Patterns of adaptation in a laboratory evolved thermophilic enzyme.
The heat sensitive psychrophilic protease subtilisin S41 was previously subjected to three rounds of mutagenesis/recombination and screening, resulting in variant 3-2G7, whose half-life at 60 degrees C is approx. 500 times that of wild-type. Here we report the results of five additional generations of laboratory evolution starting from 3-2G7. The half-life of 8th generation enzyme 8-4A9 at 60 degrees C is 1200 times that of wild-type, and slightly more than twice that of 3-2G7. This half-life is >20-fold greater than those of homologous mesophilic subtilisins SSII and BPN'. Circular dichroism melting curves indicate that subtilisin 8-4A9 unfolds at temperatures approx. 25 degrees C higher than wild-type. It is also substantially more resistant to proteolysis at 30 degrees C. Nearly half of the 13 amino acid substitutions accumulated in 8-4A9 involve the mutation of serine residues. This mirrors a pattern observed in natural proteins, where serines are statistically less prevalent in thermophilic enzymes compared to mesophilic ones.
Wintrode
P L
PL
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, USA.
Miyazaki
K
K
Arnold
F H
FH
D000596:Amino Acids / Q000737:chemistry; D002918:Chymotrypsin; D002942:Circular Dichroism; D004795:Enzyme Stability; D006207:Half-Life; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics*; D013696:Temperature; D014357:Trypsin
D000596:Amino Acids; D002918:Chymotrypsin; D014357:Trypsin; D020860:Subtilisin