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- Sunner, Hampus, 1981, et al.
(author)
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Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester
- 2015
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In: Molecules. - : MDPI AG. - 1420-3049 .- 1420-3049 .- 1431-5157. ; 20:10, s. 17807-17817
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Journal article (peer-reviewed)abstract
- Research on glucuronoyl esterases (GEs) has been hampered by the lack of enzyme assays based on easily obtainable substrates. While benzyl d-glucuronic acid ester (BnGlcA) is a commercially available substrate that can be used for GE assays, several considerations regarding substrate instability, limited solubility and low apparent affinities should be made. In this work we discuss the factors that are important when using BnGlcA for assaying GE activity and show how these can be applied when designing BnGlcA-based GE assays for different applications: a thin-layer chromatography assay for qualitative activity detection, a coupled-enzyme spectrophotometric assay that can be used for high-throughput screening or general activity determinations and a HPLC-based detection method allowing kinetic determinations. The three-level experimental procedure not merely facilitates routine, fast and simple biochemical characterizations but it can also give rise to the discovery of different GEs through an extensive screening of heterologous Genomic and Metagenomic expression libraries.
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| 2. |
- Udatha, Gupta, 1984, et al.
(author)
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Deciphering the signaling mechanisms of the plant cell wall degradation machinery in Aspergillus oryzae
- 2015
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In: BMC Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 9:1, s. 20-
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Journal article (peer-reviewed)abstract
- Background: The gene expression and secretion of fungal lignocellulolytic enzymes are tightly controlled at the transcription level using independent mechanisms to respond to distinct inducers from plant biomass. An advanced systems-level understanding of transcriptional regulatory networks is required to rationally engineer filamentous fungi for more efficient bioconversion of different types of biomass. Results: In this study we focused on ten chemically defined inducers to drive expression of cellulases, hemicellulases and accessory enzymes in the model filamentous fungus Aspergillus oryzae and shed light on the complex network of transcriptional activators required. The chemical diversity analysis of the inducers, based on 186 chemical descriptors calculated from the structure, resulted into three clusters, however, the global, metabolic and extracellular protein transcription of the A. oryzae genome were only partially explained by the chemical similarity of the enzyme inducers. Genes encoding enzymes that have attracted considerable interest such as cellobiose dehydrogenases and copper-dependent polysaccharide mono-oxygenases presented a substrate-specific induction. Several homology-model structures were derived using ab-initio multiple threading alignment in our effort to elucidate the interplay of transcription factors involved in regulating plant-deconstructing enzymes and metabolites. Systematic investigation of metabolite-protein interactions, using the 814 unique reactants involved in 2360 reactions in the genome scale metabolic network of A. oryzae, was performed through a two-step molecular docking against the binding pockets of the transcription factors AoXlnR and AoAmyR. A total of six metabolites viz., sulfite (H2SO3), sulfate (SLF), uroporphyrinogen III (UPGIII), ethanolamine phosphate (PETHM), D-glyceraldehyde 3-phosphate (T3P1) and taurine (TAUR) were found as strong binders, whereas the genes involved in the metabolic reactions that these metabolites appear were found to be significantly differentially expressed when comparing the inducers with glucose. Conclusions: Based on our observations, we believe that specific binding of sulfite to the regulator of the cellulase gene expression, AoXlnR, may be the molecular basis for the connection of sulfur metabolism and cellulase gene expression in filamentous fungi. Further characterization and manipulation of the regulatory network components identified in this study, will enable rational engineering of industrial strains for improved production of the sophisticated set of enzymes necessary to break-down chemically divergent plant biomass.
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| 3. |
- Xiros, Charilaos, 1973, et al.
(author)
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Hydrolysis and Fermentation for Cellulosic Ethanol Production
- 2015
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In: Advances in Bioenergy: The Sustainability Challenge. - Oxford, UK : John Wiley & Sons, Ltd. ; , s. 11-31, s. 11-31
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Book chapter (other academic/artistic)abstract
- This chapter summarizes the hydrolysis technologies and bioconversion processes employed for cellulosic ethanol production. Hydrolysis process involves pre-treatment methods (first-stage hydrolysis) and enzymatic hydrolysis (second-stage hydrolysis). Although cellulose is mainly present as crystalline fibers that are highly resistant to hydrolysis, its content in biomass is typically larger compared to hemicellulose and, as a result, cellulases are the key enzymes for bioethanol production. The major bioconversion processes are: The separate (or sequential) hydrolysis and fermentation (SHF), the simultaneous saccharification and fermentation (SSF) and the consolidated bioconversion process (CBP). Many yeast species have been reported to convert simple sugars to ethanol under anaerobic conditions. S. cerevisiae, Pichia stipitis, P. kudriavzevii (Candida krusei), Kluveromyces marxianus, C. shehatae, C. tropicalis, C. guilliermondii, and Pachysolen tannophilus are among the most often used yeast species in biomass-derived sugars-to-ethanol conversion processes.
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