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When cells are exposed to freezing temperatures, high concentrations of cryoprotective agents (CPA) prevent ice crystal formation, thus enhancing cell survival. However, high concentrations of CPAs can also cause cell toxicity. Exopolysaccharides (EPSs) from polar marine environments exhibit lower toxicity and display effects similar to traditional CPA. In this study, we sought to address these issues by i) selecting strains that produce EPS with novel cryoprotective activity, and ii) optimizing culture conditions for EPS production. Sixty-six bacteria producing mucous substances were isolated from the Ross Sea (Antarctic Ocean) using solid marine agar plates. Among them, Pseudoalteromonas sp. RosPo-2 was ultimately selected based on the rheological properties of the produced EPS (p-CY02). Cryoprotective activity experiments demonstrated that p-CY02 exhibited significantly cryoprotective activity at a concentration of 0.8% (w/v) on mammalian cells (HaCaT). This activity was further improved when combined with various concentrations of dimethyl sulfoxide (DMSO) compared to using DMSO alone. Moreover, the survival rate of HaCaT cells treated with 5% (v/v) DMSO and 0.8% (w/v) p-CY02 was measured at 87.9 ± 2.8% after freezing treatment. This suggests that p-CY02 may be developed as a more effective, less toxic, and novel non-permeating CPA. To enhance the production of EPS with cryoprotective activity, Response Surface Methodology (RSM) was implemented, resulting in a 1.64-fold increase in production of EPS with cryoprotective activity.

Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme. KEY POINTS: • We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.