RESUMO
Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose which have large practical application in the process of refining biomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t1/2 =6-8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420â nm is formed when reduced LPMO-CuI reacts with sub-equimolar amounts of H2 O2 . UV/Vis absorption spectroscopy, electron paramagnetic resonance, resonance Raman and stopped-flow spectroscopy suggest that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). Additionally, activity assays in the presence of sub-equimolar amounts of H2 O2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly, this suggests that the long-lived copper-dependent intermediate could be part of the catalytic mechanism for LPMOs. The observed intermediate offers a new perspective into the oxidative reaction mechanism of TaLPMO9A and hence for the biomass oxidation and the reactivity of copper in biological systems.
Assuntos
Cobre/química , Oxigenases de Função Mista/metabolismo , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/química , Cinética , Oxigenases de Função Mista/química , Oxirredução , Thermoascus/enzimologiaRESUMO
We report here the differences in sperm functional attributes and sperm-oviduct binding index in bulls with different field fertility ratings. Cryopreserved spermatozoa from Murrah buffalo bulls (n=9) with different fertility ratings were evaluated for membrane integrity, capacitation status, acrosome intactness and protein tyrosine phosphorylation status. Frozen--thawed spermatozoa were incubated with oviduct explants for 1h under 5% CO2, 38.5°C with 95% relative humidity and the number of spermatozoa bound to the unit area of oviduct explants (binding index; BI) was assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescent staining. The proportion of membrane-intact and acrosome-intact spermatozoa was significantly (P<0.05) higher and the proportion of capacitated spermatozoa was significantly (P<0.05) lower in high-fertile bulls compared with medium- and low-fertile bulls. The relationship between BI and bull fertility was significant and positive (r=0.69; P=0.04). BI was negatively and significantly (r=-0.83; P=0.01) related to membrane-compromised spermatozoa. It was concluded that the sperm-oviduct explant binding index was positively related to (1) the proportion of membrane-intact spermatozoa in a given semen sample and (2) invivo fertility of the buffalo bull, indicating the possibility of developing a fertility prediction tool using a sperm-oviduct explant binding model, once validated on a greater number of bulls.
Assuntos
Fertilidade/fisiologia , Oviductos/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Acrossomo/fisiologia , Animais , Búfalos , Feminino , Masculino , Fosforilação , Análise do Sêmen , Capacitação Espermática/fisiologiaRESUMO
Natural photosynthesis is an effective route for the clean and sustainable conversion of CO2 into high-energy chemicals. Inspired by the natural process, a tandem photoelectrochemical (PEC) cell with an integrated enzyme-cascade (TPIEC) system was designed, which transfers photogenerated electrons to a multienzyme cascade for the biocatalyzed reduction of CO2 to methanol. A hematite photoanode and a bismuth ferrite photocathode were applied to fabricate the iron oxide based tandem PEC cell for visible-light-assisted regeneration of the nicotinamide cofactor (NADH). The cell utilized water as an electron donor and spontaneously regenerated NADH. To complete the TPIEC system, a superior three-dehydrogenase cascade system was employed in the cathodic part of the PEC cell. Under applied bias, the TPIEC system achieved a high methanol conversion output of 220â µm h-1 , 1280â µmol g-1 h-1 using readily available solar energy and water.
Assuntos
Dióxido de Carbono/metabolismo , Técnicas Eletroquímicas , Metanol/metabolismo , Oxirredutases/metabolismo , Dióxido de Carbono/química , Metanol/química , Modelos Moleculares , Oxirredução , Oxirredutases/química , Processos FotoquímicosRESUMO
We reported previously that an N-acylthiourea derivative (TM-2-51) serves as a potent and isozyme-selective activator for human histone deacetylase 8 (HDAC8). To probe the molecular mechanism of the enzyme activation, we performed a detailed account of the steady-state kinetics, thermodynamics, molecular modeling, and cell biology studies. The steady-state kinetic data revealed that TM-2-51 binds to HDAC8 at two sites in a positive cooperative manner. Isothermal titration calorimetric and molecular modeling data conformed to the two-site binding model of the enzyme-activator complex. We evaluated the efficacy of TM-2-51 on SH-SY5Y and BE(2)-C neuroblastoma cells, wherein the HDAC8 expression has been correlated with cellular malignancy. Whereas TM-2-51 selectively induced cell growth inhibition and apoptosis in SH-SY5Y cells, it showed no such effects in BE(2)-C cells, and this discriminatory feature appears to be encoded in the p53 genotype of the above cells. Our mechanistic and cellular studies on HDAC8 activation have the potential to provide insight into the development of novel anticancer drugs.
