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.
尽管ω-转氨酶被认为是手性胺合成中极具工业潜力的生物催化剂，但是由于受限于酶的热不稳定及不利的反应平衡，能直接适用于工业的野生酶极少。为了发现工业适应潜力的新型ω-转氨酶，文中设计了包括底物、序列、克隆、酶活、转化率及酶学的工业适应性选择流程，并对各筛选步骤中存在的问题进行了研究，进而从土壤宏基因组中筛选出了一种来源于柄杆菌属的新型ω-转氨酶ATA-W12，以异丙胺为供体在1 mL 反应体系中转化了85.84% 1-Boc-3-吡咯烷酮 (20 mmol/L) 和67.42% 1-Boc-3-哌啶酮 (20 mmol/L)。酶学筛选发现，ATA-W12在40 ℃孵育168 h 活力维持不变；优选反应条件为pH 8.5、40 ℃；这些特征利于工业用理想氨基供体异丙胺的使用。笔者已采用ATA-W12 实现了100 g/L 光学纯(S)-(+)-1-Boc-3-氨基哌啶的50 mL 实验室规模制备，为进一步工业化生产打下基础。.
Keywords: adaptive selection; chiral amine synthesis; isopropylamine; ω-transaminase.