Alteration of specific activity and stability of thermostable neutral protease by site-directed mutagenesis.
On the basis of three-dimensional information, many amino acid substitutions were introduced in the thermostable neutral protease (NprM) of Bacillus stearothermophilus MK232 by site-directed mutagenesis. When Glu at position 143 (Glu-143), which is one of the proposed active sites, was substituted for by Gln and Asp, the proteolytic activity disappeared. F114A (Phe-114 to Ala), Y110W (Tyr-110 to Trp), and Y211W (Tyr-211 to Trp) mutant enzymes had higher activity (1.3- to 1.6-fold) than the wild-type enzyme. When an autolysis site, Tyr-93, was replaced by Gly and Ser, the remaining activities of those mutant enzymes were higher than that of the wild-type enzyme.
Kubo
M
M
Biotechnology Research Laboratory, TOSOH Corporation, Kanagawa, Japan.
Mitsuda
Y
Y
Takagi
M
M
Imanaka
T
T
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics*; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation
D001426:Bacterial Proteins; D010450:Endopeptidases; C065625:neutral protease, Bacillus stearothermophilus