RESUMO
OBJECTIVE: Cloning and secretory expression of an amidase from Kluyvera cryocrescens and characterization of its potential in preparation of chiral amino acids. RESULTS: An amidase belonging to the Ntn-hydrolase superfamily was identified from Kluyvera cryocrescens ZJB-17005 (Kc-Ami). The maximum activity of Kc-Ami was observed at pH 8.5 and 55 °C. Remarkably, Kc-Ami showed an excellent enantioselectivity (99% ee) using rac-4-(hydroxy(methyl)phosphoryl)-2-(2-phenylacetamido) butanoic acid as substrate. Kc-Ami remained stable at pH 7.0-9.0 and exhibited prominent thermostability with a half-life time of 59.1, 47.4 and 20.4 h at 50, 55 and 60 °C, respectively. Kc-Ami could be appllied to synthesize chiral amino acids and its derivatives with excellent enantioselectivity (> 99% ee). The synthesized chiral amino acids could contain short or long side chain, and further the side chain could be replaced with -OH, -COOH or benzene ring. CONCLUSIONS: Kc-Ami exhibited remarkable thermostability and excellent enantioselectivity for synthesizing chiral amino acids and its derivatives. This specific characteristic provides great potential for industrial application in preparation of chiral amino acids and its derivatives.
Assuntos
Amidoidrolases/genética , Amidoidrolases/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Clonagem Molecular/métodos , Kluyvera/enzimologia , Amidoidrolases/química , Aminoácidos/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Kluyvera/genética , Modelos Moleculares , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Carbapenemase-producing bacteria cause difficult-to-treat infections related to increased mortality in health care settings. Their occurrence has been reported in raw sewage, sewage-impacted rivers, and polluted coastal waters, which may indicate their spread to the community. We assessed the variety and concentration of carbapenemase producers in coastal waters with distinct pollution levels for 1 year. We describe various bacterial species producing distinct carbapenemases not only in unsuitable waters but also in waters considered suitable for primary contact.
Assuntos
Proteínas de Bactérias/genética , Klebsiella pneumoniae/genética , Água do Mar/microbiologia , Microbiologia da Água , beta-Lactamases/genética , Acinetobacter/enzimologia , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Aeromonas/enzimologia , Aeromonas/genética , Aeromonas/isolamento & purificação , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Brasil , Citrobacter/enzimologia , Citrobacter/genética , Citrobacter/isolamento & purificação , Enterobacter/enzimologia , Enterobacter/genética , Enterobacter/isolamento & purificação , Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Kluyvera/enzimologia , Kluyvera/genética , Kluyvera/isolamento & purificação , Pseudomonas/enzimologia , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Recreação , Serratia/enzimologia , Serratia/genética , Serratia/isolamento & purificação , beta-Lactamases/classificação , beta-Lactamases/metabolismoAssuntos
Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Kluyvera/efeitos dos fármacos , Kluyvera/genética , Plasmídeos/genética , beta-Lactamases/genética , Sequência de Bases , Infecções por Enterobacteriaceae/microbiologia , Feminino , Genoma Bacteriano , Humanos , Itália , Kluyvera/enzimologia , Plasmídeos/isolamento & purificação , Reto/microbiologia , Sequenciamento Completo do GenomaRESUMO
Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and ß chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1 Å, α = 104.1, ß = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2 Å, ß = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.
Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Kluyvera/genética , Mutação/genética , Penicilina Amidase/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Clonagem Molecular/métodos , Cristalização , Cristalografia por Raios X , Kluyvera/enzimologia , Penicilina Amidase/biossíntese , Penicilina Amidase/química , Dobramento de ProteínaRESUMO
A one-pot, two-step enzymatic synthesis of amoxicillin from penicillin G, using penicillin acylase, is presented. Immobilized penicillin acylase from Kluyvera citrophila was selected as the biocatalyst for its good pH stability and selectivity. Hydrolysis of penicillin G and synthesis of amoxicillin from the 6-aminopenicillanic acid formed and D-p-hydroxyphenylglycine methyl ester were catalyzed in situ by a single enzyme. Zinc ions can react with amoxicillin to form complexes, and the yield of 76.5% was obtained after optimization. In the combined one-pot synthesis process, zinc sulfate was added to remove produced amoxicillin as complex for shifting the equilibrium to the product in the second step. By controlling the conditions in two separated steps, the conversion of the first and second step was 93.8% and 76.2%, respectively. With one-pot continuous procedure, a 71.5% amoxicillin yield using penicillin G was obtained.
