Recombinant production of a highly efficient photolyase from Thermus thermophilus

Abstract

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋_𝑚𝑎𝑥 ) of 2.0 g L^-1 was reached after 5 h of cultivation, corresponding to 𝑃_X = 0.4 g L^-1 h. The µ_𝑚𝑎𝑥 corresponded to 1.0 h^-1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25 mg L^-1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15 µg mL^-1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase k_M of 9.5 nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products.

Keywords: CDP-DNA; Thermus thermophilus; active photoprotection; photodamage DNA; photolyase.