Assuntos
Cristalografia por Raios X , Ativação Enzimática/genética , Histona Desacetilases/biossíntese , Neuroblastoma/enzimologia , Proteínas Repressoras/biossíntese , Apoptose/efeitos dos fármacos , Benzamidas/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/administração & dosagem , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Cinética , Modelos Moleculares , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Feniltioureia/administração & dosagem , Feniltioureia/análogos & derivados , Proteínas Repressoras/química , Proteínas Repressoras/genética , Termodinâmica , Proteína Supressora de Tumor p53/biossínteseRESUMO
The study determined whether feeding during lactation affects the suppressive effect of maternal dietary lipotropes (i.e., methionine, choline, folate, and vitamin B12) on mammary carcinogenesis. Pregnant Sprague-Dawley rats were randomly allocated to the control diet during pregnancy and lactation (CC), lipotropes-fortified diet during pregnancy (LC), lipotropes-fortified diet during pregnancy plus lactation (LL), or lipotropes-fortified diet during lactation (CL). Randomly selected female offspring from each group were injected intraperitoneally with 50 mg/kg body weight of N-nitroso-N-methylurea at 50 days of age to induce mammary tumors. The LC and LL diets significantly increased tumor latency and survival (P < 0.05). Tumor volumes were significantly suppressed in LC and LL offspring as compared with the CC and CL pups (3759.1 ± 563.0 and 3603.7 ± 526.1 vs. 7465.0 ± 941.1 and 5219.3 ± 759.8 mm(3), respectively; P < 0.05). Both LC and LL lowered tumor multiplicity as compared with CC and CL (P < 0.05). The LC and LL diets repressed transcription of histone deacetylase (HDAC) 1 as well as total HDAC enzyme activity as compared with CC and CL diets (P < 0.05). Data suggest that the tumor suppressive effect of maternal dietary lipotropes is primarily in utero and may be linked to regulation of proteins involved in chromatin remodeling.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dieta , Lactação , Neoplasias Mamárias Animais/prevenção & controle , Troca Materno-Fetal , Animais , Animais Recém-Nascidos , Colina/administração & dosagem , Feminino , Ácido Fólico/administração & dosagem , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Neoplasias Mamárias Animais/induzido quimicamente , Neoplasias Mamárias Animais/enzimologia , Metionina/administração & dosagem , Metilnitrosoureia/administração & dosagem , Gravidez , Ratos , Ratos Sprague-Dawley , Vitamina B 12/administração & dosagemRESUMO
Among the different histone deacetylase (HDAC) isozymes, HDAC8 is the most highly malleable enzyme, and it exhibits the potential to accommodate structurally diverse ligands (albeit with moderate binding affinities) in its active site pocket. To probe the molecular basis of this feature, we performed detailed thermodynamic studies of the binding of structurally similar ligands, which differed with respect to the "cap", "linker", and "metal-binding" regions of the suberoylanilide hydroxamic acid (SAHA) pharmacophore, to HDAC8. The experimental data revealed that although the enthalpic (ΔH°) and entropic (ΔS°) changes for the binding of individual SAHA analogues to HDAC8 were substantially different, their binding free energies (ΔG°) were markedly similar, conforming to a strong enthalpy-entropy compensation effect. This effect was further observed in the temperature-dependent thermodynamics of binding of all SAHA analogues to the enzyme. Notably, in contrast to other metalloenzymes, our isothermal titration calorimetry experiments (performed in different buffers of varying ionization enthalpies) suggest that depending on the ligand, its zinc-binding group may or may not be deprotonated upon the binding to HDAC8. Furthermore, the heat capacity changes (ΔCp°) associated with the ligand binding to HDAC8 markedly differed from one SAHA analogue to the other, and such features could primarily be rationalized in light of the dynamic flexibility in the enzyme structure in conjunction with the reorganization of the active site resident water molecules. Arguments are presented that although the binding thermodynamic features described above would facilitate identification of weak to moderately tight-binding HDAC8 inhibitors (by a high-throughput and/or virtual screening of libraries of small molecules), they would pose major challenges for the structure-based rational design of highly potent and isozyme-selective inhibitors of human HDAC8.
