Abstract：
Recent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy-intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet-based microfluidic systems have emerged as a powerful high-throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet-based microfluidic systems, we present an absorbance-activated droplet sorter that allows of droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105-member aldehyde dehydrogenase library towards D-glyceraldehyde using a NADH mediated coupled assay that generates WST-1 formazan as the colorimetric product. We successfully identify a variant with a 51% improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.
摘要：
近几十年来，生物催化剂的商业用途急剧增加，从能源密集型传统化学品向更可持续的方法过渡。当前的酶工程技术，比如定向进化，需要生成和测试大型突变文库以鉴定优化的变体。不幸的是,传统的筛选方法无法以稳健和及时的方式筛选这样的大型图书馆。基于液滴的微流体系统已经成为一种强大的高通量工具，用于以千赫速率进行文库筛选。不幸的是,几乎所有报道的系统都基于荧光检测，将其使用限制在有限数量的天然转化荧光底物或需要使用替代底物的酶类型。为了扩大使用基于液滴的微流体系统进化的酶的范围，我们提出了一种吸光度激活的液滴分选器，它允许以千赫速率进行液滴分选，而无需对微流体系统进行光学监测。为了证明分拣机的实用性，我们使用NADH介导的偶联测定法快速筛选了一个105个成员的醛脱氢酶文库，该测定法产生WST-1甲作为比色产物。我们成功地鉴定了一种变体,其催化效率提高了51%,并且在宽底物谱上的总体活性显著增加。
