A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485 nm. Site-directed saturation mutagenesis libraries of d-threonine aldolase from Alcaligenes xylosoxidans CGMCC 1.4257 (AxDTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants AxDTA_D321C and AxDTA_N101G were identified. Substrate specificity of AxDTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore, AxDTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral β-hydroxy-α-amino acids.

建立了一种快速，可靠的苏氨酸醛缩酶（TAs）对醛和甘氨酸醛缩反应活性测定方法。这种2,4-二硝基苯肼（DNPH）方法具有高灵敏度和低背景干扰，可以通过分光光度法进行测量，以用于TA的高通量筛选和表征。对于4-甲基磺酰基苯甲醛（MSB），在约485 nm处观察到最大吸收峰。构建了来自木杯产碱霉（Alcaligenes xylosoxidans） CGMCC 1.4257（Ax DTA）的d-苏氨酸醛缩酶的定点饱和诱变文库，并使用该DNPH方法筛选了针对MSB的醛醇活性增加。两个有益的变体Ax DTA _D321C和确定了斧头DTA _N101G。使用这种DNPH方法可以轻松地表征Ax DTA及其变体对19个具有不同取代基的醛的底物特异性。此外，Ax DTA变体在醛醇缩合反应中显示出增强的催化性能和选择性。因此，我们的研究提供了一种快速筛选和表征TA的方法，在制备手性β-羟基-α-氨基酸方面具有潜在的应用前景。