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

• Published in: Protein engineering, design and selection design and selection, 2004-02, Vol.17 (2), p.133-140
• Main Authors: Suen, Wen‐Chen, Zhang, Ningyan, Xiao, Li, Madison, Vincent, Zaks, Aleksey
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Candida antarctica; Candida antarctica lipase B/enantioselectivity/family shuffling/lipase/thermostability; Chlorobenzenes - chemistry; Chlorobenzenes - metabolism; Cloning, Molecular; Cryptococcus - enzymology; Cryptococcus - genetics; Cryptococcus tsukubaensis; DNA, Fungal - genetics; Enzyme Activation; Enzyme Stability; Fungal Proteins; Gene Expression Regulation, Enzymologic; Gene Library; Glutarates - chemistry; Glutarates - metabolism; Hydrolysis; Hyphozyma; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Mitosporic Fungi - enzymology; Mitosporic Fungi - genetics; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Engineering - methods; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Analysis; Sequence Homology, Amino Acid; Substrate Specificity
• ISSN: 1741-0126; 1741-0134
• DOI: 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 C.antarctica ATCC 32657 and a 13‐fold higher activity than the most active parent derived from C.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 C.antarctica ATCC 32657 by 11‐fold and 6.4°C, respectively, which closely approached the stability characteristics of the most thermostable parent derived from Hyphozyma sp. CBS 648.91.