RESUMO
The present study was undertaken to develop a self-micellizing solid dispersion (SMSD) of tacrolimus (TAC) to improve the biopharmaceutical properties of TAC. An SMSD formulation of TAC (SMSD/TAC) and amorphous solid dispersion formulation of TAC (ASD/TAC) were prepared with Soluplus® , an amphiphilic copolymer, and hydroxypropyl cellulose, respectively. Physicochemical properties were characterized in terms of morphology, crystallinity, storage stability, interaction of TAC with Soluplus® , and micelle-forming potency; pharmacokinetic behavior was also evaluated in rats. Tacrolimus in both formulations was in an amorphous state. After storage at 40°C/75% relativity humidity for 4 weeks, there were no significant changes in the crystallinity of TAC between nonaged and aged SMSD/TAC, whereas slight recrystallization was observed in aged ASD/TAC. The results of circular dichroism (CD) and infrared spectroscopic analyses were indicative of the potent drug-polymer interaction in SMSD/TAC, possibly leading to the prevention of recrystallization. Compared with other TAC samples, SMSD/TAC exhibited significant improvement in the dissolution behavior of TAC through the immediate formation of fine micelles. After the oral administration of TAC samples (10 mg TAC/kg) to rats, there was marked enhancement in systemic exposure to TAC with both formulations; in particular, SMSD/TAC achieved an increase in bioavailability ca. 20-fold higher than crystalline TAC. The SMSD approach might provide an effective dosage form for TAC with enhanced physicochemical stability and oral absorption.
Assuntos
Polietilenoglicóis , Tacrolimo , Ratos , Animais , Ratos Sprague-Dawley , Solubilidade , Micelas , Disponibilidade Biológica , Administração OralRESUMO
The aim of the present study was to develop an injectable hydrogel (HG) formulation of fuzapladib sodium (FZP), an animal drug for acute pancreatitis (AP), with the use of polyethyleneoxide (PEO) and polylysine (pLys), a cationic polymer. A mixture of pLys and FZP was added to PEO to prepare an HG formulation, and the formulation was optimized by release test and viscosity measurements. Circular dichroism (CD) and infrared absorption (IR) spectral analyses were applied to clarify the intermolecular interactions between FZP and pLys. The pharmacokinetic behavior of FZP was evaluated after a subcutaneous administration of FZP samples (2.0 mg-FZP/kg) to rats. Although the immediate release of FZP was observed for the HG formulation, the addition of pLys at a 20-fold amount of FZP or higher led to the sustained release of FZP. Considering release behavior, the concentration of pLys was optimized as 100-fold that of FZP in the HG formulation. CD and IR spectroscopic analyses of FZP and/or pLys demonstrated an intermolecular interaction between FZP and pLys, as evidenced by the slight spectral transition. After a subcutaneous administration of HG formulation containing pLys to rats, compared with FZP alone, significant differences were observed in the pharmacokinetic behavior with a decrease of Cmax from 2.3 to 0.9 mg/mL and slower elimination kinetics. HG formulation using pLys might be a viable dosage option for FZP for the treatment of AP in animals.
