(Hyper)thermophilic enzymes: Production and purification

Pierpaolo Falcicchio, Mark Levisson, Servé W.M. Kengen, Sotirios Koutsopoulos*, John van der Oost

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterAcademicpeer-review

3 Citations (Scopus)

Abstract

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where “normal” proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

Original languageEnglish
Title of host publicationProtein Downstream Processing
EditorsNikolaos E. Labrou
PublisherHumana Press
Pages469-478
Number of pages10
ISBN (Electronic)9781071607756
ISBN (Print)9781071607749
DOIs
Publication statusPublished - 1 Nov 2020

Publication series

NameMethods in Molecular Biology
Volume2178
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Biocatalysis
  • Heterologous production
  • His-tag
  • Immobilized metal affinity chromatography (IMAC)
  • Protein purification
  • Size exclusion chromatography (SEC)
  • Thermal stability
  • Thermozymes

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