RESUMO
BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.
Assuntos
Carboidratos/química , Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Álcool Desidrogenase/biossíntese , Álcool Desidrogenase/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Proteína Morfogenética Óssea 7/biossíntese , Proteína Morfogenética Óssea 7/isolamento & purificação , Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Clonagem Molecular , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/isolamento & purificação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Humanos , Hidrolases/biossíntese , Hidrolases/isolamento & purificação , Corpos de Inclusão/metabolismo , Lipase/biossíntese , Lipase/isolamento & purificação , Proteínas Ligantes de Maltose , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/isolamento & purificaçãoRESUMO
Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity.
Assuntos
Álcool Desidrogenase/ultraestrutura , Aldeído Oxirredutases/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Acetilcoenzima A/química , Álcool Desidrogenase/isolamento & purificação , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/isolamento & purificação , Aldeído Oxirredutases/metabolismo , Microscopia Crioeletrônica , Ensaios Enzimáticos , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Etanol/química , Mutação , Conformação Proteica em alfa-Hélice/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
The recovery of enzymes from a reaction medium can be achieved in a convenient way by using magnetic nanoparticles (MNP) as carriers. Here, we present MNP with a polyelectrolyte brush composed of poly(ethylene imine) (PEI) to provide a benign environment for the immobilized enzyme molecules. Yeast alcohol dehydrogenase (ADH) has been tested for enzymatic activity when it is free in solution or adsorbed on the PEI brush-MNP. Furthermore, the effect of pressure on the enzymatic activity has been studied to reveal activation volumes, which are a sensitive probe of the transition state geometry. The results of this study indicate that the secondary structure of ADH is pressure-stable up to 9â¯kbar. The enzymatic activity of ADH can be analyzed using Michaelis-Menten kinetics free in solution and adsorbed on the PEI brush-MNP. Remarkably, no significant changes of the Michaelis constant and the activation volume are observed upon adsorption. Thus, it can be assumed that the turnover number of ADH is also the same in the free and adsorbed state. However, the maximum enzymatic rate is reduced when ADH is adsorbed, which must be explained by a lower effective enzyme concentration due to steric hindrance of the enzyme inside the PEI brush of the MNP. In this way, the pressure experiments carried out in this study enable a distinction between steric and kinetic effects on the enzymatic rate of adsorbed ADH.
Assuntos
Álcool Desidrogenase/química , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Polieletrólitos/química , Polietilenoimina/química , Proteínas de Saccharomyces cerevisiae/química , Adsorção , Álcool Desidrogenase/isolamento & purificação , Ensaios Enzimáticos , Enzimas Imobilizadas/isolamento & purificação , Etanol/química , Cinética , NAD/química , Pressão , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
With the aim of applying redox-neutral cascade reactions in organic media, fusions of a typeâ II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes. The lyophilized cell-free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane-1,4-diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert-butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ-butyrolactone, resulting in about 27 % analytical yield.
Assuntos
Álcool Desidrogenase/química , Oxigenases de Função Mista/química , Enzimas Multifuncionais/química , Proteínas Recombinantes de Fusão/química , 4-Butirolactona/síntese química , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Animais , Escherichia coli/genética , Liofilização , Cavalos , Cinética , Éteres Metílicos/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/isolamento & purificação , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Rhodococcus/enzimologia , Solventes/químicaRESUMO
The catalytic zincs in complexes of horse liver and yeast alcohol dehydrogenases (ADH) with NAD+ and the substrate analogue, 2,2,2-trifluoroethanol, are ligated to two cysteine residues and one histidine residue from the protein and the oxygen from the alcohol. The zinc facilitates deprotonation of the alcohol and is essential for catalysis. In the yeast apoenzyme, the zinc is coordinated to a nearby glutamic acid, which is displaced by the alcohol in the complex with NAD+. Some homologous medium chain dehydrogenases have a cysteine replaced by aspartic or glutamic acid residues. How an aspartic acid would affect catalysis was studied by replacing Cys-153 in Saccharomyces cerevisiae ADH1 by using site-directed mutagenesis. The C153D enzyme was about as stable as the wild-type enzyme, if EDTA was not included in the buffers. The substitution increased affinity for NAD+ by 3-fold, but did not affect NADH binding. At pH 7.3, the turnover number for ethanol oxidation (V1/Et) decreased by 7-fold and catalytic efficiency decreased 18-fold (V1/EtKb), but turnover for acetaldehyde reduction (V2/Et) was the same as for wild-type enzyme and catalytic efficiency decreased 8-fold (V2/EtKp). Deuterium isotope effects of 3.0 on V1/Et and 3.8 on V1/EtKb for ethanol oxidation suggest that hydride transfer is more rate-limiting for turnover for the C153D enzyme than by wild-type enzyme. The patterns of pH dependence for V1/EtKb for ethanol oxidation were similar for both enzymes in the pH range from 7 to 9. The C153D substitution decreased binding of trifluoroethanol by 5-fold and of pyrazole by 65-fold. Substrate specificities for C153D and wild-type ADHs for primary alcohols have similar patterns. Efficiency for secondary alcohols decreased only about 4-fold, and efficiencies for 1,2-propanediol and acetone were about the same as for wild-type enzyme. The C153D substitution modestly affects catalysis by altering ligand exchange on the zinc or local structure. Structures and mechanisms for acid-base catalysis in related medium chain dehydrogenases with substitutions of the homologous cysteine are reviewed and analyzed.
