Mining and Characterization of Amylosucrase from Calidithermus terrae for Synthesis of α-Arbutin Using Sucrose

α-Arbutin is the fourth generation whitening factor in the field of cosmetics, which can block the synthesis of melanin in epidermal cells and has the advantages of good stability and less toxic side effects. Moreover, α-arbutin has potential application value in food, medicine, and other fields. However, the extraction yield from plant tissues is relatively low, which restricts its application value. Currently, enzymatic catalysis is universally deemed the safest and most efficient method for α-arbutin synthesis. Amylosucrase (ASase), one of the most frequently employed glycosyltransferases, has been extensively reported for α-arbutin synthesis. To discover new resources of amylosucrase (ASase), this study synthesized α-arbutin using low-cost sucrose as a glycosyl donor. Probe sequences were used to identify homologous sequences from different microbial strains in protein databases as candidate ASases. Recombinant plasmids were constructed, and the enzymes were successfully expressed in <i>Escherichia coli</i>, followed by the enzymatic synthesis of α-arbutin. One ASase from <i>Calidithermus terrae</i>, named CtAs, was selected for its effective α-arbutin synthesis. The expression conditions for CtAs were optimized, its enzymatic properties were analyzed, and the conditions for the enzymatic synthesis of α-arbutin were further refined to improve its molar yield. The optimal induction conditions for CtA expression were achieved by adding IPTG at a final concentration of 0.5 mmol/L to LB medium when OD₆₀₀ reached 1.0, followed by an incubation at 20 °C and 200 r/min for 18 h. The optimal temperature and pH for CtAs were found to be 42 °C and 9.5, respectively, with good stability across the pH range of 5.0-12.0. CtAs was activated by Na⁺, K⁺, Mg²⁺, EDTA, methanol, and ethanol, but inhibited by Ca²⁺, Zn²⁺, Ba²⁺, and Ni²⁺. The kinetic parameters were <i>V_max</i> = 6.94 μmol/min/mL, <i>K_m</i> = 89.39 mmol/L, <i>K_cat</i> = 5183.97 min^-1, and <i>K_cat</i>/<i>K_m</i> = 57.99 L/(mmol·min). At 42 °C and pH 9.5, the hydrolysis/polymerization/isomerization reaction ratios were 23.27:32.96:43.77 with low sucrose concentrations and 38.50:37.12:24.38 with high sucrose concentrations. The optimal conditions for the enzymatic synthesis were determined to be at 25 °C and pH 5.0 using sucrose at a final concentration of 42 mmol/L and hydroquinone at 6 mmol/L (donor-to-acceptor ratio of 7:1), with the addition of 200 μL (0.2 mg/mL) of purified enzyme and 0.10 mmol/L ascorbic acid, under dark conditions for 6 h. The final molar yield of α-arbutin was 62.78%, with a molar conversion rate of hydroquinone of 74.60%, nearly doubling the yield compared to pre-optimization.