RESUMO
The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias , Mananas/metabolismo , Manosidases/química , beta-Manosidase , Sequência de Aminoácidos , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Galactose/análogos & derivados , Manosidases/genética , Manosidases/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosAssuntos
Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/enzimologia , Manosidases/isolamento & purificação , Sequência de Aminoácidos , Configuração de Carboidratos , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Galactose/análogos & derivados , Mananas/química , Manosidases/química , Manosidases/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , beta-ManosidaseAssuntos
Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/isolamento & purificação , alfa-Galactosidase/isolamento & purificação , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismoAssuntos
Celulase/isolamento & purificação , Pyrococcus furiosus/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Celulase/biossíntese , Celulase/química , Celulase/metabolismo , Primers do DNA , Hidrólise , Dados de Sequência Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoAssuntos
Hidrolases de Éster Carboxílico/isolamento & purificação , Sulfolobus/enzimologia , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Carboxilesterase , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The P. furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed beta-1,4 but not beta-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only beta-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage beta-glucans such as barley beta-glucan and lichenan. Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (-I, -II, and -III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100 degreesC and 6.0, respectively; a half-life of 40 h at 95 degreesC; and a denaturing temperature of 112 degreesC as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.
Assuntos
Celulase/metabolismo , Pyrococcus furiosus/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Celulase/genética , Celulose/química , Celulose/metabolismo , Mapeamento Cromossômico , Primers do DNA/genética , DNA Arqueal/genética , Evolução Molecular , Glucanos/química , Glucanos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Dados de Sequência Molecular , Pyrococcus furiosus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
An open reading frame coding for a putative protein-serine/threonine phosphatase was identified in the hyperthermophilic archaeon Pyrodictium abyssi TAG11 and named Py-PP1. Py-PP1 was expressed in Escherichia coli, purified from inclusion bodies, and biochemically characterized. The phosphatase gene is part of an operon which may provide, for the first time, insight into a physiological role for archaeal protein phosphatases in vivo.
Assuntos
Proteínas Arqueais/genética , Desulfurococcaceae/genética , Fosfoproteínas Fosfatases/genética , Sequência de Aminoácidos , Clonagem Molecular , Desulfurococcaceae/enzimologia , Escherichia coli , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.
Assuntos
Crenarchaeota/genética , DNA Polimerase Dirigida por DNA/genética , Genes Arqueais , Sequência de Aminoácidos , Proteínas Arqueais/biossíntese , Proteínas Arqueais/classificação , Proteínas Arqueais/genética , Temperatura Baixa , Crenarchaeota/enzimologia , DNA Polimerase Dirigida por DNA/biossíntese , DNA Polimerase Dirigida por DNA/classificação , Estabilidade Enzimática , Evolução Molecular , Exodesoxirribonuclease V , Exodesoxirribonucleases/análise , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.
Assuntos
Archaea/enzimologia , DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Clonagem Molecular , DNA Polimerase Dirigida por DNA/isolamento & purificação , Óperon Lac/genética , Dados de Sequência Molecular , Mutação/genética , TemperaturaRESUMO
Aldose reductase, purified to homogeneity from bovine kidney, is converted in a temperature-dependent process from a low-Km/low-Vmax form to a high-Km/high-Vmax form of the enzyme. Activation, which results in significant changes in the protein secondary structure, as detected by fluorescence spectroscopy, circular dichroism, and thiol modification with 5,5'-dithiobis(2-nitrobenzoic acid), has no effect on the apparent Mr, pI, or homogeneity of the enzyme, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose isoelectric focusing. Vmax, which varied less than 3-fold for a series of aldehyde substrates with either activation state of the enzyme, increased an average of (17 +/- 4)-fold upon activation of the enzyme. V/Kaldehyde increased or decreased up to 4-fold, depending on the substrate. Activation desensitized the enzyme to inhibition by aldose reductase inhibitors, with the apparent Ki value increasing from 2-fold for Epalrestat [ONO-2235, (E)-3-(carboxymethyl)-(E)-5-[2-methyl-3-phenylpropenylidene]-rhoda nine] to 200-fold for AL-1576 (spiro [2,7-difluorofluorene-9,4'-imidazolidine]-2',5'-dione). Biphasic double-reciprocal plots for the aldehyde substrates and biphasic Dixon plots for inhibition by AL-1576 and Statil [ICI-128,436; 3-[(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-l-ylacetic acid], observed during the course of activation, are quantitatively accounted for by the individual contributions of the two enzyme forms. On the basis of an analysis of the kinetic data, a mechanism is proposed in which isomerization of the free enzyme limits the rate of the forward reaction for the unactivated enzyme and is the primary step affected by activation.
