Directed Evolution of Novel Enzyme Activities
Final rept. 15 Apr 1999-14 Apr 2002
CALIFORNIA INST OF TECH PASADENA
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We report here the directed evolution of the two valuable oxidases horseradish peroxidase HRP and laccase. We achieved functional expression of HRP in S. cerevisiae, and in E. coli to an extent that allows the improvement of this important catalyst. Mutagenesis, recombination and screening were successful in increasing total activity 40 fold by improving expression, catalytic activity and thermostability. Mutants were produced at up to 600 UIOD in Pichia pastoris. We also used computation-guided saturation mutagenesis to create higher active and stabilized HRP mutants more efficiently. We expressed the laccase from Myceliophthora themophila in Saccharomyces cerevisiae. We employed directed evolution to improve catalysis as well as expression level to a 170 fold higher total activity. Our evolved yeast mutant has the highest functional expression ever reported for laccase in a nonfungal system. Kinetic characterization of the purified mutants showed that the substrate specificity was left unchanged. The compromised thermostability of the mutants was regained in only one generation of stability screening. For the functional improvement of laccase we developed high throughput assays. PAH bioremediation can be directly assessed with anthracene and for highest sensitivity 0.3 mUml Poly-R, a readily soluble surrogate substrate for PAHs can be used.
- Anatomy and Physiology