Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000, Ljubljana, Slovenia.
c-LEcta GmbH, Perlickstraße 5, 04103, Leipzig, Germany.
N Biotechnol. 2018 Dec 25;47:18-24. doi: 10.1016/j.nbt.2018.05.004. Epub 2018 May 18.
The industrial importance of optically pure compounds has thrown a spotlight on ω-transaminases that have shown a high potential for the synthesis of bioactive compounds with a chiral amine moiety. The implementation of biocatalysts in industrial processes relies strongly on fast and cost effective process development, including selection of a biocatalyst form and the strategy for its immobilization. Here, microscale reactors with selected surface-immobilized amine-transaminase (ATA) in various forms are described as platforms for high-throughput process development. Wild type ATA (ATA-wt) from a crude cell extract, as well as Escherichia coli cells intracellularly overexpressing the enzyme, were immobilized on the surfaces of meander microchannels of disposable plastics by means of reactor surface silanization and glutaraldehyde bonding. In addition, a silicon/glass microchannel reactor was used for immobilization of an ATA-wt, genetically engineered to contain a silica-binding module (SBM) at the N-terminus (N-SBM-ATA-wt), leading to immobilization on the non-modified inner microchannel surface. Microreactors with surface-immobilized biocatalysts were coupled with a quenching system and at-line HPLC analytics and evaluated based on continuous biotransformation, yielding acetophenone and l-alanine. E. coli cells and N-SBM-ATA-wt were efficiently immobilized and yielded a volumetric productivity of up to 14.42 g L h, while ATA-wt small load resulted in two orders of magnitude lower productivity. The miniaturized reactors further enabled in-operando characterization of biocatalyst stability, crucial for successful transfer to a production scale.
具有光学纯化合物的工业重要性使人们对 ω-转氨酶产生了浓厚的兴趣，这些酶在合成具有手性胺部分的生物活性化合物方面显示出了很高的潜力。生物催化剂在工业过程中的应用强烈依赖于快速且具有成本效益的工艺开发，包括选择生物催化剂形式及其固定化策略。在这里，描述了具有各种形式的表面固定化胺转氨酶（ATA）的微尺度反应器作为高通量工艺开发的平台。野生型 ATA（ATA-wt）来自粗细胞提取物，以及在细胞内过表达该酶的大肠杆菌细胞，通过反应器表面硅烷化和戊二醛键合固定在一次性塑料的曲折微通道表面上。此外，使用硅/玻璃微通道反应器固定化 ATA-wt，该酶经过基因工程改造，在 N 端含有硅结合模块（SBM）（N-SBM-ATA-wt），导致固定在未经修饰的内微通道表面上。表面固定化生物催化剂的微反应器与淬灭系统和在线 HPLC 分析相结合，并根据连续生物转化进行评估，生成苯乙酮和 l-丙氨酸。大肠杆菌细胞和 N-SBM-ATA-wt 被有效地固定化，产率高达 14.42 g L h，而 ATA-wt 小负载导致产率低两个数量级。这些小型化反应器还能够在操作过程中对生物催化剂稳定性进行表征，这对于成功转移到生产规模至关重要。