Assuntos
Histona Desacetilases/química , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Calorimetria , Domínio Catalítico , Desenho de Fármacos , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Ligantes , Modelos Moleculares , Estrutura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Eletricidade Estática , Termodinâmica , VorinostatRESUMO
Significant differences in biochemical parameters between normal and tumor tissues offer an opportunity to chemically design drug carriers which respond to these changes and deliver the drugs at the desired site. For example, overexpression of the matrix metalloproteinase-9 (MMP-9) enzyme in the extracellular matrix of tumor tissues can act as a trigger to chemically modulate the drug delivery from the carriers. In this study, we have synthesized an MMP-9-cleavable, collagen mimetic lipopeptide which forms nanosized vesicles with the POPC, POPE-SS-PEG, and cholesteryl-hemisuccinate lipids. The lipopeptide retains the triple-helical conformation when incorporated into these nanovesicles. The PEG groups shield the substrate lipopeptides from hydrolysis by MMP-9. However, in the presence of elevated glutathione levels, the PEG groups are reductively removed, exposing the lipopeptides to MMP-9. The resultant peptide-bond cleavage disturbs the vesicles' lipid bilayer, leading to the release of encapsulated contents. These PEGylated nanovesicles are capable of encapsulating the anticancer drug gemcitabine with 50% efficiency. They were stable in physiological conditions and in human serum. Effective drug release was demonstrated using the pancreatic ductal carcinoma cells (PANC-1 and MIAPaCa-2) in two-dimensional and three-dimensional "tumor-like" spheroid cultures. A reduction in tumor growth was observed after intravenous administration of the gemcitabine-encapsulated nanovesicles in the xenograft model of athymic, female nude mice.
Assuntos
Antineoplásicos/química , Metaloproteinase 9 da Matriz/metabolismo , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias Pancreáticas/tratamento farmacológico , Polietilenoglicóis/química , Vesículas Transportadoras/química , Animais , Antineoplásicos/administração & dosagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Matriz Extracelular/metabolismo , Feminino , Glutationa/metabolismo , Humanos , Hidrólise , Bicamadas Lipídicas/metabolismo , Lipopeptídeos/administração & dosagem , Lipopeptídeos/química , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Fosfatidilcolinas/administração & dosagem , Fosfatidilcolinas/química , Polietilenoglicóis/administração & dosagem , GencitabinaRESUMO
l-Arabinitol 4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of l-arabinitol oxidation by NAD(+), catalyzed by HjLAD, was studied within the pH range of 7.0-9.5 at 25°C. The turnover number (kcat) and the catalytic efficiency (kcat/Km) were 4200min(-1) and 290mM(-1)min(-1), respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare l-sugar l-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%.
Assuntos
Hypocrea/enzimologia , Desidrogenase do Álcool de Açúcar/metabolismo , Xilulose/biossíntese , Biocatálise , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Desidrogenase do Álcool de Açúcar/isolamento & purificação , Xilulose/químicaRESUMO
L-Xylulose is a potential starting material for therapeutics. However, its translation into clinical practice has been hampered by its inherently low bioavailability. In addition, the high cost associated with the production of L-xylulose is a major factor hindering its rapid deployment beyond the laboratory. In the current study, L-arabinitol 4-dehydrogenase from Hypocrea jecorina (HjLAD), which catalyzes the conversion of L-arabinitol into L-xylulose, was immobilized onto various carriers, and the immobilized enzymes were characterized. HjLAD covalently immobilized onto silicon oxide nanoparticles showed the highest immobilization efficiency (94.7 %). This report presents a comparative characterization of free and immobilized HjLAD, including its thermostability and kinetic parameters. The thermostability of HjLAD immobilized on silicon oxide nanoparticles was more than 14.2-fold higher than free HjLAD; the t1/2 of HjLAD at 25 °C was enhanced from 190 min (free) to 45 h (immobilized). In addition, the immobilized HjLAD retained 94 % of its initial activity after 10 cycles. When the immobilized HjLAD was used to catalyze the biotransformation of L-arabinitol to L-xylulose, 66 % conversion and a productivity of 7.9 g · h(-1) · L(-1) were achieved. The enhanced thermostability and reusability of HjLAD suggest that immobilization of HjLAD onto silicon oxide nanoparticles has the potential for use in the industrial production of rare sugars.
