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

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... t showed significantly improved stability (88C higher compared with the wild type) without compromising specific activity. Subsequent iterative saturation mutagenesis gave an esterase mutant with a 98C increased melting point, but unchanged catalytic properties.