RESUMO

This work aims to demonstrate the enzymatic production of fatty acid ethyl ester biodiesel from highly acidic feedstock in a single-step reaction, without co-solvents and avoiding the inhibition of the enzyme by ethanol and glycerol. Additionally, an empirical equation is proposed to predict the kinetics of the production reaction as a function of the used feedstock and catalyst concentration. Biodiesel production from highly acidic feedstock perform via simultaneous esterification of free fatty acids and transesterification of triacylglycerols. Enzymatic catalysis is one of the most promising alternative technologies for the biodiesel production. Increasing of the enzymatic bioactivity is crucial for the success of such process in industrial scale. Currently, stepwise addition of the alcohol or the use of co-solvents have been proposed to avoid enzyme inhibition, such strategies add downstream processes to the production. These results can be applied to the development economical-viable enzymatic production of biodiesel in industrial scale.

Assuntos

Ácidos/química , Ácidos Graxos/metabolismo , Solventes/química , Biocombustíveis , Esterificação , Ésteres , Triglicerídeos/metabolismo

RESUMO

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.

Assuntos

Peptídeos beta-Amiloides/química , Naftalenossulfonato de Anilina/química , Naftalenossulfonatos/química , Fragmentos de Peptídeos/química , Amiloide/química , Animais , Ânions/química , Benzotiazóis , Bioquímica/métodos , Linhagem Celular , Cromatografia , Vermelho Congo/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Luz , Lisina/química , Espectroscopia de Ressonância Magnética , Camundongos , Micelas , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Peso Molecular , Peptídeos/química , Fenilalanina/química , Príons/química , Estrutura Secundária de Proteína , Espalhamento de Radiação , Dodecilsulfato de Sódio/química , Espectrofotometria , Eletricidade Estática , Ácidos Sulfônicos/química , Temperatura , Sais de Tetrazólio/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Fatores de Tempo

RESUMO

Protein misfolding and aggregation have been linked to several human diseases, including Alzheimer's disease, Parkinson's disease, and systemic amyloidosis, by mechanisms that are not yet completely understood. The hallmark of most of these diseases is the formation of highly ordered and beta-sheet-rich aggregates referred to as amyloid fibrils. Fibril formation by WT transthyretin (TTR) or TTR variants has been linked to the etiology of systemic amyloidosis and familial amyloid polyneuropathy, respectively. Similarly, amyloid fibril formation by alpha-synuclein (alpha-syn) has been linked to neurodegeneration in Parkinson's disease, a movement disorder characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Here we show that consecutive cycles of compression-decompression under aggregating conditions lead to reversible dissociation of TTR and alpha-syn fibrils. The high sensitivity of amyloid fibrils toward high hydrostatic pressure (HHP) indicates the existence of packing defects in the fibril core. In addition, through the use of HHP we are able to detect differences in stability between fibrils formed from WT TTR and the familial amyloidotic polyneuropathy-associated variant V30M. The fibrils formed by WT alpha-syn were less susceptible to pressure denaturation than the Parkinson's disease-linked variants, A30P and A53T. This finding implies that fibrils of alpha-syn formed from the variants would be more easily dissolved into small oligomers by the cellular machinery. This result has physiological importance in light of the current view that the pathogenic species are the small aggregates rather the mature fibrils. Finally, the HHP-induced formation of fibrils from TTR is relatively fast (approximately 60 min), a quality that allows screening of antiamyloidogenic drugs.

Assuntos

Amiloide/química , Amiloide/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Pré-Albumina/química , Pré-Albumina/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Amiloide/genética , Amiloidose/genética , Amiloidose/metabolismo , Variação Genética , Humanos , Pressão Hidrostática , Técnicas In Vitro , Substâncias Macromoleculares , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Pré-Albumina/genética , Sinucleínas , Água/química , alfa-Sinucleína

RESUMO

The formation of amyloid aggregates is the hallmark of the amyloidogenic diseases. Transthyretin (TTR) is involved in senile systemic amyloidosis (wild-type protein) and familial amyloidotic polyneuropathy (point mutants). Through the use of high hydrostatic pressure (HHP), we compare the stability among wild-type (wt) TTR, two disease-associated mutations (V30M and L55P) and a trans-suppressor mutation (T119M). Our data show that the amyloidogenic conformation, easily populated in the disease-associated mutant L55P, can be induced by a cycle of compression-decompression with the wt protein rendering the latter highly amyloidogenic. After decompression, the recovered wt structure has weaker subunit interactions (loosened tetramer, T(4)(*)) and presents a stability similar to L55P, suggesting that HHP induces a defective fold in the wt protein, converting it to an altered conformation already present in the aggressive mutant, L55P. On the other hand, glucose, a chemical chaperone, can mimic the trans-suppression mutation by stabilizing the native state and by decreasing the amyloidogenic potential of the wt TTR at pH 5.0. The sequence of pressure stability observed was: L55P

Assuntos

Amiloide/química , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Pré-Albumina/química , Pré-Albumina/genética , Água/química , Relação Dose-Resposta a Droga , Glucose/farmacologia , Humanos , Modelos Químicos , Mutação , Pressão , Conformação Proteica , Temperatura , Termodinâmica