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Applied and Environmental Microbiology, June 2001, p. 2445-2452, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2445-2452.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

Yuji Kannan,1 Yuichi Koga,1 Yohei Inoue,1 Mitsuru Haruki,1 Masahiro Takagi,2 Tadayuki Imanaka,3 Masaaki Morikawa,1 and Shigenori Kanaya1,*

Department of Material and Life Science1 and Department of Biotechnology,2 Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, and Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501,3 Japan

Received 29 December 2000/Accepted 16 March 2001

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly-82 to Gly316), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.

* Corresponding author. Mailing address: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone and fax: 81-(0)6-6879-7938. E-mail: kanaya{at}ap.chem.eng.osaka-u.ac.jp

Applied and Environmental Microbiology, June 2001, p. 2445-2452, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2445-2452.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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