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PEDS Advance Access originally published online on April 28, 2004
Protein Engineering Design and Selection 2004 17(3):261-266; doi:10.1093/protein/gzh034
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© 2004 Oxford University Press

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

Yuko Doi1, Hidetoshi Akiyama1, Yoshiteru Yamada1, Ch’ng Ewe Ee2, Byung Rho Lee1, Masamichi Ikeguchi1 and Eiji Ichishima1,2,3

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

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

Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermolabile Zn2+- protease from Penicillium citrinum with a unique substrate profile. We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein. We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the 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.

Received December 18, 2003; revised March 26, 2004; accepted April 1, 2004 Edited by Taiji Imoto


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