Assuntos
Pancreatite , Polilisina , Ratos , Animais , Polilisina/química , Hidrogéis , Preparações de Ação Retardada/química , Antígeno-1 Associado à Função Linfocitária , Doença Aguda , LeucócitosRESUMO
PURPOSE: The aim of the present study was to develop a new multi-unit dosage formulation, Universal ORbicular Vehicle (UniORV), to improve the biopharmaceutical properties of tacrolimus (TAC). METHODS: TAC-loaded UniORV (UO/TAC) was produced by the dripping and gelling of a solution comprising TAC, gelatin, starch syrup, and triethyl citrate at 0.5 w/w% drug loading. Its microstructure was elucidated by polarized light microscopy and the Raman mapping technique. The pharmacokinetic profiles of TAC after the oral administration of UO/TAC were evaluated in rats and healthy humans. RESULTS: The dissolution behavior of UO/TAC was similar to that of commercial capsules, and the formation of nanoparticles was detected by TEM in dissolved media. In a stability study on UO/TAC, only 2.6 and 4.7% decreases in TAC concentrations were observed at 40± 2°C/75 ± 5% relative humidity for 4 months and at 50± 2°C for 2 months, respectively. A pharmacokinetic study on rats revealed a 30-fold higher AUC than that with crystalline TAC. A randomized double-blind crossover study on 8 healthy males showed that UniORV achieved a 1.4-fold increase in AUC and 34% decrease in inter-individual variation from the reference formulation. CONCLUSION: The new dosage form UniORV is a promising approach to improve the dissolution, amorphous stability, and biopharmaceutical properties of TAC, which is a poorly water-soluble drug.
Assuntos
Imunossupressores/administração & dosagem , Imunossupressores/farmacocinética , Veículos Farmacêuticos/química , Tacrolimo/administração & dosagem , Tacrolimo/farmacocinética , Administração Oral , Adulto , Animais , Citratos/química , Estudos Cross-Over , Método Duplo-Cego , Composição de Medicamentos , Liberação Controlada de Fármacos , Gelatina/química , Humanos , Imunossupressores/sangue , Masculino , Ratos , Ratos Sprague-Dawley , Amido/química , Tacrolimo/sangue , Adulto JovemRESUMO
Solution structures of DNA duplexes containing oxanine (Oxa, O) opposite a cytosine (O:C duplex) and opposite a thymine (O:T duplex) have been solved by the combined use of (1)H NMR and restrained molecular dynamics calculation. One mismatch pair was introduced into the center of the 11-mer duplex of [d(GTGACO(6)CACTG)/d(CAGTGX(17)GTCAC), X = C or T]. (1)H NMR chemical shifts and nuclear Overhauser enhancement (NOE) intensities indicate that both the duplexes adopt an overall right-handed B-type conformation. Exchangeable resonances of C(17) 4-amino proton of the O:C duplex and of T(17) imino proton of O:T duplex showed unusual chemical shifts, and disappeared with temperature increasing up to 30 °C, although the melting temperatures were >50 °C. The O:C mismatch takes a wobble geometry with positive shear parameter where the Oxa ring shifted toward the major groove and the paired C(17) toward the minor groove, while, in the O:T mismatch pair with the negative shear, the Oxa ring slightly shifted toward the minor groove and the paired T(17) toward the major groove. The Oxa mismatch pairs can be wobbled largely because of no hydrogen bond to the O1 position of the Oxa base, and may occupy positions in the strands that optimize the stacking with adjacent bases.
Assuntos
Pareamento Incorreto de Bases , DNA/química , Nucleosídeos de Purina/química , Citosina/química , Desoxirribonucleosídeos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Timina/químicaRESUMO
Inexpensive and safe energy-storage batteries with high energy densities are in high demand (e.g., for electric vehicles and grid-level renewable energy storage). This study focused on using NaFeCl4, comprising ubiquitous elements, as an electrode material for all-solid-state sodium-ion batteries. Monoclinic NaFeCl4, expected to be the most resource-attractive Fe redox material, is also thermodynamically stable. The Fe2+/3+ redox reaction of the monoclinic NaFeCl4 electrode has a higher potential (3.45 V vs. Na/Na+) than conventional oxide electrodes (e.g., Fe2O3 with 1.5 V vs. Na/Na+) because of the noble properties of chlorine. Additionally, NaFeCl4 exhibits unusually high deformability (99% of the relative density of the pellet) upon uniaxial pressing (382 MPa) at 298 K. NaFeCl4 operates at 333 K in an electrode system containing no electrolyte, thereby realizing next-generation all-solid-state batteries with high safety. A high energy density per positive electrode of 281 Wh kg-1 was achieved using only a simple powder press.