Assuntos
Álcool Desidrogenase/metabolismo , Ácido Aspártico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Zinco/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Sítios de Ligação , Domínio Catalítico , Cisteína/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Especificidade por Substrato , Zinco/químicaRESUMO
Enzymes originating from hostile environments offer exceptional stability under industrial conditions and are therefore highly in demand. Using single-cell genome data, we identified the alcohol dehydrogenase (ADH) gene, adh/a1a, from the Atlantis II Deep Red Sea brine pool. ADH/A1a is highly active at elevated temperatures and high salt concentrations (optima at 70 °C and 4 m KCl) and withstands organic solvents. The polyextremophilic ADH/A1a exhibits a broad substrate scope including aliphatic and aromatic alcohols and is able to reduce cinnamyl-methyl-ketone and raspberry ketone in the reverse reaction, making it a possible candidate for the production of chiral compounds. Here, we report the affiliation of ADH/A1a to a rare enzyme family of microbial cinnamyl alcohol dehydrogenases and explain unique structural features for halo- and thermoadaptation.
Assuntos
Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Sais/metabolismo , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Oceano Índico , Sais/química , TemperaturaRESUMO
This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10% (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, and maximum activity was detected with benzyl alcohol and 2-phenyl-1-propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1-phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts.
Assuntos
Álcool Desidrogenase/metabolismo , Álcoois/metabolismo , Proteínas Arqueais/metabolismo , Halobacteriaceae/enzimologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Álcool Benzílico/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Genes Arqueais , Haloferax volcanii/genética , Temperatura Alta , Cinética , NAD/metabolismo , NADP/metabolismo , Propanóis/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Enzyme application has gained importance over the past decade in bioprocess, biomedical, and pharmaceutical fields. We found that polyglycerol dendrimers (PGDs), which are biocompatible molecules, can recover alcohol dehydrogenase (ADH) from aqueous solution under elevated temperature. A low concentration of PGD (5 wt.%) is sufficient for the recovery of high enzymatic activity, although a high concentration (25-75 wt.%) of glycerol is generally required to stabilize ADH. The enzymatic activity of ADH in suspension with PGDs is over 60% but it is only 10% in that with glycerol. The results of osmolarity and spin-lattice relaxation time (T1) of water measurements in the presence of PGDs suggest that increased amounts of bound water to PGD molecules trigger aggregation along with the direct interaction with ADH. PGDs therefore represent good potential additives for direct recovery of enzymes from aqueous solutions.
