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Team:UC Davis/Protein Engineering Design
From 2014.igem.org
Why Aldehyde Dehydrogenases?
The aldehyde dehydrogenase family of enzymes (EC: 1.2.1.3, 1.2.1.5) was selected for use with our electrochemical biosensor. This family of enzymes catalyzes the reaction of aliphatic, straight chain aldehydes and the oxidized form of beta-nicotinamide adenine dinucleotide (NAD+) to produce the corresponding carboxylic acid and the reduced form of beta-nicotinamide adenine dinucleotide (NADH).
SCHEME OF REACTION
The aldehyde dehydrogenase enzyme family was perfect our engineering and electrochemical applications:
SCHEME OF REACTION
The aldehyde dehydrogenase enzyme family was perfect our engineering and electrochemical applications:
- This enzyme uses NAD+ as a coenzyme and produces NADH in a 1:1 molar ratio with the amount of aldehyde catalyzed. The concentration of NADH can be readily measured with a spectrophotometer reading absorbance at 340nm, allowing us to easily measure the rate of the reaction catalyzed by an aldehyde dehydrogenase enzyme.
- The active site of aldehyde dehydrogenase is in the center of a long tunnel, where NAD+ enters from one side and the aldehyde substrate enters from the other side. This tunnel gives us a large amount of flexibility in engineering amino acid residues which will affect the catalytic efficiency of this enzyme toward certain aldehyde species.
- We identified several commercial electrodes which oxidize NADH back to NAD+ and produce a current. Using a potentiostat, we can convert this current into a voltage and use a computer to determine the concentration of aldehydes in a sample. This cyclic conversion of NAD+/NADH allow us to use an electrochemical approach to quantify the concentration of aldehydes in a sample.
