Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 °C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 °C and a shift of 10 °C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29¿42 °C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Gatti Lafranconi, P., Caldarazzo, S., Villa, A., Alberghina, L., Lotti, M. (2008). Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase. FEBS LETTERS, 582(15), 2313-2318 [10.1016/j.febslet.2008.05.037].
Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase
ALBERGHINA, LILIA;LOTTI, MARINA
2008
Abstract
Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 °C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 °C and a shift of 10 °C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29¿42 °C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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