Cell-free translation of biofuel enzymes

Methods Mol Biol. 2014;1118:71-95. doi: 10.1007/978-1-62703-782-2_5.

Abstract

In nature, bacteria and fungi are able to utilize recalcitrant plant materials by secreting a diverse set of enzymes. While genomic sequencing efforts offer exhaustive lists of genes annotated as potential polysaccharide-degrading enzymes, biochemical and functional characterizations of the encoded proteins are still needed to realize the full potential of this natural genomic diversity. This chapter outlines an application of wheat germ cell-free translation to the study of biofuel enzymes using genes from Clostridium thermocellum, a model cellulolytic organism. Since wheat germ extract lacks enzymatic activities that can hydrolyze insoluble polysaccharide substrates and is likewise devoid of enzymes that consume the soluble sugar products, the cell-free translation reactions provide a clean background for production and study of the reactions of biofuel enzymes. Examples of assays performed with individual enzymes or with small sets of enzymes obtained directly from cell-free translation are provided.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Biofuels / microbiology*
  • Biomass
  • Carbohydrate Metabolism
  • Cell-Free System
  • Cellulose / metabolism
  • Cloning, Molecular
  • Clostridium thermocellum / enzymology*
  • Clostridium thermocellum / genetics*
  • DNA Primers / genetics
  • Genetic Engineering
  • Hydrolysis
  • Phosphoric Acids / metabolism
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • Protein Biosynthesis*
  • Triticum / metabolism

Substances

  • Biofuels
  • DNA Primers
  • Phosphoric Acids
  • Cellulose
  • phosphoric acid