Related Experiment Video
Updated: Jun 7, 2026

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
Published on: June 24, 2016
Mutational analysis of phenylalanine ammonia lyase to improve reactions rates for various substrates
Sebastian Bartsch1, Uwe T Bornscheuer
1Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany.
Abstract:
Phenylalanine ammonia lyases (PAL) catalyze the reversible, non-reductive amination of trans-cinnamic acid to l-phenylalanine in the presence of high ammonia concentrations. Since neither cofactor recycling nor other additives are needed and by this asymmetric synthesis theoretical yields of 100% can be reached, it is an interesting reaction for industrial processes. In this study we demonstrate the superior properties of p-nitro-cinnamic acid (p-n-CA) in the amination reaction using the PAL from Petroselinum crispum (pcPAL). By focused-directed evolution, three mutants were identified showing increased reaction rates and decreased substrate inhibition. Together, the F137V mutant with p-n-CA showed a 15-fold increased reaction rate compared with the pcPAL WT with the natural cinnamic acid. The high reaction rates were also proven in preparative scale experiments. Activities towards other p-substituted cinnamic acids showing different electronic effects of the substituent were analyzed. Focused-directed evolution around the carboxylic acid- and amine-binding site always decreased PAL activity, due to a sensitive H-bond network.
Related Concept Videos
Introduction to Mechanisms of Enzyme Catalysis
Amino Acid Biosynthetic Pathways
Enzyme Kinetics
Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is...
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Enzymes
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...

