Cell-Free Translation of Biofuel Enzymes

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.

Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

“…For final gene assembly, the corresponding error-free fragments were PCR-amplified, purified, and assembled with pEU-JGI using Gibson assembly (39). pEU-JGI is a derivative of pEU-HSBC that contains SmaI restriction sites flanking the SgfI and PmeI delimited sacB/ chloramphenicol acetyltransferase selection cassette (40). The full-length genes underwent an additional round of sequence verification, and the correct clones were archived in 96-well plates ready for DNA purification and cell-free translation.…”

“…The full-length genes underwent an additional round of sequence verification, and the correct clones were archived in 96-well plates ready for DNA purification and cell-free translation. Sequence-verified plasmids were purified as previously described, and the purified plasmids were adjusted to 1 g/l for use in coupled cell-free transcription and translation reactions (40). Translation reactions were analyzed by SDS-PAGE, and the concentration of each translated protein was quantitated using Gel Dock (Bio-Rad).…”

“…The reaction was initiated by addition of ϳ0.1 g of purified enzyme and was incubated for up to 1 h at 50°C. Reducing end products were determined using the dinitrosalicylic acid (DNS) assay (40,41). After the desired time of reaction, 1 volume of the assay mixture was added to 2 volumes of DNS reagent, mixed, and then heated at 95°C for 5 min.…”

“…The reactions were carried out at 50°C for up to 24 h. A unit of activity is defined as the release of 1 mol of reducing sugar per minute from laminarin as determined by using the DNS assay (40,41). For calculation of specific activity, the concentration of each translated protein was determined using SDS-PAGE and Gel Dock (Bio-Rad).…”

“…The reaction was started by mixing 4 l of an individual translation reaction with 10 mg/ml of laminarin in 50 mM phosphate buffer, pH 6.0. After 10 min, reducing end products were determined using the DNS assay (40,41). The pH optima were determined at 50°C using the same enzyme and substrate concentrations as above for the temperature optima studies, but the buffer was varied as follows: 50 mM sodium citrate (pH 4.0 and 5.0); 50 mM phosphate (pH 6.0); Tris-HCl (pH 7.0 and 8.0); 50 mM CHES (pH 9.0 and 10.0); and 50 mM CAPS (pH 11.0).…”