PDF(Pubmed)
Abstract：
In the last decades, biocatalysis has offered new perspectives for the synthesis of (chiral) amines, which are essential building blocks for pharmaceuticals, fine and bulk chemicals. In this regard, amidases have been employed due to their broad substrate scope and their independence from expensive cofactors. To expand the repertoire of amidases, tools for their rapid identification and characterization are greatly demanded. In this work an ultra-high throughput growth selection assay based on the production of the folate precursor p-aminobenzoic acid (PABA) is introduced to identify amidase activity. PABA-derived amides structurally mimic the broad class of commonly used chromogenic substrates derived from p-nitroaniline. This suggests that the assay should be broadly applicable for the identification of amidases. Unlike conventional growth selection assays that rely on substrates as nitrogen or carbon source, our approach requires PABA in sub-nanomolar concentrations, making it exceptionally sensitive and ideal for engineering campaigns that aim at enhancing amidase activities from minimally active starting points, for example. The presented assay offers flexibility in the adjustment of sensitivity to suit project-specific needs using different expression systems and fine-tuning with the antimetabolite sulfathiazole. Application of this PABA-based assay facilitates the screening of millions of enzyme variants on a single agar plate within two days, without the need for laborious sample preparation or expensive instruments, with transformation efficiency being the only limiting factor. KEY POINTS: • Ultra-high throughput assay (tens of millions on one agar plate) for amidase screening • High sensitivity by coupling selection to folate instead of carbon or nitrogen source • Highly adjustable in terms of sensitivity and expression of the engineering target.
摘要：
在过去的几十年里，生物催化为（手性）胺的合成提供了新的视角，这是制药的重要组成部分，精细和散装化学品。在这方面,酰胺酶由于其广泛的底物范围及其与昂贵的辅因子的独立性而被采用。为了扩大酰胺酶的范围，他们的快速识别和表征工具是非常需要的。在这项工作中，引入了基于叶酸前体对氨基苯甲酸（PABA）生产的超高通量生长选择测定法，以鉴定酰胺酶活性。PABA衍生的酰胺在结构上模拟了通常使用的衍生自对硝基苯胺的显色底物的广泛类别。这表明该测定法应广泛适用于酰胺酶的鉴定。与依赖底物作为氮源或碳源的常规生长选择测定不同，我们的方法需要亚纳米摩尔浓度的PABA，使其非常敏感和理想的工程活动，旨在提高酰胺酶的活性从最低活性的起点，例如。所提出的测定法在使用不同的表达系统和使用抗代谢物磺胺噻唑进行微调时，可以灵活地调整灵敏度以适应项目特定的需求。这种基于PABA的测定的应用有助于在两天内在单个琼脂平板上筛选数百万个酶变体。无需费力的样品制备或昂贵的仪器，转化效率是唯一的限制因素。关键点:•用于酰胺酶筛选的超高通量测定(在一个琼脂平板上数千万)•通过将选择偶联至叶酸而不是碳或氮源的高灵敏度•在工程靶标的灵敏度和表达方面高度可调。