Assuntos
Enzimas Imobilizadas/metabolismo , Nanopartículas/química , Dióxido de Silício/química , Desidrogenase do Álcool de Açúcar/metabolismo , Xilulose/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Cinética , Desidrogenase do Álcool de Açúcar/química , Temperatura , Trichoderma/enzimologiaRESUMO
Nicotinamide adenine dinucleotide (NADH) oxidase from Streptococcus pyogenes (SpNox) is a flavoprotein harboring one molecule of noncovalently bound flavin adenine dinucleotide. It catalyzes the oxidation of NADH by reducing molecular O2 to H2O directly through a four-electron reduction. In this study, we selected the lysine residues on the surface of SpNox and mutated them into arginine residues to study the effect on the enzyme activity. A single-point mutation (K184R) at the surface of SpNox enhanced NADH oxidase activity by approximately 50 % and improved thermostability with 46.6 % longer half life at 30 °C. Further insights into the function of residue K184 were obtained by substituting it with other nonpolar, polar, positively charged, and negatively charged residues. To elucidate the role of this residue, computer-assisted molecular modeling and substrate docking were performed. The results demonstrate that even a single mutation at the surface of the enzyme induces changes in the interaction at the active site and affects the activity and stability. Additionally, the data also suggest that the K184R mutant can be used as an effective biocatalyst for NAD(+) regeneration in L-rare sugar production.
Assuntos
Lisina/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Streptococcus pyogenes/enzimologia , Substituição de Aminoácidos , Estabilidade Enzimática , Flavoproteínas/química , Flavoproteínas/genética , Flavoproteínas/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , NAD/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , Oxirredução , Oxigênio/metabolismo , Mutação Puntual , Streptococcus pyogenes/genética , Temperatura , Água/metabolismoRESUMO
A highly efficient ß-1,4-mannanase-secreting strain, Pholiota adiposa SKU0714, was isolated and identified on the basis of its morphological features and sequence analysis of internal transcribed spacer rDNA. P. adiposa ß-1,4-mannanase was purified to homogeneity from P. adiposa culture supernatants by one-step chromatography on a Sephacryl gel filtration column. P. adiposa ß-1,4-mannanase showed the highest activity toward locust bean gum (V max = 1,990 U/mg protein, K m = 0.12 mg/mL) ever reported. Its internal amino acid sequence showed homology with hydrolases from the glycoside hydrolase family 5 (GH5), indicating that the enzyme is a member of the GH5 family. The saccharification of commercial mannanase and P. adiposa ß-1,4-mannanase-pretreated rice straw by Celluclast 1.5L (Novozymes) was compared. In comparison with the commercial Novo Mannaway(®) (113 mg/g-substrate), P. adiposa ß-1,4-mannanase-pretreated rice straw released more reducing sugars (141 mg/g-substrate). These properties make P. adiposa ß-1,4-mannanase a good candidate as a new commercial ß-1,4-mannanase to improve biomass pretreatment.
