title: Further stabilization of Leu¹⁵⁵ mutant thermolysins by mutation of an autodegradation site.
abstract: The autodegradation-resistant mutant thermolysins (TLNs), L155A (Leu(155) to Ala) and L155S (Leu(155) to Ser), were previously constructed by site-directed mutagenesis to enhance thermostability. These mutations suppressed autodegradation at position 154-155, resulting in increased thermostability. However, a new autodegradation site became apparent in these mutant TLNs, at position 155-156. In this study, further stabilization of the mutant TLNs to suppress this new autodegradation was attempted by the substitution of Ile(156) to Asp and Val (L155A-I156N, L155A-I156V, L155S-I156N, and L155S-I156V). SDS-PAGE analysis showed that the autodegradation at 155-156 of all double-mutant TLNs was suppressed. Thermostability at 80 °C was enhanced in all double-mutant TLNs (half-life at 80 °C: WT, 18.3 min; L155A, 25.0 min; L155S, 24.0 min; L155A-I156N, 60.8 min; L155A-I156V, 62.4 min; L155S-I156N, 93.3 min; and L155S-I156V, 40.0 min), and k (cat)/K (m) values were: WT, 220; L155A, 240; L155S, 123; L155A-I156N, 62; L155A-I156V, 760; L155S-I156N, 240; and L155S-I156V, 520 min(-1) mM(-1).
authors.affiliation: Department of Biotechnology, Faculty of Life Sciences, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga-ken 525-8577, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Matsumiya
authors.forename: Naoko
authors.initials: N
authors.lastname: Murata
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
authors.forename: Motoki
authors.initials: M
authors.lastname: Kubo
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013820:Thermolysin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013820:Thermolysin; D007930:Leucine