Introduction of a stabilizing 10 residue beta-hairpin in Bacillus subtilis neutral protease.
A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4 degree C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1 degrees C was achieved.
Eijsink
V G
VG
Department of Molecular Genetics, University of Groningen, Haren, The Netherlands.
Vriend
G
G
van den Burg
B
B
van der Zee
J R
JR
Veltman
O R
OR
Stulp
B K
BK
Venema
G
G
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005818:Genetic Engineering; D006860:Hydrogen Bonding; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013499:Surface Properties
D008666:Metalloendopeptidases; C044194:microbial metalloproteinases