Biocatalysis and Enzyme Engineering Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
Biocatalysis and Enzyme Engineering Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
Bioorg Chem. 2019 Mar;84:32-40. doi: 10.1016/j.bioorg.2018.11.017. Epub 2018 Nov 19.
Hydroxynitrile lyase (HNL) catalyzed enantioselective CC bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity. We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ∼99% ee and ∼60% conversion. Addition of organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.
羟腈酶（HNL）催化的对映选择性 CC 键形成是合成手性氰醇的有效方法，手性氰醇是合成许多精细化学品、农药和药物的重要构建块。固定化 HNL 已知可提供稳健性、可重复使用性，在某些情况下还可提高活性和选择性。我们通过交联酶聚集体（CLEA）方法优化了 Baliospermium montanum HNL（BmHNL）的固定化制备，并通过 SEM 对其进行了表征。通过 CLEA-BmHNL 从苯甲醛中获得最高（S）-扁桃腈转化率和对映体过量值（ee），对生物催化参数进行了优化。优化的反应参数为：反应时间 20min，CLEA-BmHNL 7U，底物 1.2mM，柠檬酸缓冲液 pH4.2300mM，在此条件下（S）-扁桃腈的转化率约为 60%，ee 值约为 99%。与其他 CLEA-HNL 不同，在 CLEA-BmHNL 生物催化中添加有机溶剂并没有提高产物的对映体过量值（ee）或转化率。CLEA-BmHNL 可成功重复使用 8 个连续循环，转化率或产物形成没有损失，5 个循环后对映选择性略有损失。在最佳生物催化条件下，11 种不同的手性氰醇的合成收率高达 99%，ee 值为 59%，但不同产物的转化率和 ee 值有所不同。与使用纯化的 BmHNL 相比，CLEA-BmHNL 提高了（S）-扁桃腈合成的对映选择性。以前未用 BmHNL 测试的 9 种醛首次使用 CLEA-BmHNL 转化为相应的（S）-氰醇。在本研究报告的 11 种（S）-氰醇合成中，有 8 种不是任何 CLEA-HNL 合成的。总的来说，这项研究展示了稳定、稳健和可重复使用的生物催化剂即 CLEA-BmHNL 的制备、表征及其在手性芳香族氰醇合成中的生物催化应用。