Assuntos
Aldeído Redutase/metabolismo , Rim/enzimologia , Desidrogenase do Álcool de Açúcar/metabolismo , Aldeído Redutase/isolamento & purificação , Animais , Bovinos , Cromatografia , Cromatografia em Gel , Cromatografia por Troca Iônica , Dicroísmo Circular , Durapatita , Ativação Enzimática , Hidroxiapatitas , Cinética , Matemática , Modelos Teóricos , Peso Molecular , Conformação Proteica , Especificidade por SubstratoRESUMO
Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific for human ALR2 by Western blot analysis of human muscle homogenates. The human enzyme was detected, by reaction with the antiserum (alpha-BKALR2), in homogenates of adrenal gland, muscle, lens, brain, testes, kidney, and placenta, but not in erythrocytes or leukocytes. The amount of enzyme in each tissue was determined by densitometric analysis of autoradiographs of Western blots probed with alpha-BKALR2 and [125I]protein A. Standard curves of radiographic intensity versus amount of purified human muscle ALR2 were linear in the 20 to 200-ng range; a similar sensitivity was seen in tissue homogenates containing up to 675 micrograms total protein. The results presented here for the ALR2 level in human tissues (adrenal greater than muscle greater than lens approximately brain approximately testes greater than kidney approximately placenta) are in agreement with literature values for those tissues from which the enzyme has previously been purified. A notable exception was the absence of detectable ALR2 in human erythrocytes. A quantitative comparison of immunoradiographic response showed that bovine kidney ALR2 was about sevenfold more reactive with a alpha-BKALR2 compared to the human muscle enzyme.
Assuntos
Aldeído Redutase/análise , Desidrogenase do Álcool de Açúcar/análise , Aldeído Redutase/imunologia , Aldeído Redutase/metabolismo , Anticorpos , Autorradiografia/métodos , Western Blotting/métodos , Feminino , Humanos , Radioisótopos do Iodo , Cinética , Masculino , Músculos/enzimologia , Especificidade de Órgãos , GravidezRESUMO
Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific by Western blot analysis of kidney homogenates. In addition, the antiserum (alpha-BKALR2) reacts with a single electrophoretic species in homogenates from rabbit, porcine, and human kidney. ALR2 has been detected in homogenates of bovine kidney, heart, brain and lens, and estimation of the enzyme level in these tissues was accomplished by densitometric analysis of Western blots. Standard curves using highly purified bovine kidney ALR2 were linear in the range of 5-100 ng; a similar sensitivity was seen in tissue homogenates. The results presented here for the ALR2 level in bovine tissues (kidney greater than heart greater than brain greater than lens) are in agreement with literature values for those tissues from which the enzyme has previously been purified. The interspecies similarity in electrophoretic mobility and the retention of antibody reactivity suggest extensive phylogenetic epitope conservation in mammalian aldose reductase.
Assuntos
Aldeído Redutase/imunologia , Epitopos/genética , Desidrogenase do Álcool de Açúcar/imunologia , Aldeído Redutase/análise , Aldeído Redutase/genética , Animais , Autorradiografia , Bovinos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoquímica , Rim/enzimologia , Especificidade de Órgãos , Coelhos , Especificidade da Espécie , SuínosRESUMO
5,10-Methenyltetrahydrofolate synthetase (EC 6.3.3.2), which catalyzes the ATP- and Mg2+ -dependent isomerization of 5-formyl- to 5,10-methenyltetrahydrofolate, has been purified 10,000-fold from Lactobacillus casei using sequential affinity chromatography on immobilized 5-formyltetrahydrofolate and ATP. The enzyme is homogeneous when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is monomeric with a molecular mass of 23,000 Da, and contains a high proportion of hydrophobic amino acids and a single cysteine residue. At 30 degrees C, the turnover number is 88 min-1, and the Km values at pH 6 for 5-formyltetrahydrofolate and Mg-ATP are 0.6 and 1.0 microM, respectively. The enzyme is specific for (6S)-5-formyltetrahydrofolate, but ATP can be replaced by other nucleoside 5'-triphosphates with varying efficiency. The purified enzyme is markedly stabilized by the non-ionic detergent, Tween 20.