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Determination of distribution of pharmacologically active compounds and their biological degradation during waste water treatment processes
Palyzová, Andrea ; Kyslík, Pavel ; Marešová, Helena
The project is focused on the development of technology for the degradation of persistent active pharmacologically substances (APIs) in wastewater. These substances are biologically active, due to their physicochemical properties often difficult to degrade, their consumption in human and veterinary medicine is difficult to control. By using it alone, even non-metabolized drugs enter the environment. End-user disposal of unused drugs is virtually uncontrollable and is becoming another important source of pollution. APIs are a common nodal point for the main sources of environmental contamination. Unfortunately, in the case of the API series, current WWTP systems are not even capable of capturing, let alone degradation, and are therefore distributed back to the environment via surface waters. The development of persistent APIs degradation technology at the WWTP level is the aim of the project. Its implementation will significantly reduce the level of surface water pollution and thus increase the sustainability of the use of water resources.
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Study enantioselectivity and synthesis of β-lactam antibiotics catalyzed by penicilin G acylase: Biocatalysis and in-silico experiments
Grulich, Michal ; Kyslík, Pavel (advisor) ; Kotík, Michal (referee) ; Ettrich, Rüdiger (referee)
11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...
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A heterologous expression of alpha-amino acid ester hydrolase from the strain Achromobacter sp. CCM 4824 in Escherichia coli BL21(DE3)
Schneiderová, Michaela ; Kyslík, Pavel (advisor) ; Lichá, Irena (referee)
On the chromosomal DNA of the microorganism Achromobacter sp. CCM 4824, which was gained in the Laboratory of enzyme technology MBU AVCR v.v.i., there was identified a gene coding an enzyme capable of hydrolyzation of semi-synthetic β-lactam antibiotics ampicillin and cephalosporin with a D-phenylglycine as a side chain. This enzyme belongs to a group of α-amino acid esterases, which are interesting because of a potential use in kinetically controled synthesis of β-lactam antibiotics. In several aspects α-amino acid esterases might be better than actually used penicillin acylases and that is why these enzymes are subjects of a big interest. The gene for α-amino acid esterase coded by chromosomal DNA was cloned, characterized and heterologously expressed in constructed highly-producing bacterial system Escherichia coli BL21(DE3)JM5. Produced enzyme was purified and its properties important for possible use in the production of semi-synthetic β-lactam antibiotics were determined. Key words: alpha-amino acid ester hydrolase, Achromobacter sp., recombinant expression system, β-lactam antibiotics
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Integrated development of a bioprocess: From the soil enzyme to the yeast production platform
Borčinová, Martina ; Kyslík, Pavel (advisor) ; Uhlík, Ondřej (referee) ; Hasal, Pavel (referee)
For a sustainable future, there is a call to increase the market share of bio-based technologies and materials. Microbial-based technologies have the potential and the ability to contribute substantively on many levels to global efforts to achieve sustainability. Development and utilization of microbial technologies is, however, an extensive process involving numerous steps, including the discovery of novel technologies and the development of industrially viable production systems. In the presented thesis, individual steps of microbial biotechnology development were addressed. In the first part of the study, a variety of methodological approaches were employed in order to study the effect of the anthropogenic activity (i.e., decades lasting production of penicillin G) on the structure of soil microbial communities. Moreover, both cultivable and non-cultivable fractions of populations were subjected to functional screening in order to unravel the biotechnological potential of the microorganisms in terms of production of enzymes involved in biotransformation of beta-lactam antibiotics: penicillin G acylase (PGA) and alpha amino acid ester hydrolase (AEH). Our results indicated that the impacted communities harbour a microbial community with increased diversity and richness. However, on the...
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Noel peniclilin G acylase from bacterial strain Achromobacter sp.CCM 4824
Škrob, František ; Kyslík, Pavel (advisor) ; Gabriel, Jiří (referee) ; Králová, Blanka (referee)
83 6. Závěr 1. Kultivací bakteriálního kmene Achromobacter sp. CCM 4824 v bioreaktoru byla získána biomasa s penicilinacylasovou aktivitou jako výchozí materiál pro purifikaci enzymu. Specifická aktivita biomasy měřená se substrátem penicilin G byla 19 U·g-1 sušiny. 2. Byla vyvinuta metoda purifikace nové PGA. Specifická aktivita purifikovaného enzymu byla kolem 30 U·mg-1 proteinu s celkovým výtěžkem kolem 10 % a stupněm přečištění kolem 300. 3. Bylo zjištěno, že purifikovaný enzym se skládá ze dvou nekovalentně spojených, nestejně velkých podjednotek α a β. Přesné molekulové hmotnosti obou podjednotek byly stanoveny pomocí hmotnostní spektrometrické analýzy: α = 27,02 kDa a β = 62,45 kDa. Molekulová hmotnost β-podjednotky přibližně odpovídá hmotnostem β- podjednotek známých PGA, naopak menší α-podjednotka svou molekulovou hmotností α-podjednotky známých PGA převyšuje přibližně o 3 kDa. 4. Pomocí nativní elektroforézy a izoelektrické fokusace bylo zjištěno, že purifikovaná PGA obsahovala dvě izoformy o izoelektrických bodech pI 8,0 a 8,2. Substrátová specifita obou izoforem byla stejná, proto byla směs izoforem považována za jeden enzym. 5. Nový enzym se od známých PGA odlišuje svojí substrátovou specifitou: hydrolyzuje téměř dvojnásobnou rychlostí semisyntetická β-laktamová antibiotika, která mají na...
