Cellulases are of considerable current interest for converting the cellulosic content of biomass to fermentable sugars for biofuels production. To facilitate the enzymatic conversion of biomass to sugars, pre-treatment processes at high temperatures, low pH, and even in ionic liquids are candidates to reduce the recalcitrance of the lignocellulosic substrate. Cellulases that function at high temperatures and are stable to pre-treatment conditions offer the potential advantages of lower cooling costs, higher solids loadings, reduced risk of microbial contamination, and higher reaction rates. Hyperthermophilic archaea and bacteria are potential sources of extremely stable cellulosic enzymes. To this end, bioprospecting for thermophilic, cellulose degraders and their enzymes has resulted in the discovery of a unique cellulase, designated EBI244. The 842 amino-acid cellulase contains a catalytic domain homologous to the glycosyl hydrolase family 5 domain; it has a pH optimum around 6.0, a temperature optimum near 100�C, and is active against soluble and crystalline cellulose substrates. EBI244 is at least four-times more active against ionic liquid pre-treated Avicel than untreated Avicel, and remains active in a 50% ionic liquid solution. The catalytic and structural properties of EBI244 expressed in different hosts are also being compared. These results demonstrate the discovery of a novel hyperthermophilic cellulase from a hyperthermophilic enrichment, and establish that hyperthermophilic archaea are capable of growth on crystalline cellulose.Another approach to develop more stable cellulases is to generate new enzymes by protein engineering. We have thus employed several protein engineering strategies to create cellulases with improved thermostability. In the process we have also discovered an important post-translational modification essential to maximize the thermostability of the exoglucanase Cel7A. These results will be discussed in connection with cellulase structure-stability relationships and the complementarity of bioprospecting and protein engineering for the discovery and generation of new cellulases.

Tapping new sources of cellulases: Extremophiles add to the fray

248th National Meeting (2014)

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