J. Biochem, 2004, Vol. 135, No. 4 547-553
© 2004 The Japanese Biochemical Society
BIOTECHNOLOGY |
Analysis of Autodegradation Sites of Thermolysin and Enhancement of Its Thermostability by Modifying Leu155 at an Autodegradation Site
1 Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga-ken 525-8577; and 2 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502
The relationship between the autodegradation and thermostability of thermolysin (TLN) was studied. Four autodegradation sites in TLN were identified in the presence of Ca2+. One of the sites was identified as Gly154-Leu155, and Leu155 was substituted with various amino acids, X = Ala, Ser, Phe, and Gly, by site-directed mutagenesis. The thermostability at 80°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 Gly154-Leu155 bond to the X155-Ile156 one with the mutation at Leu155. Furthermore, the Ile164-Asp165 bond was recognized newly as an autodegradation site in the mutant TLNs for the production of AF3'.
* To whom correspondence should be addressed. Tel.: +81-77-561-3901, Fax: +81-77-561-3901, E-mail: kubo{at}se.ritsumei.ac.jp
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