*4.2. Preparation of Recombinant DgAS Enzyme*

*DgAS* was amplified from the genome of *Deinococcus geothermalis* with polymerase chain reaction (PCR) with a primer set: forward 5 -cccgaattcgCTGAAAGACGTGCTCACTTCTGAAC-3 and reverse 5 -aaactcgagTTATGCTGGAGCCTCCCCGGCGGTC-3 , which contained EcoRI and XhoI restriction sites, respectively. The amplified DNA fragment (1.95 kb) was digested with EcoRI and XhoI, and ligated into the corresponding sites on the *pET-Duet-1* expression plasmid to form *pETDuet-DgAS* (Figure 1a), which was then transformed into *E. coli* (DE3) with the electroporation method [20]. The recombinant *E. coli* (DE3) was cultured in Luria-Bertani (LB) medium to optical density at 560 nm (OD560) of 0.6, and then induced with 0.2 mM of IPTG. After further cultivation at 18 ◦C for 20 h, the cells were centrifuged at 4500× *g* and 4 ◦C for 20 min. The cell pellet was washed, and spun down twice with 50 mM of phosphate buffer (PB) at pH 6.8, and then broken with sonication via a Branson S-450D Sonifier (Branson Ultrasonic Corp., Danbury, CT, USA). The sonication program was carried out for five cycles of 5 sec on and 30 sec off at 4 ◦C. The mixture was then centrifuged at 15,000× *g* and 4 ◦C for 20 min to remove the cell debris. The supernatant containing the produced DgAS fused with a His-tag in its N-terminal was applied in a Ni2<sup>+</sup> <sup>a</sup>ffinity column (10 i.d. <sup>×</sup> 50 mM, Ni Sepharose 6 Fast Flow, GE Healthcare, Chicago, IL, USA). The His-tag fused DgAS was washed with PB with 25 mM of imidazole and eluted with PB containing 250 mM of imidazole. The elution was then concentrated and desalted through Macrosep 10 K centrifugal filters (Pall, Ann Arbor, MI, USA). The concentration of the purified DgAS was determined with the Bradford method [20], and analyzed with SDS-PAGE (Figure 1b). The purified DgAS was stored in a final concentration of 50% glycerol at –80 ◦C before use.

#### *4.3. Biotransformation of 8-OHDe by the Purified DgAS Enzyme*

Biotransformation was carried out in 1 mL of reaction mixture containing 0.1 mg/mL of 8-OHDe, 12.5 μg/mL of DgAS, 150 mM of sucrose, and 50 mM of Tris, pH 8.0. The reaction was performed at 40 ◦C for 30 min. After reaction, the reaction mixture was analyzed with UPLC. To optimize the major compound production, 1 mg/mL of 8-OHDe and 125 μg of DgAS were used as the substrate at different temperatures, pH levels, and sucrose concentrations. To optimize the pH, 50 mM of acetate buffer (pH 5 and pH 6), phosphate buffer (pH 7), and Tris buffer (pH 8 and pH 9) were used.

## *4.4. UPLC*

UPLC was performed with an Acquity® UPLC system (Waters, Milford, MA, USA). The stationary phase was the Kinetex® C18 column (1.7 <sup>μ</sup>m, 2.1 i.d. <sup>×</sup> 100 mM, Phenomenex Inc., Torrance, CA, USA), and the mobile phase was 1% acetic acid in water (A) and methanol (B). The linear gradient elution condition was 0 min with 36% B to 7 min with 81% B at a flow rate of 0.2 mL/min. The detection condition was set at 254 nm.
