Thermostabilization of an esterase by alignment-guided focussed directed evolution.
Site-saturation libraries of the Pseudomonas fluorescens esterase were created targeting three surface positions to increase its thermostability on the basis of the B-factor iterative test principle. All three positions were saturated simultaneously using our recently developed protocol for the design of 'small, but smart' mutant libraries bearing only consensus-like mutations. Hence, the library size could be significantly reduced while ensuring a high hit rate. Variants could be identified that showed significantly improved stability (8° C higher compared with the wild type) without compromising specific activity. Subsequent iterative saturation mutagenesis gave an esterase mutant with a 9° C increased melting point, but unchanged catalytic properties.
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany.
Helge
H
Jochens
Dirk
D
Aerts
Uwe T
UT
Bornscheuer
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution; D004795:Enzyme Stability / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011551:Pseudomonas fluorescens / Q000201:enzymology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013696:Temperature
D001426:Bacterial Proteins; D004950:Esterases