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Engineering glycoside hydrolase stability by the introduction of zinc binding.

  • Author(s): Ellinghaus, Thomas L;
  • Pereira, Jose H;
  • McAndrew, Ryan P;
  • Welner, Ditte H;
  • DeGiovanni, Andy M;
  • Guenther, Joel M;
  • Tran, Huu M;
  • Feldman, Taya;
  • Simmons, Blake A;
  • Sale, Kenneth L;
  • Adams, Paul D
  • et al.
Abstract

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.

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