Assuntos
Amoxicilina/metabolismo , Kluyvera/enzimologia , Penicilina Amidase/metabolismo , Zinco/metabolismo , Biotecnologia/métodos , Glicina/análogos & derivados , Glicina/metabolismo , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/metabolismo , Penicilina Amidase/isolamento & purificação , Penicilina G/metabolismo , Tecnologia Farmacêutica/métodosRESUMO
L-DOPA (L-dihydroxyphenylalanine) is a promising drug for Parkinson's disease and thereby has a growing annual demand. Tyrosine phenol-lyase (TPL)-based catalysis is considered to be a low-cost yet efficient route for biosynthesis of L-DOPA. TPL is a tetrameric enzyme that catalyzes the synthesis of L-DOPA from pyrocatechol, sodium pyruvate, and ammonium acetate. The implementation of TPL for L-DOPA production has been hampered and the need for the most efficient TPL source with higher L-DOPA production and substrate conversion rate is prevailing. This study involves identifying a novel TPL from Kluyvera intermedia (Ki-TPL) and displayed a robust expression in Escherichia coli. The recombinant strain YW000 carrying Ki-TPL proved strong catalytic activity with a highest L-DOPA yield compared with 16 other TPLs from different organisms. With a further aim to improve this efficiency, random mutagenesis of Ki-TPL was performed and a mutant namely YW021 was obtained. The whole cells of YW021 as biocatalyst yielded 150.4 g L-1 of L-DOPA with a 99.99 % of pyrocatechol conversion at the optimum condition of pH 8.0 at 25 °C, which is the highest level reported to date. Further, the homology modeling and structural analysis revealed the mutant residues responsible for the extensive L-DOPA biosynthesis.
Assuntos
Biocatálise , Escherichia coli/metabolismo , Kluyvera/enzimologia , Levodopa/química , Tirosina Fenol-Liase/genética , Acetatos/química , Catecóis/química , Clonagem Molecular , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Mutagênese , Mutação , Fosfato de Piridoxal/química , Ácido Pirúvico/química , Sódio/química , TemperaturaRESUMO
ISEcp1B is an insertion element associated with the emerging expanded-spectrum beta-lactamase bla(CTX-M) genes in Enterobacteriaceae. Because ISEcp1B-bla(CTX-M)positive strains may be identified from humans and animals, the ability of this insertion sequence to mobilize the bla(CTX-M-2) gene was tested from its progenitor Kluyvera ascorbata to study the effects of amoxicillin/clavulanic and cefquinome as enhancers of transposition. These beta-lactam molecules are administered parenterally to treat infected animals. ISEcp1B-mediated mobilization of the bla(CTX-M-2) gene from K. ascorbata to a plasmid location in Escherichia coli J53 was studied. Transposition assays were performed with overnight cultures with amoxicillin/clavulanic acid and cefquinome at concentrations expected to mimic those found in feces after parenteral administration (0.4-0.008 mg L(-1) and 0.32-0.064 mg L(-1), respectively). Amoxicillin/clavulanic acid and cefquinome did not modify the transposition frequency (1.85+/-1.7 x 10(-7)) whereas ceftazidime (0.5 mg L(-1)), used as a control, did (5.2+/-2.7 x 10(-5)). Therefore, it is likely that neither amoxicillin/clavulanic acid nor cefquinome concentrations as found in the gut flora may enhance mobilization of the bla(CTX-M) genes in Enterobacteriaceae.
