Abstract: A range of Cel7 putative cellobiohydrolase genes were identified using genome mining and homologous sequence alignment to Cel7A from Trichoderma reesei. A representative subset of these genes from across a broad diversity of evolutionarily disparate sources were cloned and expressed in T. reesei using a constitutive promotor and a common secretion signal. The purified recombinant enzymes were tested for efficacy on various substrates. The top performers were subjected to structural studies and subsites likely to confer enhanced performance were predicted using homology modeling and comparisons of natural sequence diversity. Once identified, the subsites were genetically introduced individually and combinatorically into the best in class Cel7A backbone we have found to date and then expressed in T. reesei and tested. A triple mutant was determined to have the highest cellulase activity we have measured in a cellobiohydrolase to date.