Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling

• Published in: Protein Engineering Design and Selection
• Main Authors: Suen, Wen-Chen, Zhang, Ningyan, Xiao, Li, Madison, Vincent, Zaks, Aleksey
• Format: Text
• Language: English
• Published: Oxford University Press 2004
• Subjects: Article; Antarc*; Antarctica
• Online Access: http://peds.oxfordjournals.org/cgi/content/short/gzh017v1; https://doi.org/10.1093/protein/gzh017

DNA family shuffling was used to create chimeric lipase B proteins with improved activity toward the hydrolysis of diethyl 3-[3', 4'-dichlorophenyl]-glutarate (DDG). Three homologous lipases from Candida antarctica ATCC 32657, Hyphozyma sp. CBS 648.91, and Crytococcus tsukubaensis ATCC 24555 were cloned and shuffled to generate a diverse gene library. A high-throughput screening assay was developed and used successfully to identify chimeric lipase B proteins having a 20-fold higher activity toward DDG than lipase B from Candida antarctica ATCC 32657 and a 13-fold higher activity than the most active parent derived from Crytococcus tsukubaensis ATCC 24555. In addition, the stability characteristics of several highly active chimeric proteins were also improved as a result of family shuffling. For example, the half life at 45°C and melting point (Tm) of one chimera exceeded those of lipase B from Candida antarctica ATCC 32657 by 11-fold and 6.4°C, which closely approached the stability characteristics of the most thermostable parent derived from Hyphozyma sp. CBS 648.91