Assuntos
Elementos de DNA Transponíveis/fisiologia , beta-Lactamases/genética , beta-Lactamas/farmacologia , Animais , Conjugação Genética , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Kluyvera/efeitos dos fármacos , Kluyvera/enzimologia , Kluyvera/genética , Kluyvera/crescimento & desenvolvimento , Plasmídeos/genética , beta-Lactamases/metabolismoRESUMO
In the past decade CTX-M enzymes have become the most prevalent extended-spectrum beta-lactamases, both in nosocomial and in community settings. The insertion sequences (ISs) ISEcp1 and ISCR1 (formerly common region 1 [CR1] or orf513) appear to enable the mobilization of chromosomal beta-lactamase Kluyvera species genes, which display high homology with blaCTX-Ms. These ISs are preferentially linked to specific genes: ISEcp1 to most blaCTX-Ms, and ISCR1 to blaCTX-M-2 or blaCTX-M-9. The blaCTX-M genes embedded in class 1 integrons bearing ISCR1 are associated with different Tn402-derivatives, and often with mercury Tn21-like transposons. The blaCTX-M genes linked to ISEcp1 are often located in multidrug resistance regions containing different transposons and ISs. These structures have been located in narrow and broad host-range plasmids belonging to the same incompatibility groups as those of early antibiotic resistance plasmids. These plasmids frequently carry aminoglycoside, tetracycline, sulfonamide or fluoroquinolone resistance genes [qnr and/or aac(6')-Ib-cr], which would have facilitated the dissemination of blaCTX-M genes because of co-selection processes. In Escherichia coli, they are frequently carried in well-adapted phylogenetic groups with particular virulence-factor genotypes. Also, dissemination has been associated with different clones (CTX-M-9 or CTX-M-14 producers) or epidemic clones associated with specific enzymes such as CTX-M-15. All these events might have contributed to the current pandemic CTX-M beta-lactamase scenario.
Assuntos
Bactérias/enzimologia , Infecções Bacterianas/microbiologia , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/epidemiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana Múltipla , Doenças Endêmicas , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/enzimologia , Genes Bacterianos , Saúde Global , Humanos , Integrons , Kluyvera/enzimologia , Plasmídeos/genética , beta-Lactamases/genéticaAssuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Kluyvera/efeitos dos fármacos , Kluyvera/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/genética , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Infecções por Enterobacteriaceae/microbiologia , Humanos , Kluyvera/genética , Kluyvera/isolamento & purificação , Testes de Sensibilidade Microbiana , Análise de Sequência de DNA , beta-Lactamases/metabolismoRESUMO
This study characterised the genetic environment of the chromosomally encoded bla(KLUA-9) gene from a clinical Kluyvera ascorbata isolate and performed a kinetic characterisation of KLUA-9. Purified KLUA-9 showed the highest catalytic efficacies towards benzylpenicillin, ampicillin, piperacillin, first-generation cephalosporins, cefuroxime and cefoperazone; like other 'cefotaximases', it showed a much higher rate of hydrolysis of cefotaxime than ceftazidime, whilst dicloxacillin, cefoxitin and imipenem behaved as poor substrates. A 9kb insert from K. ascorbata was cloned (Escherichia coli KK68C1) and sequenced. bla(KLUA-9) and its 266bp upstream flanking region (almost identical to the integron-associated bla(CTX-M-2)) are preceded by an aspat variant, a ypdABC-like operon and two open reading frames with unknown functions. Unlike ISCR1-associated bla(CTX-M-2) genes, we failed to detect the putative orf513 recombination sites. Instead, we were able to localise the 5bp target sites for insertion of ISEcp1B, suggesting that this element could be responsible for future (or still undetected) mobilisation of bla(KLUA-9) to more efficiently transferred elements.