Assuntos
Álcool Desidrogenase/química , Dendrímeros/química , Glicerol/química , Polímeros/química , Temperatura , Água/química , Álcool Desidrogenase/isolamento & purificaçãoRESUMO
NAD(P)-dependent group III alcohol dehydrogenases (ADHs), well known as iron-activated enzymes, generally lose their activities under aerobic conditions due to their oxygen-sensitivities. In this paper, we expressed an extremely thermostable group III ADH from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (PhADH) heterologously in Escherichia coli. When purified from a culture medium containing nickel, the recombinant PhADH (Ni-PhADH) contained 0.85 ± 0.01 g-atoms of nickel per subunit. Ni-PhADH retained high activity under aerobic conditions (9.80 U mg-1), while the enzyme expressed without adding nickel contained 0.46 ± 0.01 g-atoms of iron per subunit and showed little activity (0.27 U mg-1). In the presence of oxygen, the activity of the Fe2+-reconstituted PhADH prepared from the Ni-PhADH was gradually decreased, whereas the Ni2+-reconstituted PhADH maintained enzymatic activity. These results indicated that PhADH with bound nickel ion was stable in oxygen. The activity of the Ni2+-reconstituted PhADH prepared from the expression without adding nickel was significantly lower than that from the Ni-PhADH, suggesting that binding a nickel ion to PhADH in this expression system contributed to protecting against inactivation during the expression and purification processes. Unlike other thermophilic group III ADHs, Ni-PhADH showed high affinity for NAD(H) rather than NADP(H). Furthermore, it showed an unusually high k cat value toward aldehyde reduction. The activity of Ni-PhADH for butanal reduction was increased to 60.7 U mg-1 with increasing the temperature to 95 °C. These findings provide a new strategy to obtain oxygen-sensitive group III ADHs.
Assuntos
Álcool Desidrogenase/genética , Regulação Enzimológica da Expressão Gênica/genética , Oxigênio/metabolismo , Reação em Cadeia da Polimerase , Pyrococcus horikoshii/enzimologia , Álcool Desidrogenase/isolamento & purificação , Álcool Desidrogenase/metabolismo , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
BACKGROUND: The non-conventional yeast Arxula adeninivorans uses 1-butanol as a carbon source and has recently attracted attention as a promising organism for 1-butanol production. Alcohol dehydrogenases (adhp) are important catalysts in 1-butanol metabolism, but only Aadh1p from Arxula has been characterized. This enzyme is involved in ethanol synthesis but has a low impact on 1-butanol degradation. RESULTS: In this study, we identified and characterized a second adhp from A. adeninivorans (Aadh2p). Compared to Saccharomyces cerevisiae ADHs' (ScAdh) protein sequences it originates from the same ancestral node as ScAdh6p, 7p and 4p. It is also localized in the cytoplasm and uses NAD(H) as cofactor. The enzyme has its highest activity with medium chain-length alcohols and maximum activity with 1-butanol with the catalytic efficiency of the purified enzyme being 42 and 43,000 times higher than with ethanol and acetaldehyde, respectively. Arxula adeninivorans strain G1212/YRC102-AADH2, which expresses the AADH2 gene under the control of the strong constitutive TEF1 promoter was constructed. It achieved an ADH activity of up to 8000 U/L and 500 U/g dry cell weight (dcw) which is in contrast to the control strain G1212/YRC102 which had an ADH activity of up to 1400 U/L and 200 U/g dcw. Gene expression analysis showed that AADH2 derepression or induction using non-fermentable carbon-sources such as ethanol, pyruvate, glycerol or 1-butanol did occur. Compared to G1212/YRC102 AADH2 knock-out strain had a slower growth rate and lower 1-butanol consumption if 1-butanol was used as sole carbon source and AADH2-transformants did not grow at all in the same conditions. However, addition of the branched-chain amino acids leucine, isoleucine and valine allowed the transformants to use 1-butanol as carbon source. The addition of these amino acids to the control strain and Δaadh2 mutant cultures had the effect of accelerating 1-butanol consumption. CONCLUSIONS: Our results confirm that Aadh2p plays a major role in A. adeninivorans 1-butanol metabolism. It is upregulated by up to 60-fold when the cells grow on 1-butanol, whereas only minor changes were found in the relative expression level for Aadh1p. Thus the constitutive overexpression of the AADH2 gene could be useful in the production of 1-butanol by A. adeninivorans, although it is likely that other ADHs will have to be knocked-out to prevent 1-butanol oxidation.