RESUMO
RNA polymerases (RNAPs) transcribe genes through the barrier of nucleoproteins and site-specific DNA-binding proteins on their own or with the aid of accessory factors. Proteins are often covalently trapped on DNA by DNA damaging agents, forming DNA-protein cross-links (DPCs). However, little is known about how immobilized proteins affect transcription. To elucidate the effect of DPCs on transcription, we constructed DNA templates containing site-specific DPCs and performed in vitro transcription reactions using phage T7 RNAP. We show here that DPCs constitute strong but not absolute blocks to in vitro transcription catalyzed by T7 RNAP. More importantly, sequence analysis of transcripts shows that RNAPs roadblocked not only by DPCs but also by the stalled leading RNAP become highly error prone and generate mutations in the upstream intact template regions. This contrasts with the transcriptional mutations induced by conventional DNA lesions, which are delivered to the active site or its proximal position in RNAPs and cause direct misincorporation. Our data also indicate that the trailing RNAP stimulates forward translocation of the stalled leading RNAP, promoting the translesion bypass of DPCs. The present results provide new insights into the transcriptional fidelity and mutual interactions of RNAPs that encounter persistent roadblocks.
Assuntos
Bacteriófago T7/enzimologia , Bacteriófago T7/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Gênica/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Catálise , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Mutagênese/genética , Mutação/genéticaRESUMO
Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on T(m) values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar T(m) values were separated and detected quantitatively by using a packed column carrying two different DNA probes.
Assuntos
DNA/análise , Técnicas Analíticas Microfluídicas/métodos , Sondas de DNA/química , Técnicas Analíticas Microfluídicas/instrumentação , TemperaturaRESUMO
Reactive oxygen species produced by phagocytosing neutrophils are essential for innate host defense against invading microbes. Previous observations revealed that antibody-catalyzed ozone formation by human neutrophils contributed to the killing of bacteria. In this study, we discovered that 4 amino acids themselves were able to catalyze the production of an oxidant with the chemical signature of ozone from singlet oxygen in the water-oxidation pathway, at comparable level to antibodies. The resultant oxidant with the chemical signature of ozone exhibited significant bactericidal activity in our distinct cell-free system and in human neutrophils. The results also suggest that an oxidant with the chemical signature of ozone produced by neutrophils might potentiate a host defense system, when the host is challenged by high doses of infectious agents. Our findings provide biological insights into the killing of bacteria by neutrophils.
Assuntos
Aminoácidos/metabolismo , Antibacterianos/metabolismo , Neutrófilos/metabolismo , Ozônio/metabolismo , Adulto , Aminoácidos/química , Catálise , Escherichia coli/metabolismo , Doença Granulomatosa Crônica/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Oxidantes/biossíntese , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Oxigênio Singlete/metabolismoRESUMO
DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA , DNA/metabolismo , Desoxirribonucleotídeos/genética , Proteínas/metabolismo , Recombinação Genética , Animais , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteína BRCA2/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromossomos/metabolismo , Cricetinae , DNA/química , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Decitabina , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Formaldeído/farmacologia , Histonas/metabolismo , Humanos , Peso Molecular , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/química , Rad51 Recombinase/metabolismoRESUMO
Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO(2)-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5'-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa>Gua>adenine (Ade) approximately thymine (Thy)>cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.
Assuntos
DNA Ligases/química , Enzimas de Restrição do DNA/química , DNA de Cadeia Simples/química , Polinucleotídeo 5'-Hidroxiquinase/química , Nucleosídeos de Purina/química , Pareamento de Bases , Citosina/química , Clivagem do DNA , DNA de Cadeia Simples/genética , Fosforilação , Nucleosídeos de Purina/genéticaRESUMO
T4 DNA ligase-based mismatch detection methods have been proposed as useful strategies for single nucleotide polymorphism (SNP) analyses. However, there is a critical problem for cytosine/thymine (C/T) SNP analyses: guanine:thymine (G:T) mismatch is not distinguished from guanine:cytosine (G:C). Here we employed chemically modified nucleobases, such as oxanine and hypoxanthine, at the end of a ligation fragment and analyzed their influence on the ligation efficiency between G:C and G:T. Successful ligation for G:C and no ligation for G:T were observed when oxanine was employed adjacent to guanine in the ligation junction. This ligation method using an oxanine-containing fragment has strong potentials for the accurate analysis of C/T SNPs.