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Study enantioselectivity and synthesis of β-lactam antibiotics catalyzed by penicilin G acylase: Biocatalysis and in-silico experiments
Grulich, Michal ; Kyslík, Pavel (advisor) ; Kotík, Michal (referee) ; Ettrich, Rüdiger (referee)
11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...
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A heterologous expression of alpha-amino acid ester hydrolase from the strain Achromobacter sp. CCM 4824 in Escherichia coli BL21(DE3)
Schneiderová, Michaela ; Kyslík, Pavel (advisor) ; Lichá, Irena (referee)
On the chromosomal DNA of the microorganism Achromobacter sp. CCM 4824, which was gained in the Laboratory of enzyme technology MBU AVCR v.v.i., there was identified a gene coding an enzyme capable of hydrolyzation of semi-synthetic β-lactam antibiotics ampicillin and cephalosporin with a D-phenylglycine as a side chain. This enzyme belongs to a group of α-amino acid esterases, which are interesting because of a potential use in kinetically controled synthesis of β-lactam antibiotics. In several aspects α-amino acid esterases might be better than actually used penicillin acylases and that is why these enzymes are subjects of a big interest. The gene for α-amino acid esterase coded by chromosomal DNA was cloned, characterized and heterologously expressed in constructed highly-producing bacterial system Escherichia coli BL21(DE3)JM5. Produced enzyme was purified and its properties important for possible use in the production of semi-synthetic β-lactam antibiotics were determined. Key words: alpha-amino acid ester hydrolase, Achromobacter sp., recombinant expression system, β-lactam antibiotics
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Morphine alkaloid metabolism in bacteria
Zahradník, Jiří ; Kyslík, Pavel (advisor) ; Lichá, Irena (referee)
Morphine alkaloids and their derivatives are pharmaceutically important substances. Huge production and consumption of these compounds predetermines them to be significant pollutants in the environment. Some of them have been detected in surface waters. The aim of this study was to characterize effects of morphine alkaloids on the physiology of three model organisms: Agrobacterium sp. R89-1, Escherichia coli XL-1 (Blue), and Raoultella sp. kDF8, and elucidation of the mechanisms leading to toxicity. The biotransformation potential and utilization ability were characterized for model organisms. It was demonstrated that the microorganism Agrobacterium sp. R89-1 is capable of rapid biotransformation of codeine to its 14-OH derivatives. The manifestation of morphine compounds toxic effects for the strain R89-1 is the highest. In contrast, microorganism Raoultella sp. KDF8 is able to utilize codeine as a carbon and energy source. The accumulation of 14-OH-derivatives was not observed. Escherichia coli XL-1 (Blue) is not able to biotransform or utilize codeine. Α, β-unsaturated ketones (morphinone, codeinone, 14-OH-morphinone and 14-OH-codeinone) were found as a most toxic intermediates of codeine metabolism. Bacterial cell growth (strains R89-1 and KDF8) in the presence of codeine is characteristic with...
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Microbial consortia and metagenome of industrially polluted soil: occurrence of genes encoding AEH
Pitkina, Anastasiya ; Kyslík, Pavel (advisor) ; Lichá, Irena (referee)
Soils contain highly diverse consortia of bacteria making them very attractive starting points for both culture-dependent and metagenomic discovery efforts. The present diploma thesis analyses the composition of the microbial community from pharmaceutically polluted soil, with the employment of next-generation Illumina sequencing of 16S rDNA region. This analysis revealed high complexity of the soil microbial environment and confirmed that anthropogenic activity (represented by production of beta- lactam antibiotics) influences the variability and abundance of the species, yet without reducing the microbial diversity. In the second part of the thesis, isolation and heterologous expression of a novel gene encoding alpha-amino acid ester hydrolase (AEH) from a cultivable soil microorganism B. cereus is described. AEHs possess industrial potential for biocatalytic synthesis of semi-synthetic beta-lactam antibiotics, which are presently of great clinical importance. Powered by TCPDF (www.tcpdf.org)
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