Assuntos
Biomassa , Pholiota/enzimologia , beta-Manosidase/metabolismo , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Of the different hydroxamate-based histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic acid (SAHA) has been approved by the Food and Drug Administration for the treatment of T-cell lymphoma. Interestingly, a structurally similar inhibitor, trichostatin A (TSA), which has a higher in vitro inhibitory potency against HDAC8, reportedly shows poor efficacy in clinical settings. To gain molecular insight into this discriminatory feature, we performed transient kinetic and isothermal titration calorimetric studies for the interaction of SAHA and TSA with the recombinant form of human HDAC8. The transient kinetic data revealed that the binding of both inhibitors to the enzyme showed biphasic profiles, which represented an initial encounter of the enzyme with the inhibitor followed by the isomerization of the transient enzyme-inhibitor complexes. The temperature-dependent transient kinetic studies with these inhibitors revealed that the bimolecular process is primarily dominated by favorable enthalpic changes, as opposed to the isomerization step, which is solely contributed by entropic changes. The standard binding enthalpy (ΔH°) of SAHA, deduced from the transient kinetic as well as the isothermal titration calorimetric experiments, was 2-3 kcal/mol higher than that of TSA. The experimental data presented herein suggest that SAHA serves as a preferential (target-specific and -selective) HDAC8 inhibitor as compared to TSA. Arguments that the detailed kinetic and thermodynamic studies may guide the rational design of HDAC inhibitors as therapeutic agents are presented.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Calorimetria , Inibidores de Histona Desacetilases/química , Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/química , Cinética , Temperatura , TermodinâmicaRESUMO
The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with a broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the zinc dependence of MDR superfamily proteins, have not yet have been adequately investigated. Using a density functional theory-based screening strategy, we have identified a strictly conserved glycine residue (Gly) in the zinc-dependent MDR superfamily. To elucidate the role of this conserved Gly in MDR, we carried out a comprehensive structural, functional, and computational analysis of four MDR enzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry, electron paramagnetic resonance (EPR), quantum mechanics, and molecular mechanics analysis. Gly substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved Gly resulted in alterations in the coordination of the catalytic zinc ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (K(d)) toward the zinc ion. The Gly mutants also showed different spectroscopic properties in EPR compared with those of the wild type, indicating that the binding geometries of the zinc to the zinc binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic zinc ion during catalysis of the MDR superfamily enzymes.
Assuntos
Álcool Desidrogenase/química , Proteínas Fúngicas/química , Glicina/química , Neurospora crassa/enzimologia , Zinco/química , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Motivos de Aminoácidos , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicina/genética , Glicina/metabolismo , Mutagênese Sítio-Dirigida , Neurospora crassa/genética , Estrutura Terciária de Proteína , Zinco/metabolismoRESUMO
An endo-1,4-ß-xylanase gene, xylcg, was cloned from Chaetomium globosum and successfully expressed in Escherichia coli. The complete gene of 675 bp was amplified, cloned into the pET 28(a) vector, and expressed. The optimal conditions for the highest activity of the purified recombinant XylCg were observed at a temperature of 40 °C and pH of 5.5. Using oat-spelt xylan, the determined K m, V max, and k cat/K m values were 0.243 mg ml⻹, 4,530 U mg⻹ protein, and 7,640 ml s⻹ mg⻹, respectively. A homology model and sequence analysis of XylCg, along with the biochemical properties, confirmed that XylCg belongs to the GH11 family. Rice straw pretreated with XylCg showed 30 % higher conversion yield than the rice straw pretreated with a commercial xylanase. Although xylanases have been characterized from fungal and bacterial sources, C. globosum XylCg is distinguished from other xylanases by its high catalytic efficiency and its effectiveness in the pretreatment of lignocellulosic biomass.
Assuntos
Chaetomium/enzimologia , Endo-1,4-beta-Xilanases/metabolismo , Escherichia coli/metabolismo , Avena/química , Biomassa , Chaetomium/genética , Clonagem Molecular , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/isolamento & purificação , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Lignina/metabolismo , Modelos Moleculares , Oryza/química , Caules de Planta/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura , Xilanos/metabolismoRESUMO
Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.