Assuntos
Citosina , DNA Ligases/química , DNA Ligases/genética , Guanina , Polimorfismo de Nucleotídeo Único , Nucleosídeos de Purina/química , Pareamento Incorreto de Bases , Sequência de BasesRESUMO
The relationship of chemical structures of 6-formylpterin (6FP) and its derivatives with scavenging activity of singlet oxygen ((1)O(2)) was examined. First, effects of pterin derivatives on (1)O(2) released from activated human neutrophils were examined. The neutrophils, stimulated with opsonized zymosan, released (1)O(2) that was detected by chemiluminescence using a (1)O(2) specific probe, trans-1-(2'-methoxyvinyl)pyrene. 6FP and its derivatives suppressed the (1)O(2) release. 6FP and other commercially available pterin derivatives, such as biopterin and neopterin, which have different substitutions at the 6-position, suppressed the (1)O(2) release with similar extent. On the other hand, newly synthesized pterin derivatives, which have different substitutions at the 2- and/or 3-position, such as 2-amino-6-formyl-3-methylpteridin-4-one, suppressed the (1)O(2) release in a dose-dependent manner and more potently than 6FP. Then, the (1)O(2) scavenging activity of pterin derivatives was examined photochemically by direct analysis of near-infrared luminescence at 1270 nm, the most sensitive method for the detection of (1)O(2). When rose Bengal, a photosensitizer, in D(2)O solution, was irradiated by 514 nm laser beam, the emission spectrum of (1)O(2) was observed. 6FP suppressed this emission spectrum of (1)O(2), and the newly synthesized pterin derivatives with different substituent at the 2- and/or 3-position suppressed the spectrum more potently than 6FP. The order of potency was similar to that obtained from biological assays. These findings indicate that the substitutions at the 2- and/or 3-position play an important role in (1)O(2) scavenging activity of pterin derivatives.
Assuntos
Sequestradores de Radicais Livres/farmacologia , Neutrófilos/efeitos dos fármacos , Pterinas/farmacologia , Oxigênio Singlete/metabolismo , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/síntese química , Sequestradores de Radicais Livres/química , Humanos , Neutrófilos/metabolismo , Pterinas/síntese química , Pterinas/química , Rosa Bengala , Relação Estrutura-Atividade , Zimosan/farmacologiaRESUMO
The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP(+)-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.
Assuntos
D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Etanol/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Madeira/metabolismo , Xilose/metabolismoRESUMO
Oxanine having an O-acylisourea structure was explored to see if its reactivity with amino group is useful in DNA microarray fabrication. By the chemical synthesis, a nucleotide unit of oxanine (Oxa-N) was incorporated into the 5'-end of probe DNA with or without the -(CH2)n- spacers (n = 3 and 12) and found to immobilize the probe DNA covalently onto the NH2-functionalized glass slide by one-pot reaction, producing the high efficiency of the target hybridization. The methylene spacer, particularly the longer one, generated higher efficiency of the target recognition although there was little effect on the amount of the immobilized DNA oligomers. The post-spotting treatment was also carried out under the mild conditions (at 25 or 42 degrees C) and the efficiencies of the immobilization and the target recognition were evaluated similarly, and analogous trends were obtained. It has also been determined under the mild conditions that the humidity and time of the post-spotting treatment, pH of the spotting solution and the synergistic effects with UV-irradiation largely contribute to the desired immobilization and resulting target recognition. Immobilization of DNA oligomer by use of Oxa-N on the NH2-functionalized surface without any activation step would be employed as one of the advanced methods for generating DNA-conjugated solid surface.
