Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase.
Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees 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 degrees C and a shift of 10 degrees 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 degrees 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.
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, Milan, Italy.
Pietro
P
Gatti-Lafranconi
Serena M
SM
Caldarazzo
Alessandra
A
Villa
Lilia
L
Alberghina
Marina
M
Lotti
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003080:Cold Temperature; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D006358:Hot Temperature; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D044206:Pseudomonas fragi / Q000201:enzymology*
D001426:Bacterial Proteins; D008049:Lipase