Abstract:
:Specific activity was compared between wild-type (WT) neutral protease from Bacillus stearothermophilus and mutant protease (M1; Gly144 replaced by Ala144) with enhanced thermostability. When casein was used as a substrate, M1 showed 1.5-times higher specific activity than that of WT. In contrast, the specific activities of M1 for soluble reduced lysozyme and insulin B chain were lower than those of WT by 17.2 and 13.2%, respectively. After digestion of the insulin A chain by these enzymes, the peptide products were purified and the N-terminal amino acid sequences were determined. WT enzyme cleaved insulin A chain at three sites, whereas no digestion was observed with M1. Using Z-Gly-Leu-NH2 as a substrate, the kinetic parameters were determined. The Km values are nearly equal for both enzymes, whereas the kcat of M1 (240 min-1) was much smaller compared to the WT (830 min-1). The data indicate that the mutation (addition of a methyl group) exerts an effect by changing both the catalytic velocity and thermostability.
journal_name
FEBS Lettjournal_title
FEBS lettersauthors
Takagi M,Imanaka Tdoi
10.1016/0014-5793(89)81006-3subject
Has Abstractpub_date
1989-08-28 00:00:00pages
43-6issue
1-2eissn
0014-5793issn
1873-3468pii
0014-5793(89)81006-3journal_volume
254pub_type
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