RESUMO
Thermochemical pretreatment and enzymatic hydrolysis are the areas contributing most to the operational costs of second generation ethanol in lignocellulosic biorefineries. The improvement of lignocellulosic enzyme cocktails has been significant in the recent years. Although the needs for the reduction of the energy intensity and chemical consumption in the pretreatment step are well known, the reduction of the severity of the process strongly affects the enzymatic hydrolysis yield. To explore the formulation requirements of the well known cellulolytic cocktail from Myceliophthora thermophila on mild pretreated raw materials, this cocktail was tested on steam exploded corn stover without acid impregnation. The low hemicellulose yield and significant accumulation of xylobiose compared with the standard pretreated material obtained with dilute acid impregnation evidenced a clear limitation in the conversion of xylan to xylose. In order to complement the beta-xylosidase limitation, a selection of enzymes was expressed and tested in this fungus. A controlled expression of xylosidases from Aspergillus nidulans, Aspergillus fumigatus, and Fusarium oxysporum allowed recovering hemicellulose yields reached with standard acid treated material. The results underline the need of parallel development of the pretreatment process with the optimization of the formulation of the enzymatic cocktails.
Assuntos
Proteínas Fúngicas/química , Lignina/química , Componentes Aéreos da Planta/química , Xilosidases/metabolismo , Zea mays/química , Ativação Enzimática , Hidrólise , Especificidade por SubstratoRESUMO
Most organisms performing oxygenic photosynthesis contain either cytochrome c(6) or plastocyanin, or both, to transfer electrons from cytochrome b(6)-f to photosystem I. Even though plastocyanin has superseded cytochrome c(6) along evolution, plants contain a modified cytochrome c(6), the so called cytochrome c(6A), whose function still remains unknown. In this article, we describe a second cytochrome c(6) (the so called cytochrome c(6)-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c(6A) than to cyanobacterial cytochrome c(6). In this article, we conclude that the cytochrome c(6)-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c(6) and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c(6) and plastocyanin. In any way, the Cyt c(6)-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.
Assuntos
Proteínas de Bactérias/metabolismo , Citocromos c6/metabolismo , Nostoc/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Citocromos c6/química , Citocromos c6/genética , Citocromos c6/isolamento & purificação , DNA Bacteriano/química , Transporte de Elétrons , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Luz , Modelos Moleculares , Dados de Sequência Molecular , Nostoc/genética , Nostoc/fisiologia , Oxirredução , Fotossíntese/fisiologia , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Little information is yet available on the alpha-amylases of cyanobacteria. Here, the presence of an alpha-amylase in the cyanobacterium Nostoc sp. PCC 7119 is first demonstrated. A gene (amy1) encoding a cytoplasmic alpha-amylase (Amy1) protein has been identified, cloned, and overexpressed in Escherichia coli cells. The recombinant protein is a 56.7-kDa monomer, which has been purified to electrophoretic homogeneity by affinity chromatography. The substrate specificity and end product analyses confirm that it is a calcium-dependent alpha-amylase enzyme, which exhibits its maximum activity at 31 degrees C and at pH between 6.5 and 7.5. The Amy1 protein breaks down mainly starch, is also able to cleave glycogen and dextrin, and exhibits no activity against xylan or pullulan. So the enzyme cannot efficiently attack the maltodextrins with degrees of polymerization below that of maltooctaose. Maltotriose, maltose, and maltotetraose are the major products of the enzymatic reaction with starch as substrate. The enzyme shows a very high turnover number against soluble potato starch (3,420 +/- 270 s(-1)), as compared with other alpha-amylases reported in the literature. The high catalytic efficiency and relatively low optimum temperature of the Nostoc Amy1 protein make this previously unexplored group of cyanobacterial enzymes of great interest for further physiological studies and industrial applications.
