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

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2005, p. 5433-5439, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5433-5439.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L {alpha}-Glucan Phosphorylase

Michiyo Yanase,* Hiroki Takata, Kazutoshi Fujii, Takeshi Takaha, and Takashi Kuriki

Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan

Received 29 November 2004/ Accepted 16 January 2005

The thermostability of potato type L {alpha}-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39->Leu (F39L), Asn135->Ser (N135S), and Thr706->Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.

* Corresponding author. Mailing address: Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan. Phone: 81-6-6477-8425. Fax: 81-6-6477-8362. E-mail: yanase-michiyo{at}glico.co.jp.

Applied and Environmental Microbiology, September 2005, p. 5433-5439, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5433-5439.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»