage or formation of acyl - β-lactam nucleus bond and is used in industry for synthesis of semi-synthetic β-lactam antibiotics, in eleven samples from 4.5 m soil horizon. Sequence analysis of PCR amplicons on metagenomic templates revealed nucleotide sequences of major part of potential structural penicillin acylase genes. After translation it has been found that the sequences are most homologous to penicillin amidase from Conexibacter woesei. Further perspective of this metagenomic study is amplification of at least one complete structural gene of environmental penicillin acylase. However, the cloned regions of the gene can also be used to create hybrid penicillin acylases using gene shuffling...
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