State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, China.
Appl Microbiol Biotechnol. 2012 Jan;93(1):143-50. doi: 10.1007/s00253-011-3444-7. Epub 2011 Jul 14.
Biocatalytic acylation of 1-β-D-arabinofuranosylcytosine (ara-C) was developed using whole cell of Aspergillus oryzae as a novel catalyst. (13)C nuclear magnetic resonance (NMR) analysis indicated that the whole-cell biocatalyst had more specific activity toward the 3'-hydroxyl group than 5'-hydroxyl group among the available hydroxyl groups in sugar moiety of ara-C. Except for glucose and maltose, 11 carbon sources supplemented to basal media, including Spans, Tweens, olive oil and oleic acid, exhibited notable enhancement effects on both the cell growth and the acylation reactions. It was suggested that the carbon sources containing controlled-release oleic acid were the important substrates for the production of fungal cell-bound lipase with specific activity, partially due to a gradual induction effect of their released oleic acid on the cell-bound lipase production. Despite the low initial reaction rate and substrate conversion, the addition of 2.0 g/l Span 80 resulted in a higher 3'-regioselectivity of the cells than 81%. By using Tween 85 at its optimum concentration of 5.0 g/l, however, the highest initial rates (3.2 mmol/l h) and substrate conversion (76%) of the whole-cell catalyzed acylation of ara-C can be achieved. It was also found that the 3'-regioselectivity of the cells showed observable increase by extending the culture time. And the activity of cell-bound lipase drastically increased in the early stage of cell growth and then declined in the late culture stage, whatever the culture media used. Our results thus indicated that A. oryzae whole cell was a promising green tool for biosynthesis of nucleoside esters with potential bioactivities.
利用米曲霉（Aspergillus oryzae）全细胞作为新型催化剂，对 1-β-D-阿拉伯呋喃糖胞嘧啶（ara-C）进行生物催化酰化反应。（13）C 核磁共振（NMR）分析表明，与 ara-C 糖部分中可用的羟基相比，全细胞生物催化剂对 3'-羟基的特异性活性更高。除葡萄糖和麦芽糖外，补充到基础培养基中的 11 种碳源，包括 Span、Tween、橄榄油和油酸，对细胞生长和酰化反应均有显著的促进作用。这表明含有控释油酸的碳源是产生具有特定活性的真菌细胞结合脂肪酶的重要底物，部分原因是其释放的油酸对细胞结合脂肪酶产生的逐渐诱导作用。尽管初始反应速率和底物转化率较低，但添加 2.0 g/L Span 80 会导致细胞的 3'-区域选择性高于 81%。然而，通过使用浓度为 5.0 g/L 的 Tween 85，可实现全细胞催化酰化 ara-C 的初始速率（3.2 mmol/L h）和底物转化率（76%）的最高值。还发现，通过延长培养时间，细胞的 3'-区域选择性可明显增加。无论使用何种培养基，细胞结合脂肪酶的活性在细胞生长的早期阶段急剧增加，然后在后期培养阶段下降。因此，我们的研究结果表明，米曲霉全细胞是一种很有前途的绿色工具，可用于生物合成具有潜在生物活性的核苷酯。
