文献类型：期刊文献

中文题名：一种新型ω-转氨酶的工业适应性选择

英文题名：Industrial adaptability selection for a novel ω-transaminase

作者：王继国[1];谢有余[1];王华磊[1];魏东芝[1]

机构：[1]华东理工大学生物工程学院,上海200237

年份：2020

卷号：36

期号：9

起止页码：1929

中文期刊名：生物工程学报

外文期刊名：Chinese Journal of Biotechnology

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD2019_2020】；PubMed;

基金：国家自然科学基金(No.21776084/B060804)资助。

语种：中文

中文关键词：手性胺合成;ω-转氨酶;异丙胺;适应性选择

外文关键词：chiral amine synthesis;ω-transaminase;isopropylamine;adaptive selection

摘要：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1mL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100g/L光学纯(S)-(+)-1-Boc-3-氨基哌啶的50mL实验室规模制备,为进一步工业化生产打下基础。
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-m L scale with isopropylamine(IPA) as amine donor. ATA-W12 maintained 100% activity at 40 ℃ for 168 h, and its optimal reaction condition is at pH 8.5 and 40 ℃. 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.