Assuntos
Biotecnologia/métodos , Enzimas Imobilizadas/metabolismo , Engenharia de Proteínas/métodos , Biocatálise , Biotecnologia/tendências , Estabilidade Enzimática , Enzimas Imobilizadas/química , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas/tendências , Solventes/química , Especificidade por Substrato , TemperaturaRESUMO
Chaetomium globosum endo-1,4-ß-xylanase (XylCg) is distinguished from other xylanases by its high turnover rate (1,860 s(-1)), the highest ever reported for fungal xylanases. One conserved amino acid, W48, in the substrate binding pocket of wild-type XylCg was identified as an important residue affecting XylCg's catalytic efficiency.
Assuntos
Chaetomium/enzimologia , Endo-1,4-beta-Xilanases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Domínio Catalítico , Endo-1,4-beta-Xilanases/genética , Cinética , Modelos Moleculares , Dados de Sequência MolecularRESUMO
The extracellular enzyme matrix metalloproteinase-9 (MMP-9) is overexpressed in atherosclerotic plaques and in metastatic cancers. The enzyme is responsible for rupture of the plaques and for the invasion and metastasis of a large number of cancers. The ability of ultrasonic excitation to induce thermal and mechanical effects has been used to release drugs from different carriers. However, the majority of these studies were performed with low frequency ultrasound (LFUS) at kilohertz frequencies. Clinical usage of LFUS excitations will be limited due to harmful biological effects. Herein, we report our results on the release of encapsulated contents from substrate lipopeptide incorporated echogenic liposomes triggered by recombinant human MMP-9. The contents release was further enhanced by the application of diagnostic frequency (3 MHz) ultrasound. The echogenic liposomes were successfully imaged employing a medical ultrasound transducer (4-15 MHz). The conditioned cell culture media from cancer cells (secreting MMP-9) released the encapsulated dye from the liposomes (30-50%), and this release is also increased (50-80%) by applying diagnostic frequency ultrasound (3 MHz) for 3 min. With further developments, these liposomes have the potential to serve as multimodal carriers for triggered release and simultaneous ultrasound imaging.
Assuntos
Lipossomos/química , Metaloproteinase 9 da Matriz/química , Ultrassom/métodos , Linhagem Celular Tumoral , Células HeLa , Humanos , Lipossomos/metabolismo , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismoRESUMO
We report the mechanistic studies of a FAD:NADH reductase (PrnF) involved in arylamine oxygenation. PrnF catalyzes the reduction of FAD via a sequential ordered bi-bi mechanism with NADH as the first substrate to bind and FADH(2) as the first product to be released. The residues Asp145 and His146 are proposed as catalytic acid/base residues for PrnF based on pH profile and molecular dynamics simulation studies. These studies provide the first detailed account of the mechanism of the flavin reductase involved in arylamine oxygenation.
Assuntos
FMN Redutase/química , FMN Redutase/metabolismo , Modelos Moleculares , Pseudomonas fluorescens/enzimologia , Aminas/metabolismo , Sítios de Ligação , Concentração de Íons de Hidrogênio , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Estrutura Molecular , Oxigênio/químicaRESUMO
Post-traumatic stress (PTSD) is considered a clinical issue that influences numerous people from diverse trades all over the world. Numerous research scholars recorded diverse complexities to estimate the severity of the PTSD symptoms in the patients. But diagnosing PTSD and obtaining accurate diagnosing techniques becomes a more complicated task. Therefore, this paper develops a speech based post-traumatic stress disorder monitoring method and the significant objective of the proposed method is to determine if the patients are affected by PTSD. The proposed approach utilizes three different steps: pre-processing or pre-emphasis, feature extraction as well as classification to evaluate the patients affected by PTSD or not. The input speech signal is initially provided to the pre-processing phase where the speech gets segmented into frames. The speech frame is then extracted and classified using XGBoost based Teamwork optimization (XGB-TWO) algorithm. In addition to this, we utilized two different types of datasets namely TIMIT and FEMH to evaluate and classify the PSTD from the speech signals. Furthermore, based on the evaluation of the proposed model to diagnose PTSD patients, various evaluation metrics namely accuracy, specificity, sensitivity, and recall are evaluated. Finally, the experimental investigation and comparative analysis are carried out and the evaluation results demonstrated that the accuracy rate achieved for the proposed technique is 98.25%.
RESUMO
Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.