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Stabilization of Enzymes by Using Thermophiles

  • Ana Luisa Ribeiro
  • Mercedes Sánchez
  • Aurelio Hidalgo
  • José BerenguerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1645)

Abstract

Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions. Metagenomic libraries from thermal environments are potential sources of thermostable enzymes of prokaryotic origin, but the number of expected hits could be quite low for enzymes handling substrates such as steroids, rarely found in prokaryotes. An alternative to metagenome screening is the selection of thermostable variants of well-known steroid-processing enzymes. Here we review and detail a protocol for such selection, where error-prone PCR (epPCR) is used to introduce random mutations into a gene to create a variants library for further selection of thermostable variants in the thermophile Thermus thermophilus. The method involves the use of folding interference vectors where the proper folding of the enzyme of interest at high temperature is linked to the folding of a reporter encoding a selectable property such as thermostable resistance to kanamycin, leading to a life-or-death selection of variants of reinforced folding independently of the activity of the enzyme.

Key words

Thermus thermophilus Enzyme variants library Thermostabilization Folding-interfering reporter 

Notes

Acknowledgments

This work has been supported by grant RTC-2014-1439-1 from the Spanish Ministry of Economy and Competence. An institutional grant from Fundación Ramón Areces to CBMSO is also acknowledged.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Ana Luisa Ribeiro
    • 1
  • Mercedes Sánchez
    • 1
  • Aurelio Hidalgo
    • 1
  • José Berenguer
    • 1
    Email author
  1. 1.Centro de Biología Molecular Severo Ochoa (UAM-CSIC), Facultad de CienciasUniversidad Autónoma de Madrid, Consejo Superior de Investigaciones CientíficasMadridSpain

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