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Enzyme Optimization and Process Development for a Scalable Synthesis of (R)-2-Methoxymandelic Acid
Organic Process Research & Development ( IF 3.1 ) Pub Date : 2021-08-12 , DOI: 10.1021/acs.oprd.1c00250 Mark E. Scott 1 , Xiaotian Wang 1 , Luke D. Humphreys 1 , Michael J. Geier 1 , Balamurali Kannan 1 , Johann Chan 2 , Gareth Brown 3 , Daniel F. A. R. Dourado 3 , Darren Gray 3 , Stefan Mix 3 , Aliaksei Pukin 3
Organic Process Research & Development ( IF 3.1 ) Pub Date : 2021-08-12 , DOI: 10.1021/acs.oprd.1c00250 Mark E. Scott 1 , Xiaotian Wang 1 , Luke D. Humphreys 1 , Michael J. Geier 1 , Balamurali Kannan 1 , Johann Chan 2 , Gareth Brown 3 , Daniel F. A. R. Dourado 3 , Darren Gray 3 , Stefan Mix 3 , Aliaksei Pukin 3
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The rational protein engineering of wild type BCJ2315 nitrilase and its use in the development of a one-pot, enantioselective, dynamic kinetic resolution for (R)-2-methoxymandelic acid is reported. Through a combination of molecular docking and B-factor analyses, a focused library of mutants was identified and screened to improve nitrilase selectivity, activity, and stability. Initial optimization revealed that the addition of sodium bisulfite prevented enzyme deactivation by the aldehyde starting material, removing the need to isolate the cyanohydrin and allowing for development of a one-pot process for mutant screening. Further optimization of the process with the preferred mutants revealed subtle interactions between temperature, pH, substrate loading, and enzyme source which ultimately led to development of a suitable lyophilized whole cell process for scale-up, affording (R)-2-methoxymandelic acid in 97% ee and 70% isolated yield on multigram scale.
中文翻译：
(R)-2-甲氧基扁桃酸的可扩展合成的酶优化和工艺开发
报道了野生型 BCJ2315 腈水解酶的合理蛋白质工程及其在 ( R )-2-甲氧基扁桃酸的一锅法、对映选择性、动态动力学拆分中的应用。通过分子对接和B的结合因子分析，确定并筛选了一个集中的突变体库，以提高腈水解酶的选择性、活性和稳定性。最初的优化表明，亚硫酸氢钠的添加防止了醛起始材料对酶的失活，消除了分离氰醇的需要，并允许开发用于突变体筛选的一锅法。使用优选突变体进一步优化该过程揭示了温度、pH、底物负载和酶源之间的微妙相互作用，这最终导致开发了适合放大的冻干全细胞过程，在97% ee 和 70% 多克规模的分离产量。
更新日期：2021-08-12
中文翻译：
(R)-2-甲氧基扁桃酸的可扩展合成的酶优化和工艺开发
报道了野生型 BCJ2315 腈水解酶的合理蛋白质工程及其在 ( R )-2-甲氧基扁桃酸的一锅法、对映选择性、动态动力学拆分中的应用。通过分子对接和B的结合因子分析，确定并筛选了一个集中的突变体库，以提高腈水解酶的选择性、活性和稳定性。最初的优化表明，亚硫酸氢钠的添加防止了醛起始材料对酶的失活，消除了分离氰醇的需要，并允许开发用于突变体筛选的一锅法。使用优选突变体进一步优化该过程揭示了温度、pH、底物负载和酶源之间的微妙相互作用，这最终导致开发了适合放大的冻干全细胞过程，在97% ee 和 70% 多克规模的分离产量。
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