Assuntos
Cefalosporinas/farmacologia , Kluyvera/enzimologia , Kluyvera/genética , beta-Lactamases/genética , Sequência de Bases , Resistência às Cefalosporinas/genética , Cefalosporinas/metabolismo , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Infecções por Enterobacteriaceae/microbiologia , Evolução Molecular , Genes Bacterianos , Humanos , Técnicas In Vitro , Cinética , Kluyvera/efeitos dos fármacos , Kluyvera/isolamento & purificação , Dados de Sequência Molecular , Plasmídeos/genética , beta-Lactamases/metabolismoRESUMO
Formation of inclusion bodies is an important obstacle to the production of active recombinant protein in Escherichia coli. Thus, soluble expression of penicillin G acylase from Kluyvera citrophila was investigated in BL21(DE3). In this study, the yield of active enzyme was significantly enhanced by the composition of the medium and induction opportunity. When 0.5 mmol/L IPTG was added to complex medium at 15 h after incubation, the volumetric and specific activities of penicillin G acylase both achieved the highest values, respectively. However, aggravation of intracellular proteolysis and decline of enzyme expression were also observed if induction occurred too much later. Ca2+ ion was another critical factor in cell growth and protein expression. When 24 mmol/L Ca2+ ion was adding to the medium at the beginning of fermentation, a greater than 2-fold increase in cell density and a 7-fold increase in volumetric activity of penicillin G acylase were reached. Nevertheless, no significant benefit for recombination protein expression was found when excess Ca2+ was added after induction time. This study demonstrates that the induction starting time and Ca2+ ion are two critical factors for the expression of active penicillin G acylase.
Assuntos
Reatores Biológicos/microbiologia , Cálcio/administração & dosagem , Técnicas de Cultura de Células/métodos , Escherichia coli/metabolismo , Kluyvera/enzimologia , Penicilina Amidase/biossíntese , Engenharia de Proteínas/métodos , Relação Dose-Resposta a Droga , Ativação Enzimática , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Kluyvera/genética , Penicilina Amidase/genética , Proteínas Recombinantes/metabolismo , Fatores de TempoAssuntos
Cromossomos Bacterianos/genética , Kluyvera/enzimologia , beta-Lactamases/genética , Ampicilina/farmacologia , Antibacterianos/farmacologia , Gentamicinas/farmacologia , Humanos , Kluyvera/efeitos dos fármacos , Kluyvera/genética , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Ácido Nalidíxico/farmacologia , Sulfametoxazol/farmacologia , Trimetoprima/farmacologiaRESUMO
Penicillin acylases are industrially important enzymes for the production of 6-APA, which is used extensively in the synthesis of secondary antibiotics. The enzyme translates into an inactive single chain precursor that subsequently gets processed by the removal of a spacer peptide connecting the chains of the mature active heterodimer. We have cloned the penicillin G acylase from Kluyvera citrophila (KcPGA) and prepared two mutants by site-directed mutagenesis. Replacement of N-terminal serine of the ß-subunit with cysteine (Serß1Cys) resulted in a fully processed but inactive enzyme. The second mutant in which this serine is replaced by glycine (Serß1Gly) remained in the unprocessed and inactive form. The crystals of both mutants belonged to space group P1 with four molecules in the asymmetric unit. The three-dimensional structures of these mutants were refined at resolutions 2.8 and 2.5 Å, respectively. Comparison of these structures with similar structures of Escherichia coli PGA (EcPGA) revealed various conformational changes that lead to autocatalytic processing and consequent removal of the spacer peptide. The large displacements of residues such as Arg168 and Arg477 toward the N-terminal cleavage site of the spacer peptide or the conformational changes of Arg145 and Phe146 near the active site in these structures suggested probable steps in the processing dynamics. A comparison between the structures of the processed Serß1Cys mutant and that of the processed form of EcPGA showed conformational differences in residues Argα145, Pheα146, and Pheß24 at the substrate binding pocket. Three conformational transitions of Argα145 and Pheα146 residues were seen when processed and unprocessed forms of KcPGA were compared with the substrate bound structure of EcPGA. Structure mediation in activity difference between KcPGA and EcPGA toward acyl homoserine lactone (AHL) is elucidated.
