ID | 12355 |
Eprint ID | 12355
|
FullText URL | |
Title Alternative | Purification, Characterization and Crystal Structure of Isoamylase from Thermophilic Bacteria Rhodothermus marinus
|
Author |
Tachibana, Akiko
Imada, Katsumi
Kinoshita, Miki
Namba, Keiichi
Tsutsumi, Noriko
Hashida, Miyoko
Sakaguchi, Hiromichi
Inagaki, Kenji
Kaken ID
researchmap
|
Abstract | The isoamylase gene from Rhodothermus marinus was cloned into and expressed in Escherichia coli
Top 10. As a result of characterization of purified R. marinus isoamylase. the enzyme had an optimum
pH of 4.0 and optimum temperature of 70℃. Thermal inactivation studies of the purified R. marinus
isoamylase revealed the enzymatic activity to be uninfluenced after one hour incubation at 60℃. These
results suggest that R. marinus isoamylase has high thermostability. The crystallization and crystal
structure analysis of R. marinus isoamylase was performed. The three-dimensional structure at 1.9Å
resolution was determined in complex with the panose. R. marinus isoamylase is composed of three
domains N, A and C, and, has a (β/α)8-barrel in domain A. The secondary structural alignments of the
R. marinus isoamylase and P. amyloderamosa isoamylase was carried out. They have the four active-site
consensus regions characteristic of the α-amylase family. And the essential residue of the α-amylase
family (D359, E395, and D467) was conserved in these enzymes. R. marinus isoamylase has shorter loops
than P. amyloderamosa isoamylase. And R. marinus isoamylase had no Ca2+ binding site. These results
are thought to be factors of thermostability of R. marinus isoamylase.
|
Abstract Alternative | Rhodothermus marinus 由来イソアミラーゼ遺伝子を組み込んだプラスミド pBX2を使用し,大腸菌 Top10株を形質転換し,16時間の前培養,24時間の本培養後,菌体破砕し,得られた無細胞抽出液を熱処理(80℃,10 min),50オ硫安分画,陰イオン交換カラムクロマトグラフィー(DEAEントヨパール),ハイドロキシアパタイトカラムクロマトグラフィーに供して本酵素の精製を行った.本精製酵素の性質検討を行った結果,本酵素の最適反応温度は70℃,pH4であり,また本酵素は60℃で1時間処理しても活性が低下することが無く,Pseudomonas amyloderamosa 由来イソアミラーゼよりも高い耐熱性を有することが判明した.本酵素の結晶化・X線結晶構造解析を行った結果,本酵素は P. amyloderamosa 由来イソアミラーゼと同様Nドメイン・AドメインCドメインの3つのドメインから構成されており,活性残基(D359,E395,D467)など活性中心付近のアミノ酸残基も P. amyloderamosa 由来イソアミラーゼと同様,高度に保存されていた.本酵素の熱安定性が P. amyloderamosa 由来イソアミラーゼよりも高
い要因として,P. amyloderamosa 由来イソアミラーゼよりもループの長さが全体的に短いことと,カルシウムイオン結合サイトの欠如が挙げられた.今後さらに構造解析を進めることにより,本酵素の熱安定性機構,反応
機構など更なる知見が得られることが期待される.
|
Keywords | isoamylase
Rhodothermus marinus
crystal structure
thermostability
|
Published Date | 2008-02
|
Publication Title |
岡山大学農学部学術報告
|
Publication Title Alternative | Scientific Reports of the Faculty of Agriculture Okayama University
|
Volume | volume97
|
Issue | issue1
|
Publisher | 岡山大学農学部
|
Publisher Alternative | Faculty of Agriculture, Okayama University
|
Start Page | 1
|
End Page | 7
|
ISSN | 0474-0254
|
NCID | AN00033029
|
Content Type |
Departmental Bulletin Paper
|
language |
Japanese
|
File Version | publisher
|
Refereed |
False
|
Eprints Journal Name | srfa
|