A number of enzymes important for biocatalyst development or as drug targets are associated with a pH shift during their catalytic reaction, owing to the concommitant release or uptake of protons. Here, we show that an enzyme assay developed using the fluorescent pH indicator HPTS can be adapted for reliable and continuous activity determination of representative enzymes from multiple EC classes that operate in the viable pH range 5.5–8.5, using ratiometric measurement (F₄₈₅/F₄₀₅). Kinetic measurements obtained with this method closely match literature values determined using other assay types. Further, the assay was employed to screen variants of transketolase from Geobacillus stearothermophilus (TK_gst) aimed at engineering substrate promiscuity and remote enantioselectivity for 3-hydroxyaldehydes. The fluorescence-based assay displayed 70-fold improved sensitivity in comparison to an absorption-based assay for transketolase screening, with a limit of detection of 0.044 mM and Z-factor of 0.52. Double-site mutagenesis at the G264 and S385 positions yielded variants with 5–15-fold increased activity on the tested 3-hydroxyaldehydes compared to the TK_gst (L382F) base variant. Although the directed evolution engineering strategy did not achieve significant remote enantioselectivity in this first round of mutagenesis, the simple fluorescence-based pH-shift assay was shown to be useful as a versatile primary high-throughput screen for in vitro enzyme engineering.

中文翻译：

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基于荧光的 pH 变化测定具有广泛的应用范围，可用于酶采矿和工程中酶活性的高通量测定


许多对于生物催化剂开发或作为药物靶点很重要的酶在催化反应过程中由于质子的伴随释放或摄取而与 pH 值变化相关。在这里，我们展示了使用荧光 pH 指示剂 HPTS 开发的酶测定法，可以使用比率测量 ( F ₄₈₅ / F ₄₀₅ ）。用这种方法获得的动力学测量结果与使用其他测定类型确定的文献值非常匹配。此外，该测定还用于筛选来自嗜热脂肪地芽孢杆菌(TK _gst ) 的转酮酶变体，旨在设计底物混杂性和 3-羟基醛的远程对映选择性。与基于吸收的转酮醇酶筛选检测相比，基于荧光的检测灵敏度提高了 70 倍，检测限为 0.044 mM， Z因子为 0.52。与 TK _gst (L382F) 碱基变体相比，G264 和 S385 位点的双位点诱变产生的变体对测试的 3-羟基醛的活性增加了 5-15 倍。尽管定向进化工程策略在第一轮诱变中没有实现显着的远程对映选择性，但简单的基于荧光的pH变化测定被证明可用作体外酶工程的多功能初级高通量筛选。