Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling
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 245...
| Published in: | Protein Engineering Design and Selection |
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| Main Authors: | , , , , |
| Format: | Text |
| Language: | English |
| Published: |
Oxford University Press
2004
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| Subjects: | |
| Online Access: | http://peds.oxfordjournals.org/cgi/content/short/gzh017v1 https://doi.org/10.1093/protein/gzh017 |
| Summary: | 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 |
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