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

• The variant10 mutant SepDERA with three amino acid substitutions (T120 C, G174I, and G213C) was harvested after three rounds of site-directed mutagenesis.
• The result suggested that the inactivation of DERA by exposure to aldehyde may result from protein structural damage.
• After reconstruction, variant10 retained 70.5% and 27.2% of its DRP cleavage activity after exposure for 2 h and 24 h, respectively, in 300 mM acetaldehyde.

DERASEP (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 DERAECO (2-deoxy-ribose aldolase cloned from Escherichia coli). However, the acetaldehyde tolerance of DERASEP 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 DERASEP 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, variant10 mutant DERASEP 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 DERASEP 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 variant10 was only half that of DERASEP. Thus, the acetaldehyde tolerance of DERASEP 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.

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