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Updated: Mar 12, 2026

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
Published on: August 8, 2016
Protein engineering of aldolase LbDERA for enhanced activity toward real substrates with a high-throughput screening
Xue-Cheng Jiao1, Jiang Pan1, Xu-Dong Kong1
1Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Abstract:
A new high-throughput method for screening 2-deoxyribose-5-phosphate aldolase variants with a higher activity toward aldol reaction of unnatural aldehydes was established for the first time by coupling with an aldehyde dehydrogenase LeADH. The error-prone PCR and site-directed saturation mutagenesis libraries of aldolase LbDERA were constructed and screened using the high-throughput method. Two improved variants, LbDERAT29L and LbDERAF163Y, were identified and combined, giving a double mutant LbDERAT29L/F163Y which showed 7-fold higher activity than the native enzyme. The crystal structure of LbDERAT29L/163Y obtained by X-ray diffraction with 1.77 Å resolution revealed the structural changes responsible for the significant activity improvement.
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