Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site.
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'.
Matsumiya
Yoshiki
Y
Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga-ken 525-8577.
Nishikawa
Kouji
K
Aoshima
Hisae
H
Inouye
Kuniyo
K
Kubo
Motoki
M
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*
D000596:Amino Acids; D004151:Dipeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013820:Thermolysin; D007930:Leucine; D002122:Calcium Chloride