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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23321 43431 2013 9 PDF Available
Title
Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions
Abstract

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 β-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 β-subunit, reduced activity and irregular stability. NHase-TH-A2 exhibited a enhanced expression of β-subunit but complete loss of activity; while NHase-TH-A3 exhibited not only a slightly enhanced expression of β-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.

► Three deformation-prone thermal-sensitive regions of NHase were identified by RMSF calculation. ► The stabilized salt-bridge interactions in thermophilic NHase were transferred into NHase-TH. ► The three salt bridges showed different results in expression, enzyme activity and stability. ► Salt bridge network changes were further modeled by molecular dynamic simulation. ► The C-terminal salt-bridge is powerful for stability intensification with unreduced activity.

Keywords
C-terminal salt-bridge strategy; Enzyme stability; Molecular dynamic simulation; Nitrile hydratase; Thermal-sensitive region
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Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 164, Issue 2, March 2013, Pages 354–362
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us