Directed evolution has emerged as one of the most effective approaches in developing and adapting biocatalysts to industrial requirements. This work shows the development of hidrophobic and thermotolerant biocatalyst useful in biorefining processes for the Petroleum Industry. We generated a second library of Cloroperoxidase enzyme gene by Error-Prone PCR (EP-PCR) with the selection of the best mutants based on their hidrophobicity, thermoresistance and stability in organic media. These proteins were more active than the first library mutants and the wildtype enzyme. We demonstrated that this directed evolution technique is efficient in generating chimeric libraries of enzymes of industrial interest.