Assuntos
Kluyvera/enzimologia , Kluyvera/genética , Penicilina Amidase/química , Penicilina Amidase/metabolismo , Processamento de Proteína Pós-Traducional , Sítios de Ligação , Cristalografia por Raios X , Kluyvera/química , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação , Penicilina Amidase/genética , Ligação Proteica , Percepção de Quorum , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Penicillin G Acylas (PGA) of Providencia rettgeri (ATCC 25599) was evolved using a modified DNA family shuffling method. The identity of pga genes from Escherichia coli, Kluyvera citrophila and Providencia rettgeri ranges from 62.5% to 96.9%. The pga genes from above three species were recombined and shuffled to create interspecies pga gene fusion libraries. By substituting assembled chimaeras for corresponding region of pETPPGA, different recombinants were constructed and expressed in E. coli JM109(DE3). Mutants with obvious beta-lactam synthetic activity were selected from the plates and the ratios of synthesis to hydrolysis (S/H) were determined subsequently. It was shown that the primary structures of selected positives exhibited significant diversity among each library. The best mutant possessed 40% higher synthetic activity than the wild type enzyme of PrPGA. It was further proved in this study that the domain of alpha subunit contributed much more to improve the specific activity of synthesis. Results showed a recombinant PGA with higher synthetic activity was acquired by the method of DNA shuffling.
Assuntos
Embaralhamento de DNA/métodos , Escherichia coli/enzimologia , Kluyvera/enzimologia , Penicilina Amidase/genética , Providencia/enzimologia , DNA Bacteriano/genética , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Kluyvera/genética , Penicilina Amidase/metabolismo , Plasmídeos/genética , Mutação Puntual , Providencia/genética , Proteínas Recombinantes de Fusão/genética , Recombinação Genética , Homologia de SequênciaRESUMO
Use of penicillin acylases for the production of semi-synthetic penicillins is well-known. Escherichia coli penicillin G acylase (EcPGA) has been extensively used for this purpose; however, Kluyvera citrophila penicillin G acylase (KcPGA) is assumed to be a better substitute, owing to its increased resilience to extreme pH conditions and ease of immobilization. In the present article we report a new dimension for the amidase activity of KcPGA by demonstrating its ability to cleave bacterial quorum sensing signal molecules, acyl homoserine lactones (AHL) with acyl chain length of 6-8 with or without oxo-substitution at third carbon position. Initial evidence of AHL degrading capability of KcPGA was obtained using CV026 based bioassay method. Kinetic studies performed at pH 8.0 and 50 °C revealed 3-oxo-C6 HSL to be the best substrate for the enzyme with V(max) and K(m) values of 21.37+0.85 mM/h/mg of protein and 0.1+0.01 mM, respectively. C6 HSL was found to be the second best substrate with V(max) and K(m) value of 10.06+0.27 mM/h/mg of protein and 0.28+0.02 mM, respectively. Molecular modeling and docking studies performed on the active site of the enzyme support these findings by showing the fitting of AHLs perfectly within the hydrophobic pocket of the enzyme active site.
Assuntos
Acil-Butirolactonas/metabolismo , Proteínas de Bactérias/fisiologia , Kluyvera/enzimologia , Penicilina Amidase/fisiologia , Percepção de Quorum/fisiologia , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Chromobacterium/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Genes Bacterianos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Kluyvera/genética , Modelos Moleculares , Penicilina Amidase/química , Penicilina Amidase/genética , Penicilinas/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
Kluyvera species strain OM3 isolated from spent mushroom substrate could produce a high level of cellulase-free xylanase (5.12 U/mL). This xylanase showed maximum activities at 70 °C and pH 8.0, which could retain 100% and 71% activity after 1h incubation at 60 °C and 70 °C, and maintain stability over a wide range of pHs (5.0-9.0), indicating its thermal and pH stability. Moreover, the xylanase could hydrolyze untreated lignocellulosics (e.g., palm oil fiber) to reducing sugars with a yield of 27.1-46.9 mg/g. A co-culture consisting of Kluyvera sp. strain OM3 and Clostridium sp. strain BOH3 could directly convert birchwood xylan to 1.2g/L butanol, which was comparable to the amount of butanol (1.7 g/L) generated via separate hydrolysis by the xylanase and fermentation by Clostridium sp. strain BOH3. This is the first report on the production, characterization of a xylanase from genus Kluyvera and its application for butanol production directly from hemicelluloses.
