Τίτλος:
Evaluation of thermal stability of cellulosomal hydrolases and their complex formation
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
Enzymatic breakdown of plant biomass is an essential step for its utilization in biorefinery applications, and the products could serve as substrates for the sustainable and environmentally friendly production of fuels and chemicals. Toward this end, the incorporation of enzymes into polyenzymatic cellulosome complexes—able to specifically bind to and hydrolyze crystalline cellulosic materials, such as plant biomass—is known to increase the efficiency and the overall hydrolysis performance of a cellulase system. Despite their relative abundance in various mesophilic anaerobic cellulolytic bacteria, there are only a few reports of cellulosomes of thermophilic origin. However, since various biorefinery processes are favored by elevated temperatures, the development of thermophilic designer cellulosomes could be of great importance. Owing to the limited number of thermophilic cellulosomes, designer cellulosomes, composed of mixtures of mesophilic and thermophilic components, have been constructed. As a result, the overall thermal profile of the individual parts and the resulting complex has to be extensively evaluated. Here, we describe a practical guide for the determination of temperature stability for cellulases in the cellulosome complexes. The approach is also appropriate for other related enzymes, notably xylanases as well as other glycoside hydrolases. We provide detailed experimental procedures for the evaluation of the thermal stability of the individual designer cellulosome components and their complexes as well as protocols for the assessment of complex integrity at elevated temperatures. © Springer Science+Business Media, LLC, part of Springer Nature 2018.
Συγγραφείς:
Kahn, A.
Galanopoulou, A.P.
Hatzinikolaou, D.G.
Moraïs, S.
Bayer, E.A.
Περιοδικό:
Methods in Molecular Biology
Εκδότης:
Humana Press Inc.
Λέξεις-κλειδιά:
balanced salt solution; calcium chloride; cellulosome; chimeric enzyme; glycosidase; hydrolase; microcrystalline cellulose; polysorbate 20; recombinant enzyme; scaffold protein; tris buffered saline; unclassified drug; xylan endo 1,3 beta xylosidase; buffer; cellulase; cellulosome; recombinant protein, assay; chemical interaction; Clostridium thermocellum; complex formation; controlled study; enzyme activity; enzyme stability; evaluation study; high temperature; native polyacrylamide gel electrophoresis; nonhuman; polyacrylamide gel electrophoresis; protein interaction; pull down assay; size exclusion chromatography; standardization; thermophilic bacterium; enzyme assay; enzyme stability; enzymology; hydrolysis; metabolism; procedures; solution and solubility; temperature, Buffers; Cellulase; Cellulosomes; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Enzyme Assays; Enzyme Stability; Hydrolysis; Recombinant Proteins; Solutions; Temperature
DOI:
10.1007/978-1-4939-7877-9_12
