Further Stabilization of Leu155 Mutant Thermolysins by Mutation of an Autodegradation Site

• Published in: Applied biochemistry and biotechnology Vol. 166; no. 3; pp. 735 - 743
• Main Authors: Matsumiya, Yoshiki, Murata, Naoko, Inouye, Kuniyo, Kubo, Motoki
• Format: Journal Article
• Language: English
• Published: New York Humana Press Inc 01.02.2012
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Bacillus - enzymology; Bacillus - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biotechnology; Chemistry; Chemistry and Materials Science; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Escherichia coli - enzymology; Escherichia coli - genetics; Half-Life; Hot Temperature; Kinetics; Leucine - chemistry; Leucine - genetics; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Proteolysis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Thermolysin - chemistry; Thermolysin - genetics; Thermolysin - metabolism; Thermostability; Autodegradation; Thermolysin; Protease
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-011-9462-1

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 .