β-glucosidase CelB from Pyrococcus furiosus: Production by Escherichia coli, purification, and in vitro evolution

Autor:	J.H.G. Lebbink, W.M. de Vos, S.W.M. Kengen, Thijs Kaper, J. van der Oost
Rok vydání:	2001
Předmět:	chemistry.chemical_classification biology Mutant Active site Heterologous biology.organism_classification medicine.disease_cause Directed evolution Enzyme Biochemistry chemistry biology.protein Pyrococcus furiosus medicine Escherichia coli Systematic evolution of ligands by exponential enrichment
DOI:	10.1016/s0076-6879(01)30389-0
Popis:	Publisher Summary One of the key enzymes of the hyperthermophilic archaeon Pyrococcus furiosus involved in growth on B-linked sugars is the inducible B-glucosidase (CelB). CelB from P. furiosus serves as a very suitable model glycosylhydrolase to study substrate specificity as well as adaptations of stability and activity to extreme temperatures. Stable production in E. coli and an efficient purification protocol allow for simple and rapid preparation of pure wildtype and mutant CelB enzymes. This chapter an presents an overview of the state of the art on this hyperthermostable enzyme, protocols for heterologous production and enzyme purification, and development and application of a directed evolution procedure that has resulted in the isolation and characterization of an active site mutant.
Databáze:	OpenAIRE
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