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Engineering enzymes towards biotherapeutic applications using ancestral sequence reconstruction

Time: Fri 2020-09-25 10.00

Location:​, Stockholm (English)

Subject area: Chemistry

Doctoral student: Natalie Hendrikse , Ytbehandlingsteknik

Opponent: Professor Reinhard Sterner, Institut für Biophysik und physikalische Biochemie, University of Regensburg

Supervisor: Associate Professor Per-Olof Syrén, Science for Life Laboratory, SciLifeLab, Ytbehandlingsteknik; Associate Professor Johan Rockberg, Proteinteknologi; Dr. Erik Nordling,

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Enzymes are versatile biocatalysts that fulfill essential functions in all forms of life and, therefore, play an important role in health and disease. One specific application of enzymes in life science is their use as biopharmaceuticals, which typically benefits from high catalytic activity and stability. Increased stability and activity are both desirable properties for biopharmaceuticals as they are directly related to dosage, which in turn affects administration time, cost of production and potency of a drug. The aim of the work presented in this thesis is to enhance the therapeutic potential of enzymes by means of enzyme engineering, in particular using ancestral sequence reconstruction. In Paper I, we established the utility of this method in a model system and obtained ancestral terpene cyclases with increased activity, stability and substrate scope. In Paper II, we described the successful crystallization of the most stable ancestral terpene cyclase, which allowed for rational design of substrate specificity. Finally, we applied the method to two therapeutically relevant enzyme families associated with rare metabolic disorders. We obtained ancestral phenylalanine/tyrosine ammonia-lyases with substantially enhanced thermostability and long-term stability in Paper III and ancestral iduronate-2-sulfatases with increased activity in Paper IV. In summary, the results presented herein highlight the potential of ancestral sequence reconstruction as a method to obtain stable enzyme scaffolds for further engineering and to enhance therapeutic properties of enzymes.