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 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 degrees C and melting point (T(m)) of one chimera exceeded those of lipase B from C.antarctica ATCC 32657 by 11-fold and 6.4 degrees C, respectively, which closely approached the stability characteristics of the most thermostable parent derived from Hyphozyma sp. CBS 648.91.
Suen
Wen-Chen
WC
Biotransformations Group, Schering-Plough Research Institute, Union, NJ 07083, USA. wen-chen.suen@spcorp.com
Zhang
Ningyan
N
Xiao
Li
L
Madison
Vincent
V
Zaks
Aleksey
A
D000595:Amino Acid Sequence; D002722:Chlorobenzenes / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D003454:Cryptococcus / Q000201:enzymology / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D005656:Fungal Proteins; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005977:Glutarates / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003904:Mitosporic Fungi / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002722:Chlorobenzenes; D004271:DNA, Fungal; D005656:Fungal Proteins; D005977:Glutarates; D011994:Recombinant Proteins; C494125:diethyl 3-(3',4'-dichlorophenyl)glutarate; D008049:Lipase; C431349:lipase B, Candida antarctica