Engineering and Kinetic Stabilization of the Therapeutic Enzyme Anabeana variabilis Phenylalanine Ammonia Lyase

作者：Jaliani Hossein Zarei; Farajnia Safar^*; Mohammadi Seyyed Abolghasem; Barzegar Abolfazl; Talebi Saeed

来源：Applied Biochemistry and Biotechnology, 2013, 171(7): 1805-1818.

DOI：10.1007/s12010-013-0450-5

• Disulfide bond
• Mutation
• Phenylalanine ammonia lyase
• Protein stability
• Rational engineering

Anabeana variabilis phenylalanine ammonia lyase has just recently been discovered and introduced in clinical trials of phenylketonuria enzyme replacement therapy for its outstanding kinetic properties. In the present study, kinetic stabilization of this therapeutically important enzyme has been explored by introduction of a disulfide bond into the structure. Site-directed mutagenesis was performed with quick-change PCR method. Recombinant wild-type and mutated enzymes were expressed in Escherichia coli, and his-tagged proteins were affinity purified. Formation of disulfide bond was confirmed by Ellman's method, and then chemical unfolding, kinetic behavior, and thermal inactivation of mutated enzyme were compared with the wild type. Based on our results, the Q292C mutation resulted in a significant improvement in kinetic stability and resistance against chemical unfolding of the enzyme while kinetic parameters and pH profile of enzyme activity were remained unaffected. The results of the present study provided an insight towards designing phenylalanine ammonia lyases with higher stability.

• 出版日期2013-12