Assuntos
1-Butanol/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Redes e Vias Metabólicas/genética , Leveduras/enzimologia , Álcool Desidrogenase/isolamento & purificação , Carbono/metabolismo , Etanol/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , NAD/metabolismo , Leveduras/genética , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismoRESUMO
Epoxy functionalized magnetic Fe3O4@SiO2 nanoparticles were successfully prepared and characterized by Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The prepared nanoparticles were used for immobilization of alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae by covalent attachment. The optimal immobilization conditions were obtained as follows: enzyme/support 4.49mg/g, pH 8.0, buffer concentration 0.05M, time 12h and temperature 30°C. Under these conditions, a high immobilization yield and efficiency of above 92% were obtained after the optimization. Broad pH tolerance and high thermostability were achieved by the immobilization. The immobilized ADH retained about 84% initial activity after five cycles. Kinetic parameters Vmax and Km of free and immobilized ADH were determined as 56.72µM/min, 44.27µM/min and 11.54mM, 31.32mM, respectively. (R)-mandelic acid synthesis with the immobilized ADH was carried out, and the yield of (R)-mandelic acid was as high as 64%. These results indicate that the ADH immobilized onto epoxy-functionalized nanoparticles is an efficient and simple way for preparation of stable ADH, and the immobilized ADH has potential applications in the production of (R)-mandelic acid.
Assuntos
Álcool Desidrogenase/química , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Glioxilatos/química , Nanopartículas de Magnetita/química , Ácidos Mandélicos/química , Ácidos Mandélicos/síntese química , Álcool Desidrogenase/isolamento & purificação , Biocatálise , Soluções Tampão , Estabilidade Enzimática , Enzimas Imobilizadas/isolamento & purificação , Resinas Epóxi/química , Óxido Ferroso-Férrico/química , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Nanopartículas de Magnetita/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Dióxido de Silício/química , Estereoisomerismo , TemperaturaRESUMO
Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHE(S77)). Interestingly, the ADHE(S77) was unable to be solubilized from membrane with detergents either 1% Triton X-100 or 1% Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH(4))(2)SO(4) without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHES77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHES77 may play a pivotal role in modulating intracellular acetaldehyde concentration.
Assuntos
Álcool Desidrogenase/isolamento & purificação , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/isolamento & purificação , Aldeído Oxirredutases/metabolismo , Citrobacter/enzimologia , Enzimas Multifuncionais/isolamento & purificação , Enzimas Multifuncionais/metabolismo , Acetaldeído/metabolismo , Acetilcoenzima A/metabolismo , Acilação , Álcool Desidrogenase/química , Álcoois/metabolismo , Aldeído Oxirredutases/química , Anaerobiose , Betaína/análogos & derivados , Biocatálise , Coenzima A/metabolismo , Detergentes , Cinética , Peso Molecular , Enzimas Multifuncionais/química , Octoxinol , Subunidades Proteicas , SolubilidadeRESUMO
Isatin-binding activity of mice liver proteins has been investigated in the samples from the control and flight groups by using the methods of biosensor and proteomic analysis. It was found the higher isatin-binding activity in mice of flight group. The content of a number of individual isatin-binding proteins in the samples of the flight groups differ slightly from the ground control. However, in samples from animals which have weekly post-flight adaptation, the level of certain proteins was significantly increased. The latter allows us to assume that the main events in the proteome of mice (at least in subproteome of isatin-binding proteins), occurs in early post-flight period.
Assuntos
Adaptação Fisiológica , Proteínas de Transporte/metabolismo , Isatina/química , Voo Espacial , Actinas/isolamento & purificação , Actinas/metabolismo , Álcool Desidrogenase/isolamento & purificação , Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/isolamento & purificação , Aldeído Desidrogenase/metabolismo , Animais , Proteínas de Transporte/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Fígado/química , Camundongos , Camundongos Endogâmicos C57BL , Peroxirredoxinas/isolamento & purificação , Peroxirredoxinas/metabolismo , Ligação Proteica , Proteoma/isolamento & purificação , Proteoma/metabolismo , Fatores de Tempo , Ausência de PesoRESUMO
In this paper, we explore the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes for biofuel cell applications. The bioanode architectures were designed with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles, along with different immobilization techniques. Three different polymeric matrices were tested (tetrabutyl ammonium bromide (TBAB)-modified Nafion; octyl-modified linear polyethyleneimine (C8-LPEI); and cellulose) in the DET studies. The modified Nafion membrane provided the best electrical communication between enzymes and the electrode surface, with catalytic currents as high as 16.8 ± 2.1 µA cm(-2). Then, a series of ferrocene redox polymers were evaluated for MET. The redox polymer 1,1'-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI) provided the best electrochemical response. Using this polymer, the electrochemical assays conducted in the presence of MWCNTs and MWCNTs-Au indicated a Jmax of 781 ± 59 µA cm(-2) and 925 ± 68 µA cm(-2), respectively. Overall, from the results obtained here, DET using the PQQ-dependent ADH and AldDH still lacks high current density, while the bioanodes that operate via MET employing ferrocene-modified LPEI redox polymers show efficient energy conversion capability in ethanol/air biofuel cells.
