基于酮基荧光探针的转氨酶高通量筛选及应用

Transaminases: high-throughput screening via a ketone-fluorescent probe and applications

摘要:

转氨酶是一类催化氨基酸和酮酸之间氨基转移反应的酶，在有机胺及其衍生物的合成中具有重要作用。然而天然酶通常对非天然底物催化效率低，限制了其广泛应用。对转氨酶的蛋白质改造可有效提升其催化性能，从而拓宽应用范围。本研究基于对甲氧基-2-氨基芐胺肟(para-methoxy-2-amino benzamidoxime,PMA)与酮类化合物之间产生荧光显色反应从而能够有效监测酮类化合物浓度变化的机制，建立了适用于转氨酶活性的高通量筛选方法，通过体系的系统优化，显著提升了筛选方法的高效性、灵敏性和精确性，以4-羟基-2-丁酮为底物，构建了放线菌(Actinobacteria sp.)来源的转氨酶突变文库，成功获得了活力提升的突变体，提高了(R)-3-氨基丁醇的酶法合成效率。本研究为高效筛选、改造转氨酶并实现其应用奠定了重要基础。

Abstract:

Transaminases are a class of enzymes that catalyze the transfer of amino between amino acids and keto acids, playing an important role in the biosynthesis of organic amines and the corresponding derivatives. However, natural enzymes often have low catalytic efficiency against non-natural substrates, which limits their widespread applications. Enzyme engineering serves as an effective approach to improve the catalytic properties and thereby expand the application scope of transaminases. In this study, a high-throughput screening method for transaminases was established based on the fluorescent color reaction between methoxy-2-aminobenzoxime (PMA) and ketones. According to the changes in fluorescence intensity, the concentration changes of ketones could be easily monitored. The efficiency, sensitivity, and accuracy of the screening method were improved by optimization of the system. With 4-hydroxy-2-butanone as the substrate, the mutant library of the transaminase fromActinobacteria sp. was established and a mutant with increased activity was successfully obtained, which improved the production efficiency of (R)-3-aminobutanol by enzyme-catalyzed synthesis. This study laid an important foundation for efficient screening, modification, and application of transaminase.