Assuntos
Biocombustíveis/microbiologia , Butanóis/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Kluyvera/enzimologia , Kluyvera/isolamento & purificação , Temperatura , Anaerobiose , Biomassa , Técnicas de Cocultura , Estabilidade Enzimática , Fermentação , Hidrólise , Kluyvera/crescimento & desenvolvimento , Lignina/metabolismo , Dados de Sequência Molecular , Filogenia , Xilanos/metabolismoRESUMO
ISEcp1B has been reported to be associated with and to mobilize the emerging expanded-spectrum beta-lactamase blaCTX-M genes in Enterobacteriaceae. Thus, the ability of this insertion sequence to mobilize the blaCTX-M-2 gene was tested from its progenitor, Kluyvera ascorbata. Insertions of ISEcp1B upstream of the blaCTX-M-2 gene in K. ascorbata reference strain CIP7953 were first selected with cefotaxime (0.5 and 2 microg/ml). In those cases, ISEcp1B brought promoter sequences enhancing blaCTX-M-2 expression in K. ascorbata. Then, ISEcp1B-mediated mobilization of the blaCTX-M-2 gene from K. ascorbata to Escherichia coli J53 was attempted. The transposition frequency of ISEcp1B-blaCTX-M-2 occurred at (6.4+/-0.5)x10(-7) in E. coli. Cefotaxime, ceftazidime, and piperacillin enhanced transposition, whereas amoxicillin, cefuroxime, and nalidixic acid did not. Transposition was also enhanced when studied at 40 degrees C.
Assuntos
Elementos de DNA Transponíveis/fisiologia , Kluyvera/genética , beta-Lactamases/genética , Kluyvera/enzimologia , Kluyvera/crescimento & desenvolvimentoRESUMO
Penicillin G acylase, from Kluyvera citrophila, was used in kinetic resolution of DL-tert-leucine. N-phenylacetylated-DL-tert-leucine, chemically synthesized from DL-tert-leucine, was enantioselctively hydrolyzed by penicillin G acylase to obtain L-tert-leucine, D-tert-leucine was prepared by acid-catalyzed hydrolysis of the remaining substrate. The total yields of D-tert-leucine and L-tert-leucine are 80.6% and 83.1%, respectively. The enantiomeric excess of the two products, D-tert-leucine and L-tert-leucine, are 98.5% and 99%. This is a practical way for the preparation of D-tert-leucine and L-tert-leucine.
Assuntos
Leucina/análogos & derivados , Leucina/síntese química , Penicilina Amidase/metabolismo , Catálise , Concentração de Íons de Hidrogênio , Hidrólise , Kluyvera/enzimologia , Leucina/química , Estrutura Molecular , Rotação Ocular , Penicilina G/metabolismoRESUMO
Penicillin G acylase (PGA) from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-DL-tert-leucine (N-Phac-DL-Tle) to produce L-tert-leucine (L-Tle). The effects of various organic cosolvents on hydrolysis of N-Phac-DL-Tle have been investigated in aqueous-cosolvent medium. It was founded that the rate of PGA-catalyzed reaction was significantly affected by the presence of 2% (v/v) organic cosolvent concentration. The initial rate fell with increasing logP of the cosolvent, but for logP values less than -0.24 the rate was faster than in purely aqueous medium. Additionally, the relative rate increases with the increase of dielectric constant (epsilon) of organic cosolvents. The yields of L-Tle in all aqueous-cosolvent systems were above 95% with the enantiomeric excess (ee) of >99%.
Assuntos
Kluyvera/enzimologia , Compostos Orgânicos/química , Penicilina Amidase/química , Solventes/química , Valina/análogos & derivados , Ativação Enzimática , Enzimas Imobilizadas/química , Cinética , Leucina/análogos & derivados , Valina/químicaRESUMO
Several genes for the enzyme penicillin G acylase, as isolated from four different micro-organisms (Alcaligenes facaelis, Escherichia coli, Kluyvera cryocrescens or Providencia rettgeri) were modified at their carboxy-termini to include His-tag fusions, then were expressed from the plasmid pET-24a(+) in E. coli JM109(DE3) cells. All fusion proteins were next purified to homogeneity in a single step by agar-based Co-IDA chromatography, and were then evaluated as catalysts for the synthesis of cephalexin by a kinetically controlled strategy. We find here that the penicillin G acylase enzyme from K. cryocrescens shows a higher intrinsic synthesis/hydrolysis ratio, when compared to three other enzymes from A. facaelis or P. rettgeri, or E. coli.