3AXD

The truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase V18Y/W203Y in apo-form

3AXD の概要

• エントリーDOI: 10.2210/pdb3axd/pdb
• 関連するPDBエントリー: 3AXE
• 分子名称: Beta-glucanase, 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL, CALCIUM ION, ... (4 entities in total)
• 機能のキーワード: glucanase, cellobiose/cellotetraose, hydrolase
• 由来する生物種: Fibrobacter succinogenes
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 56220.22

構造登録者

Huang, J.W.,Cheng, Y.S.,Ko, T.P.,Lin, C.Y.,Lai, H.L.,Chen, C.C.,Ma, Y.,Huang, C.H.,Zheng, Y.,Liu, J.R.,Guo, R.T. (登録日: 2011-04-03, 公開日: 2012-02-15, 最終更新日: 2023-11-01)

主引用文献

Huang, J.W.,Cheng, Y.S.,Ko, T.P.,Lin, C.Y.,Lai, H.L.,Chen, C.C.,Ma, Y.,Zheng, Y.,Huang, C.H.,Zou, P.,Liu, J.R.,Guo, R.T.
Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase
Appl.Microbiol.Biotechnol., 94:111-121, 2012

PubMed Abstract: 1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.

PubMed: 21959377
DOI: 10.1007/s00253-011-3586-7

実験手法

X-RAY DIFFRACTION (1.53 Å)