National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
Int J Biol Macromol. 2023 Dec 31;253(Pt 6):127348. doi: 10.1016/j.ijbiomac.2023.127348. Epub 2023 Oct 10.
The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a "best" engineered variant MwTA, which exhibited 79.2-fold higher activity than the chimeric scaffold MwTA. Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates.
（R）-ω-转氨酶作为手性胺合成的生物催化剂的应用受到立体选择性和底物谱狭窄的限制。本文提出了一种有效的（R）-ω-转氨酶设计策略，用于在手性胺合成中应用于西他列汀中间体的不对称合成。由于天然转氨酶对大体积前西他列汀酮缺乏活性，因此根据底物游走原理，首先筛选了具有催化机制和对截断前西他列汀酮活性的转氨酶支架。通过基序交换同步建立转氨酶嵌合体，同时赋予对前西他列汀酮的催化活性和（R）选择性，然后进行逐步进化。该过程产生了“最佳”工程变体 MwTA，其活性比嵌合支架 MwTA 高 79.2 倍。结构分析表明，这种增强的活性主要归因于扩大和适应性的底物口袋和隧道。新型（R）-转氨酶的工业操作稳定性不理想，预计进一步修饰该蛋白以提高其对温度、pH 和有机溶剂的耐受性，以满足可持续的工业需求。本研究强调了一种有用的工程生物催化剂的进化策略，为合成高价值药物中间体的酶赋予新的性质和功能。
