Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of β-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160 mM 6-APA, 350 mM acyl donor methylester[Symbol: see text]HCl, pH 6.3, 25 °C, reaction time of 200 min) with immobilized NPGA equaled 96.9 % and 91.1 %, respectively. The enzyme was highly thermostable with maximum activity at 60 °C (pH 8.0) and 65 °C (pH 6.0). Activity half-life at 60 °C (pH 8.0) and at 60 °C (pH 6.0) was 24 min and 6.9 h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230 mM 6-APA, 340 mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5 °C, pH 6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.
- MeSH
- Achromobacter enzymologie MeSH
- amoxicilin metabolismus MeSH
- ampicilin metabolismus MeSH
- antibakteriální látky metabolismus MeSH
- beta-laktamy metabolismus MeSH
- biotransformace MeSH
- enzymy imobilizované chemie metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- kyselina penicilanová analogy a deriváty metabolismus MeSH
- penicilinamidasa chemie izolace a purifikace metabolismus MeSH
- stabilita enzymů MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The ferric uptake regulator gene (fur), its promoter region and Fur box of pvdS gene involved in siderophore-mediated iron uptake system were sequenced in the parent strain Pseudomonas aeruginosa PAO1 and in the fur mutant FPA121 derived from the strain PAO1. We identified the gene fur 179 bearing a novel, single-point mutation that changed the amino acid residue Gln60Pro in the DNA-binding domain of the Fur protein. The synthesis of pyoverdine was studied in cultures of the strains PAO1 and FPA121 grown in iron-deplete and iron-replete (60 μmol/L FeIII) medium. The amino acid replacement in the regulatory Fur protein is responsible for the overproduction of pyoverdine in iron-deplete and iron-replete medium. No mutation was identified in the Fur box of the gene pvdS.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- kultivační média chemie MeSH
- mutační analýza DNA MeSH
- mutantní proteiny genetika metabolismus MeSH
- oligopeptidy biosyntéza MeSH
- Pseudomonas aeruginosa genetika metabolismus MeSH
- represorové proteiny genetika metabolismus MeSH
- substituce aminokyselin * MeSH
- vazebná místa MeSH
- železité sloučeniny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Production of enantiopure esomeprazole by biocatalysis is of great demand by pharmaceutical industry. A Gram-positive bacterium oxidizing omeprazole sulfide 1a (5-methoxy-2-[((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)thio]-1H-benzoimidazole) to (S)-sulfoxide esomeprazole 2a (S)-5-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl) methylsulfinyl]-3H-benzoimidazole was isolated from soil polluted with elemental sulfur. The strain exhibited the highest identity with the genus Lysinibacillus and catalyzed oxidation of 1a into enantiopure esomeprazole with conversion of 77% in a stirred bioreactor, fed-batch culture. No consecutive oxidation of (S)-sulfoxide to sulfone was observed during whole-cell catalysis. The unique characteristics of the catalyst provide a solid basis for further improvement and development of sustainable green bioprocess.
- MeSH
- Bacillus metabolismus MeSH
- bioreaktory MeSH
- biotransformace MeSH
- chromatografie na tenké vrstvě MeSH
- DNA primery MeSH
- koncentrace vodíkových iontů MeSH
- kultivační média MeSH
- omeprazol analogy a deriváty metabolismus MeSH
- oxidace-redukce MeSH
- polymerázová řetězová reakce MeSH
- sekvence nukleotidů MeSH
- stereoizomerie MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Penicillin G acylase of Escherichia coli (PGAEc) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of beta-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. RESULTS: Leader-less pga gene encoding PGAEcunder the control of AOX1 promoter was cloned in Pichia pastoris X-33. The intracellular overproduction of heterologous PGAEc(hPGAEc) was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l-1 was reached. The hPGAEc was purified, characterized and compared with the wild-type PGAEc. The alpha-subunit of the hPGAEc formed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB) to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin. CONCLUSIONS: Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGAEc in P. pastoris X-33. Aberrant processing of the precursor of PGAEc in the cytosol yielded the mature enzyme with modified traits.
- MeSH
- bioreaktory MeSH
- Escherichia coli enzymologie genetika MeSH
- fermentace MeSH
- klonování DNA MeSH
- penicilinamidasa biosyntéza genetika izolace a purifikace MeSH
- Pichia genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- proteiny z Escherichia coli biosyntéza genetika izolace a purifikace MeSH
- průmyslová mikrobiologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
