ID 105962
Title Transcription
コウネツセイ サイキン Thermus thermophilus ムキ ピロリンサン カスイ ブンカイ コウソ ノ error-prone PCR ヘンイ ドウニュウ ニヨル ネツ ギョウシュウ ヨクセイ
Title Alternative
Thermus thermophilus Inorganic Pyrophosphatase Variants by Error-prone PCR Suppressed Thermal Aggregation
Author
Sugiyama, Noriko Faculty of Integrated Arts and Sciences, The University of Tokushima
Maeda, Ayumi Faculty of Integrated Arts and Sciences, The University of Tokushima
Tanaka, Hiroshi Faculty of Integrated Arts and Sciences, The University of Tokushima
Hanafusa, Eiichiro Graduate school of Human and Natural Environment Sciences, The University of Tokushima
Kanai, Taku Graduate school of Human and Natural Environment Sciences, The University of Tokushima
Satoh, Takanori Institute of Integrated Arts and Sciences, The University of Tokushima Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Keywords
Thermophile
Pyrophosphatase
Error-prone PCR
Directed evolution
Thermus thermophilus
Content Type
Departmental Bulletin Paper
Description
Thermus thermophilus Inorganic pyrophosphatase (E.C. 3.6.1.1., Tth PPase) exhibits high thermostability, but thermal aggregation was observed on heating above 85 °C. In addition, we reported that sole cysteine in C-terminal region plays a key role in the thermostability and thermal aggregation of Tth PPase [Kohaku, Y. et al. (2008) Natl.Sci.Res., 22, 75-84]. In this study, we approached the suppression of its thermal aggregation by error-prone PCR mutagenesis of whole molecule or C-terminal region. Firstly, we obtained thermostable four variants (Q119H/L162F, L162F, K173E and K159E/A170T) by error-prone PCR mutagenesis. Moreover, we examined thermostabilities of four variants in terms of the enzyme activity, tertiary and quaternary structure.
Although conformation and quaternary structure of four variants were almost the same as those of wild type enzyme in native state, K173E and K159E/A170T variants showed higher thermostabilities than wild type in tertiary and quaternary structure. In particular, thermal aggregation of these two variants would be suppressed after heating at 85°C. Therefore, it was suggested that Lys159 and Lys173 in the molecular surface of C-terminal region may contribute to the formation of thermal aggregation of Tth PPase.
Journal Title
徳島大学総合科学部自然科学研究 = Natural Science Research, The University of Tokushima
ISSN
09146385
NCID
AN10065859
Volume
27
Issue
4
Start Page
71
End Page
79
Sort Key
71
Published Date
2013-10
Remark
P.71の著者の英語表記に誤植(誤)TAKANA(正)TANAKA
EDB ID
FullText File
language
jpn
TextVersion
Publisher
departments
Science and Technology