Engineering an enzyme to resist boiling.
In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.
Van den Burg
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B
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren The Netherlands. burgb@biol.rug.nl
Vriend
G
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Veltman
O R
OR
Venema
G
G
Eijsink
V G
VG
D019943:Amino Acid Substitution; D003198:Computer Simulation; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D013379:Substrate Specificity; D013816:Thermodynamics; D013820:Thermolysin / Q000096:biosynthesis / Q000737:chemistry
D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; D013820:Thermolysin