高通量筛选定向进化和 D-苏氨酸醛缩酶醛醇活性表征的方法。
High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase.
机构信息
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
出版信息
Appl Biochem Biotechnol. 2021 Feb;193(2):417-429. doi: 10.1007/s12010-020-03447-y. Epub 2020 Oct 5.
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 and AxDTA 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方法，用于测定苏氨酸醛缩酶（TAs）对醛和甘氨酸的醛缩合活性。这种 2,4-二硝基苯肼（DNPH）方法具有高灵敏度和低背景干扰，可用于 TAs 的高通量筛选和表征的分光光度法测量。对于 4-甲基亚磺酰基苯甲醛（MSB），最大吸收峰出现在约 485nm。利用该 DNPH 方法，对来自 Alcaligenes xylosoxidans CGMCC 1.4257（AxDTA）的 D-苏氨酸醛缩酶进行了定点饱和突变文库的构建和筛选，以提高对 MSB 的醛缩合活性。鉴定出两个有益的变体 AxDTA 和 AxDTA。利用该 DNPH 方法，轻松地对 AxDTA 和变体对具有不同取代基的 19 种醛的底物特异性进行了表征。此外，AxDTA 变体在醛缩反应中表现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 TAs 的方法，该方法在制备手性β-羟基-α-氨基酸方面具有潜在应用。
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