Wang Jiguo, Xie Youyu, Wang Hualei, Wei Dongzhi
School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Sep 25;36(9):1929-1938. doi: 10.13345/j.cjb.200033.
ω-transaminase (ω-TA) is the most promising biocatalyst for chiral amine synthesis. However, most wild-type ω-TAs cannot be applied in industry directly due to their low stability and unfavorable reaction equilibrium. In order to discover a novel ω-TA for industrial application, we designed a procedure of adaptive selection, including the screening of substrates, protein sequences and clones, enzyme activity, and product conversion and characterization, as well as trouble-shooting of each step. Through this procedure, we screened a novel ω-TA, ATA-W12 of Caulobacter sp. from a soil metagenome. The strain could convert 20 mmol/L 1-Boc-3-pyrrolidinone and 20 mmol/L 1-Boc-3-piperidone with 85.84% and 67.42% conversion rate, respectively, in a 1-mL scale with isopropylamine (IPA) as amine donor. ATA-W12 maintained 100% activity at 40 °C for 168 h, and its optimal reaction condition is at pH 8.5 and 40 °C. These excellent properties benefit the application of IPA as an ideal amino donor in industry. We scaled up the production of (S)-(+)-1-boc-3-aminopiperidine up to 50 mL (100 g/L) scale with this novel biocatalyst for its further industrial application.
ω-转氨酶（ω-TA）是用于手性胺合成最具前景的生物催化剂。然而，大多数野生型ω-TA由于其稳定性低和反应平衡不理想而无法直接应用于工业。为了发现一种适用于工业应用的新型ω-TA，我们设计了一个适应性筛选程序，包括底物、蛋白质序列和克隆的筛选、酶活性、产物转化与表征，以及每一步的故障排除。通过这个程序，我们从土壤宏基因组中筛选出一种新型ω-TA，即柄杆菌属的ATA-W12。在1 mL规模下，以异丙胺（IPA）作为胺供体时，该菌株能够分别以85.84%和67.42%的转化率转化20 mmol/L的1-Boc-3-吡咯烷酮和20 mmol/L的1-Boc-3-哌啶酮。ATA-W12在40 °C下168小时保持100%的活性，其最佳反应条件是pH 8.5和40 °C。这些优异的特性有利于IPA作为理想氨基供体在工业中的应用。我们使用这种新型生物催化剂将（S）-（+）-1-叔丁氧羰基-3-氨基哌啶的生产规模扩大到50 mL（100 g/L），以用于其进一步的工业应用。