Assuntos
Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Fontes de Energia Bioelétrica , Enzimas Imobilizadas/metabolismo , Gluconobacter/enzimologia , Cofator PQQ/metabolismo , Álcool Desidrogenase/química , Álcool Desidrogenase/isolamento & purificação , Aldeído Desidrogenase/química , Aldeído Desidrogenase/isolamento & purificação , Fontes de Energia Bioelétrica/microbiologia , Eletrodos , Transporte de Elétrons , Enzimas Imobilizadas/química , Etanol/metabolismo , Compostos Ferrosos/química , Polímeros de Fluorcarboneto/química , Gluconobacter/metabolismo , Modelos Moleculares , Nanotubos de Carbono/química , Oxirredução , Polietilenoimina/químicaRESUMO
Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.
Assuntos
Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Gluconacetobacter/enzimologia , Acetatos/análise , Álcool Desidrogenase/química , Álcool Desidrogenase/isolamento & purificação , Aldeídos/análise , Sequência de Aminoácidos , Biocatálise , Radioisótopos de Carbono/química , Cromatografia Gasosa-Espectrometria de Massas , Marcação por Isótopo , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oxirredução , Desnaturação Proteica , TemperaturaRESUMO
Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD(+)-dependent ADH activity was diminished by 20-75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.
Assuntos
Álcool Desidrogenase/metabolismo , Lepidópteros/microbiologia , Metarhizium/enzimologia , Metarhizium/crescimento & desenvolvimento , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Animais , Clonagem Molecular , Perfilação da Expressão Gênica , Inativação Gênica , Ponto Isoelétrico , Larva/microbiologia , Larva/fisiologia , Lepidópteros/fisiologia , Metarhizium/genética , Peso Molecular , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de Aminoácidos , Análise de Sobrevida , VirulênciaRESUMO
Studies on the biosynthesis of oil compounds in Perilla will help in understanding regulatory systems of secondary metabolites and in elucidating reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, an aldo-keto reductase (AKR) and a geraniol dehydrogenase (GeDH), which are thought to participate in the biosynthesis of perilla essential oil components, such as citral and perillaldehyde, were isolated from three pure lines of perilla. These enzymes shared high amino acid sequence identity within the genus Perilla, and were expressed regardless of oil type. The overall reaction from geranyl diphosphate to citral was performed in vitro using geraniol synthase and GeDH to form a large proportion of citral and relatively little geraniol as reaction products. The biosynthetic pathway from geranyl diphosphate to citral, the main compound of citral-type perilla essential oil, was established in this study.
Assuntos
Álcool Desidrogenase/isolamento & purificação , Aldeído Redutase/isolamento & purificação , Óleos Voláteis/metabolismo , Perilla/enzimologia , Ácido alfa-Linolênico/metabolismo , Monoterpenos Acíclicos , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases , Sequência de Aminoácidos , Vias Biossintéticas , Clonagem Molecular , Difosfatos , Diterpenos , Expressão Gênica , Biblioteca Gênica , Cinética , Dados de Sequência Molecular , Monoterpenos/química , Monoterpenos/metabolismo , Óleos Voláteis/química , Perilla/química , Perilla/genética , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Óleos de Plantas/química , Óleos de Plantas/metabolismo , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Análise de Sequência de DNA , Terpenos/química , Terpenos/metabolismo , Ácido alfa-Linolênico/químicaRESUMO
In this work, poly(HEMA-co-GMA) cryogel was synthesized by using cryopolymerization technique and this cryogel was functionalized with Cibacron Blue F3GA dye. Prepared dye attached cryogel was used for the reversible immobilization of alcohol dehydrogenase from its aqueous solution. Cibacron Blue F3GA attached poly(HEMA-co-GMA) cryogel was characterized by environmental scanning electron microscopy (ESEM), energy dispersive X-ray (EDX) analysis and swelling studies. Surface morphology of the cryogel was considerably porous and the pore size was found to be around 30-50 µm. Effects of medium pH, initial alcohol dehydrogenase concentration, medium temperature and ionic strength on the alcohol dehydrogenase adsorption were also investigated and maximum alcohol dehydrogenase adsorption onto the dye-attached cryogel was found to be 8.55 mg/g cryogel. Adsorbed alcohol dehydrogenase was desorbed from the cryogel by using 10 mL of NaCl solution (1.0M; in pH 4.0 acetate buffer). Synthesized cryogel was able to reuse for 25 sequential cycles and it was found that, there was no negligible decrease in the adsorption capacity of the dye-attached cryogel.
