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PEDS Advance Access published online on April 28, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh034
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Received December 18, 2003
Revised March 26, 2004
Accepted April 1, 2004

Article

Thermal stabilization of penicillolysin, a thermo-labile 19 kDa Zn2+--protease, obtained by site-directed mutagenesis

Yuko Doi 1, Hidetoshi Akiyama 1, Yoshiteru Yamada 1, Ch'ng Ewe Ee 2, Byung Rho Lee 1, Masamichi Ikeguchi 1, Eiji Ichishima 1*

1 Laboratory of Molecular Enzymology, Graduate School of Bioengineering, Soka University, Hachioji, Tokyo 192-8577, Japan
2 Laboratory of Molecular Enzymology, Graduate School of Agriculture Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan

* To whom correspondence should be addressed. E-mail: ichisima{at}t.soka.ac.jp.


  Abstract

Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermo-labile Zn2+-protease from Penicillium citrinum with a unique substrate profile. We have expressed recombinant penicillolysin in Aspergillus oryzae and have generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intent to explore thermal stabilization of this protein. We have based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from Aspergillus oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from edible mushroom Grifora frondsa. The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.

Keywords: aspzincin/ M35 protease/ mutagenesis/penicillolysin/ thermal stabilization/Zn2+-protease


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