High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase
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High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase Lei Gong 1 & Guochao Xu 1 & Xudong Cao 1 & Ruizhi Han 1 & Jinjun Dong 1 & Ye Ni 1 Received: 26 June 2020 / Accepted: 29 September 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4dinitrophenylhydrazine (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. Sitedirected 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 AxDTAD321C and AxDTAN101G 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. Keywords 2,4-Dinitrophenylhydrazine . D-threonine aldolase . Site-directed saturation mutagenesis . High-throughput screening . Substrate specificity
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003447-y) contains supplementary material, which is available to authorized users.
* Guochao Xu [email protected] * Ye Ni [email protected]
1
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122 Jiangsu, China
Applied Biochemistry and Biotechnology
Introduction The formation of carbon-carbon (C–C) bonds is one of the key reactions for organic synthesis to construct carbon backbone by connecting smaller substructures to obtain more sophisticated molecules [1]. Among all C–C formation reactions, the aldol condensation between aldehyde groups is the most common reaction in organic chemistry [2, 3]. Various natural aldolases have been reported with activity in the reversible and stereoselective addition of an affinity donor to an electrophilic acceptor such as aldehydes and amino acids [4, 5]. Threonine aldolases (TAs) are pyridoxal 5′-phosphate (PLP)-dependent aldolases with versatile performance. It has been reported that TAs display great potential in catalyzing enantio- and diastereoselective reversible addition of glycine as donor with aldehydes acceptor to form βhydroxy-α-amino acid with two new stereogenic centers in a single reaction [6]. Optically active β-hydroxy-α-amino acids have been widely used as key bui
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