Assuntos
Álcool Desidrogenase/química , Cromatografia de Afinidade/métodos , Corantes/química , Criogéis/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Triazinas/química , Adsorção , Álcool Desidrogenase/isolamento & purificação , Cromatografia de Afinidade/instrumentação , Criogéis/síntese química , Concentração de Íons de Hidrogênio , Porosidade , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
The noncanonical alcohol dehydrogenase AlkJ is encoded on the alkane-metabolizing alk operon of the mesophilic bacterium Pseudomonas putida GPo1. To gain insight into the enzymology of AlkJ, we have produced the recombinant protein in Escherichia coli and purified it to homogeneity using His6 tag affinity and size exclusion chromatography (SEC). Despite synthesis in the cytoplasm, AlkJ was associated with the bacterial cell membrane, and solubilization with n-dodecyl-ß-D-maltoside was necessary to liberate the enzyme. SEC and spectrophotometric analysis revealed a dimeric quaternary structure with stoichiometrically bound reduced flavin adenine dinucleotide (FADH2). The holoenzyme showed thermal denaturation at moderate temperatures around 35°C, according to both activity assay and temperature-dependent circular dichroism spectroscopy. The tightly bound coenzyme was released only upon denaturation with SDS or treatment with urea-KBr and, after air oxidation, exhibited the characteristic absorption spectrum of FAD. The enzymatic activity of purified AlkJ for 1-butanol, 1-hexanol, and 1-octanol as well as the n-alkanol derivative ω-hydroxy lauric acid methyl ester (HLAMe) was quantified in the presence of the artificial electron acceptors phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCPIP), indicating broad substrate specificity with the lowest activity on the shortest alcohol, 1-butanol. Furthermore, AlkJ was able to accept as cosubstrates/oxidants the ubiquinone derivatives Q0 and Q1, also in conjunction with cytochrome c, which suggests coupling to the bacterial respiratory chain of this membrane-associated enzyme in its physiological environment. Using gas chromatographic analysis, we demonstrated specific biocatalytic conversion by AlkJ of the substrate HLAMe to the industrially relevant aldehyde, thus enabling the biotechnological production of 12-amino lauric acid methyl ester via subsequent enzymatic transamination.
Assuntos
Álcool Desidrogenase/metabolismo , Aldeídos/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Pseudomonas putida/enzimologia , Álcool Desidrogenase/química , Álcool Desidrogenase/isolamento & purificação , Cromatografia de Afinidade , Coenzimas/metabolismo , Escherichia coli/genética , Desnaturação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The amplification of useful genes from metagenomes offers great biotechnological potential. We employed this approach to isolate alcohol dehydrogenase (adh) genes from Pseudomonas to aid in the synthesis of optically pure alcohols from various ketones. A PCR primer combination synthesized by reference to the adh sequences of known Pseudomonas genes was used to amplify full-length adh genes directly from 17 samples of DNA extracted from soil. Three such adh preparations were used to construct Escherichia coli plasmid libraries. Of the approximately 2800 colonies obtained, 240 putative adh-positive clones were identified by colony-PCR. Next, 23 functional adh genes named using the descriptors HBadh and HPadh were analyzed. The adh genes obtained via this metagenomic approach varied in their DNA and amino acid sequences. Expression of the gene products in E. coli indicated varying substrate specificity. Two representative genes, HBadh-1 and HPadh-24, expressed in E. coli and Pseudomonas putida, respectively, were purified and characterized in detail. The enzyme products of these genes were confirmed to be useful for producing anti-Prelog chiral alcohols.