This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Taguchi, S. Articles by Momose, H. Search for Related Content PubMed PubMed Citation Articles by Taguchi, S. Articles by Momose, H. Agricola Articles by Taguchi, S. Articles by Momose, H.

 Previous Article  |  Next Article 

Appl Environ Microbiol, February 1998, p. 492-495, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Engineering of a Cold-Adapted Protease by Sequential Random Mutagenesis and a Screening System

Seiichi Taguchi,^* Akiyoshi Ozaki, and Haruo Momose

Department of Biological Science and Technology, Science University of Tokyo, Noda-shi, Chiba 278, Japan

Received 28 August 1997/Accepted 9 November 1997

A cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46-51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the k_cat/K_m value) 100% higher than that of the wild type at 10°C when N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased K_m value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60°C and circular dichroism spectra at 10°C.

---

^* Corresponding author. Mailing address: Dept. of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba 278, Japan. Phone: 81-471-24-1501, ext. 4428. Fax: 81-471-25-1841. E-mail: staguchi{at}rs.noda.sut.ac.jp.

This article has been cited by other articles:

• Sasaki, M., Uno, M., Akanuma, S., Yamagishi, A. (2008). Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii. Protein Eng Des Sel 21: 721-727 [Abstract] [Full Text]  
• Prieto, J., Epinat, J.-C., Redondo, P., Ramos, E., Padro, D., Cedrone, F., Montoya, G., Paques, F., Blanco, F. J. (2008). Generation and Analysis of Mesophilic Variants of the Thermostable Archaeal I-DmoI Homing Endonuclease. J. Biol. Chem. 283: 4364-4374 [Abstract] [Full Text]  
• Pulido, M.A., Koga, Y., Takano, K., Kanaya, S. (2007). Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation. Protein Eng Des Sel 0: gzm006v1-11 [Abstract] [Full Text]  
• Yaish, M. W.F., Doxey, A. C., McConkey, B. J., Moffatt, B. A., Griffith, M. (2006). Cold-Active Winter Rye Glucanases with Ice-Binding Capacity. Plant Physiol. 141: 1459-1472 [Abstract] [Full Text]  
• Tindbaek, N., Svendsen, A., Oestergaard, P. R., Draborg, H. (2004). Engineering a substrate-specific cold-adapted subtilisin. Protein Eng Des Sel 17: 149-156 [Abstract] [Full Text]  
• Takase, K., Taguchi, S., Doi, Y. (2003). Enhanced Synthesis of Poly(3-hydroxybutyrate) in Recombinant Escherichia coli by Means of Error-Prone PCR Mutagenesis, Saturation Mutagenesis, and In Vitro Recombination of the Type II Polyhydroxyalkanoate Synthase Gene. J Biochem 133: 139-145 [Abstract] [Full Text]  
• Arisawa, A., Matsufuji, M., Nakashima, T., Dobashi, K., Isshiki, K., Yoshioka, T., Yamada, S., Momose, H., Taguchi, S. (2002). Streptomyces Serine Protease (DHP-A) as a New Biocatalyst Capable of Forming Chiral Intermediates of 1,4-Dihydropyridine Calcium Antagonists. Appl. Environ. Microbiol. 68: 2716-2725 [Abstract] [Full Text]  
• Kichise, T., Taguchi, S., Doi, Y. (2002). Enhanced Accumulation and Changed Monomer Composition in Polyhydroxyalkanoate (PHA) Copolyester by In Vitro Evolution of Aeromonas caviae PHA Synthase. Appl. Environ. Microbiol. 68: 2411-2419 [Abstract] [Full Text]  
• Yasugi, M., Suzuki, T., Yamagishi, A., Oshima, T. (2001). Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus. Protein Eng Des Sel 14: 601-607 [Abstract] [Full Text]  
• Suzuki, T., Yasugi, M., Arisaka, F., Yamagishi, A., Oshima, T. (2001). Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures. Protein Eng Des Sel 14: 85-91 [Abstract] [Full Text]  
• Taguchi, S., Komada, S., Momose, H. (2000). The Complete Amino Acid Substitutions at Position 131 That Are Positively Involved in Cold Adaptation of Subtilisin BPN'. Appl. Environ. Microbiol. 66: 1410-1415 [Abstract] [Full Text]  
• Song, J. K., Rhee, J. S. (2000). Simultaneous Enhancement of Thermostability and Catalytic Activity of Phospholipase A1 by Evolutionary Molecular Engineering. Appl. Environ. Microbiol. 66: 890-894 [Abstract] [Full Text]  
• Iyo, A. H., Forsberg, C. W. (1999). A Cold-Active Glucanase from the Ruminal Bacterium Fibrobacter succinogenes S85. Appl. Environ. Microbiol. 65: 995-998 [Abstract] [Full Text]