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'.
MeSH terms
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Amino Acid Sequence / physiology
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Amino Acid Substitution / genetics*
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Amino Acids / chemistry
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Amino Acids / genetics
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Bacillus / enzymology
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Binding Sites / genetics
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Calcium Chloride / chemistry
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Circular Dichroism
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Dipeptides / chemistry
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Dipeptides / metabolism
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Electrophoresis, Polyacrylamide Gel
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Enzyme Stability / genetics
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Escherichia coli / genetics
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Genetic Vectors / genetics
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Hot Temperature
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Kinetics
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Leucine / chemistry
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Leucine / genetics
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Models, Chemical
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Models, Molecular
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Mutagenesis, Site-Directed
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Peptide Fragments / analysis
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Point Mutation / genetics
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Temperature
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Thermolysin / chemistry
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Thermolysin / genetics
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Thermolysin / metabolism*
Substances
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Amino Acids
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Dipeptides
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Peptide Fragments
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Recombinant Proteins
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Thermolysin
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Leucine
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Calcium Chloride