Improving the acetaldehyde tolerance of DERASEP by enhancing the rigidity of its protein structure

DERA SEP (2-deoxy- d -ribose-5-phosphate aldolase cloned from Staphylococcus epidermidis ) shows higher catalytic activity toward 2-deoxy- d -ribose-5-phosphate (DRP) than that of DERA ECO (2-deoxy-ribose aldolase cloned from Escherichia coli ). However, the acetaldehyde tolerance of DERA SEP is still at low level, which limits its use in large-scale applications. Although the mechanism responsible for enzyme inactivation in the presence of solvents is unclear, earlier work indicated the positive correlation between the tolerance of an enzyme for organic solvents and the rigidity of its structure. The acetaldehyde tolerance of DERA SEP was improved by site-directed mutagenesis on the basis of enhancing the rigidity of the protein structure. After calculating the virtual mutation energy (stability) of the enzyme and experimental verification, variant 10 mutant DERA SEP with three amino acid substitutions (T120C, G174I, and G213C) was harvested after three rounds of site-directed mutagenesis. It showed surprisingly high tolerance to acetaldehyde, with almost 70.5% residual catalytic activity after exposure to 300   mM acetaldehyde for 2   h, while the wild-type DERA SEP retained only 11.3% activity under the same condition. Because the rigidity of the protein was increased after incorporating the mutations, the specific activity of the variant 10 was only half that of DERA SEP . Thus, the acetaldehyde tolerance of DERA SEP was efficiently increased by enhancing the rigidity of the structure. These results suggested that the inactivation of DERA by exposure to acetaldehyde may result from protein structural damage.