Assuntos
Sondas de DNA/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Nucleosídeos de Purina/química , Aminas/química , Vidro , Umidade , Concentração de Íons de Hidrogênio , Sondas de Oligonucleotídeos/química , Temperatura , Fatores de TempoRESUMO
We have developed a simple ESR spin trapping based method for hydroxyl (OH) radical scavenging-capacity determination, using iron-free OH radical source. Instead of the widely used Fenton reaction, a short (typically 5 seconds) in situ UV-photolysis of a dilute hydrogen peroxide aqueous solution was employed to generate reproducible amounts of OH radicals. ESR spin trapping was applied to quantify OH radicals; the decrease in the OH radical level due to the specimen's scavenging activity was converted into the OH radical scavenging capacity (rate). The validity of the method was confirmed in pure antioxidants, and the agreement with the previous data was satisfactory. In the second half of this work, the new method was applied to the sera of chronic renal failure (CRF) patients. We show for the first time that after hemodialysis, OH radical scavenging capacity of the CRF serum was restored to the level of healthy control. This method is simple and rapid, and the low concentration hydrogen peroxide is the only chemical added to the system, that could eliminate the complexity of iron-involved Fenton reactions or the use of the pulse-radiolysis system.
RESUMO
Cytotoxic effects of the combined use of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a radical scavenger and an approved medicine for acute brain infarction in Japan, with a pterin derivative, were examined in vitro. When pancreatic cancer cell line Panc-1 cells were incubated with 50 to 400 microM of a pterin derivative, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one (DFP), and the equivalent dose of edaravone, reactive oxygen species (ROS), were generated, and cell death was induced. ROS generation and the loss of mitochondrial membrane potential (MMP) preceding cell death were simultaneously monitored using time-lapse microscopy with an ROS-sensitive dye and a probe to monitor MMP, respectively. Cell death was also estimated quantitatively by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. ROS generation and cell death were prominent when more than 100 microM of each agent was used in combination, whereas the sole use of each agent did not show any effects even at the highest dose, 400 microM. Chemical analysis revealed that DFP and edaravone react immediately in aqueous solution and produce a new compound named DFP-E. DFP-E chemically reacted with NADH much faster than DFP and generated ROS, and biologically, it was much more cell-permeable than DFP. These findings collectively indicated that the combined use of DFP with edaravone produced DFP-E, which caused intracellular ROS generation and cell death. Cell death was observed in normal cells, and edaravone reacted with another pterin derivative to yield an ROS-generating compound. As a result, care should be taken with the clinical use of edaravone when pterin derivatives stay in the body.
Assuntos
Antipirina/análogos & derivados , Citotoxinas/metabolismo , Sequestradores de Radicais Livres/metabolismo , Líquido Intracelular/metabolismo , Pterinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antipirina/química , Antipirina/metabolismo , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Edaravone , Sequestradores de Radicais Livres/química , Humanos , Pterinas/químicaRESUMO
A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD(+)-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP(+). In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP(+)-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP(+)-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.
Assuntos
D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Etanol/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , NADP/metabolismo , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We constructed a set of recombinant Saccharomyces cerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP(+)-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.
Assuntos
D-Xilulose Redutase/metabolismo , Etanol/metabolismo , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Saccharomyces cerevisiae/enzimologia , Xilose/metabolismo , D-Xilulose Redutase/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Xilitol/metabolismoRESUMO
Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 degrees C higher thermal transition temperature (T(CD)) and 20.8 degrees C higher half denaturation temperature (T(1/2)) compared with wild-type.
Assuntos
D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Pichia/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , D-Xilulose Redutase/química , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Termodinâmica , Zinco/metabolismoRESUMO
Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.