title: Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site.
abstract: The relationship between the autodegradation and thermostability of thermolysin (TLN) was studied. Four autodegradation sites in TLN were identified in the presence of Ca(2+). One of the sites was identified as Gly(154)-Leu(155), and Leu(155) was substituted with various amino acids, X = Ala, Ser, Phe, and Gly, by site-directed mutagenesis. The thermostability at 80 degrees C increased with the amino acid substitutions in the order of Ala>Phe>Ser>Gly>Leu (WT TLN). An additional autodegradation fragment that was not observed with WT TLN appeared for all mutant TLNs examined. The autodegradation site shifted from the Gly(154)-Leu(155) bond to the X(155)-Ile(156) one with the mutation at Leu(155). Furthermore, the Ile(164)-Asp(165) bond was recognized newly as an autodegradation site in the mutant TLNs for the production of AF3'.
authors.affiliation: Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga-ken 525-8577.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Matsumiya
authors.forename: Kouji
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Hisae
authors.initials: H
authors.lastname: Aoshima
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
authors.forename: Motoki
authors.initials: M
authors.lastname: Kubo
mesh_terms: D000595:Amino Acid Sequence / Q000502:physiology; D019943:Amino Acid Substitution / Q000235:genetics*; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites / Q000235:genetics; D002122:Calcium Chloride / Q000737:chemistry; D002942:Circular Dichroism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000032:analysis; D017354:Point Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D004151:Dipeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013820:Thermolysin; D007930:Leucine; D002122:Calcium Chloride