Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions

被引:79
作者
Chen, Jie [1 ]
Yu, Huimin [1 ]
Liu, Changchun [1 ]
Liu, Jie [1 ]
Shen, Zhongyao [1 ]
机构
[1] Tsinghua Univ, Dept Chem Engn, Beijing 100084, Peoples R China
关键词
C-terminal salt-bridge strategy; Enzyme stability; Molecular dynamic simulation; Nitrile hydratase; Thermal-sensitive region; RHODOCOCCUS-RHODOCHROUS J1; DIRECTED EVOLUTION; GLUTAMATE-DEHYDROGENASE; PROTEIN STABILITY; ENZYME STABILITY; ION-PAIRS; IMMOBILIZATION; MUTATION; PERFORMANCE; SIMULATION;
D O I
10.1016/j.jbiotec.2013.01.021
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The regions and types suitable mutations for bridging salt-bridges to intensify enzyme stability are identified in this study. Using nitrile hydratase (NHase) as the model enzyme, three deformation-prone thermal-sensitive regions (A1, A2 and A3 in beta-subunit), identified by RMSF calculations of the thermophilic NHase 1V29 from Bacillus SC-105-1 and 1UGQ from Pseudonocardia thermophila JCM3095, were determined and the stabilized salt-bridge interactions were transferred into the corresponding region of industrialized mesophilic NHase-TH from Rhodococcus ruber TH. Three types of salt bridges active-center-adjacent (in A1), internal neighboring-residue-bridged (in A2) and C-terminal-residue-bridged (A3) were constructed in NHase-TH. The engineered NHase-TH-A1 showed reduced expression of beta-subunit, reduced activity and irregular stability. NHase-TH-A2 exhibited a enhanced expression of beta-subunit but complete loss of activity; while NHase-TH-A3 exhibited not only a slightly enhanced expression of beta-subunit and enzyme activity, but also a 160% increase in thermal stability, a 7% enhanced product tolerance and a 75% enhanced resistance to cell-disruption by ultrasonication. The molecular dynamic (MD) simulation revealed that NHase-TH-A3, with a moderate RMSD value, generates 10 new salt bridges in both internal-subunit and interfacial-subunit, confirming that a C-terminal salt-bridge strategy is powerful for enzyme stability intensification through triggering global changes of the salt bridge networks. (C) 2013 Elsevier B.V. All rights reserved.
引用
收藏
页码:354 / 362
页数:9
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