Abstract
The
Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in
Escherichia coli. The preparation yields 470
UL
−1 of enzyme at a specific activity of 9.4
U
mg
−1. During retroaldol cleavage of KDPG, the enzyme shows a
k
cat that decreases with decreasing temperature. A more than offsetting decrease in
K
m yields an enzyme that is more efficient at 40
°C than at 70
°C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.
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