Li Anqi, He Yamei, Chen Wenxuan, Tao Huimei, Wu Huawei, Li Shaobin
College of Life Sciences, Yangtze University, 1 South-Loop Road, Jingzhou 434025, China.
Int J Mol Sci. 2024 Dec 12;25(24):13359. doi: 10.3390/ijms252413359.
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 , followed by the enzymatic synthesis of α-arbutin. One ASase from , 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 OD 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, Mg, EDTA, methanol, and ethanol, but inhibited by Ca, Zn, Ba, and Ni. The kinetic parameters were = 6.94 μmol/min/mL, = 89.39 mmol/L, = 5183.97 min, and / = 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.
α - 熊果苷是化妆品领域的第四代美白因子，它能够阻断表皮细胞中黑色素的合成，具有稳定性好、毒副作用小的优点。此外，α - 熊果苷在食品、医药等领域也具有潜在的应用价值。然而，从植物组织中提取的产率相对较低，这限制了其应用价值。目前，酶催化被普遍认为是合成α - 熊果苷最安全、最有效的方法。淀粉蔗糖酶（ASase）是最常用的糖基转移酶之一，已被广泛报道用于α - 熊果苷的合成。为了发现淀粉蔗糖酶（ASase）的新来源，本研究以低成本的蔗糖作为糖基供体合成α - 熊果苷。使用探针序列从蛋白质数据库中的不同微生物菌株中鉴定同源序列作为候选ASase。构建重组质粒，并在[具体宿主]中成功表达这些酶，随后进行α - 熊果苷的酶促合成。从[具体来源]中筛选出一种名为CtAs的ASase，因其能有效合成α - 熊果苷。对CtAs的表达条件进行了优化，分析了其酶学性质，并进一步优化了α - 熊果苷酶促合成的条件以提高其摩尔产率。CtA表达的最佳诱导条件是当OD达到1.0时，向LB培养基中加入终浓度为0.5 mmol/L的IPTG，然后在20°C和200 r/min下孵育18 h。发现CtAs的最佳温度和pH分别为42°C和9.5，在pH 5.0 - 12.0范围内具有良好的稳定性。CtAs被Na、K、Mg、EDTA、甲醇和乙醇激活，但被Ca、Zn、Ba和Ni抑制。动力学参数为Vmax = 6.94 μmol/min/mL，Km = 89.39 mmol/L，kcat = 5183.97 min，kcat/Km = 57.99 L/(mmol·min)。在42°C和pH 9.5条件下，低蔗糖浓度时水解/聚合/异构化反应比率为23.27:32.96:43.77，高蔗糖浓度时为38.50:37.12:24.38。酶促合成的最佳条件确定为在25°C和pH 5.0下，使用终浓度为42 mmol/L的蔗糖和6 mmol/L的对苯二酚（供体与受体比例为7:1），加入200 μL（0.2 mg/mL）纯化酶和0.10 mmol/L抗坏血酸，在黑暗条件下反应6 h。α - 熊果苷的最终摩尔产率为62.78%，对苯二酚的摩尔转化率为74.60%，与优化前相比产率几乎提高了一倍。
