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A robust cosolvent-compatible halohydrin dehalogenase by computational library design
Hesam Arabnejad1, Marco Dal Lago2, Peter A Jekel1
1Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Protein Engineering, Design & Selection : PEDS
|December 22, 2016
Summary
Researchers enhanced halohydrin dehalogenase (HheC) enzyme stability for organic cosolvent reactions using computational design. A 12-fold mutant (HheC-H12) showed increased melting temperature and cosolvent resistance, preserving catalytic activity.
Area of Science:
- Enzyme engineering
- Computational biochemistry
- Biocatalysis
Background:
- Halohydrin dehalogenase (HheC) is a valuable biocatalyst.
- Organic cosolvents often limit enzyme applicability.
- Enzyme stabilization is crucial for industrial applications.
Purpose of the Study:
- To computationally design and experimentally validate stabilizing mutations for HheC.
- To improve HheC's performance in organic cosolvents.
- To create a more robust enzyme for industrial biocatalysis.
Main Methods:
- Computational library design (Framework for Rapid Enzyme Stabilization by Computational libraries).
- In silico evaluation: energy calculations, disulfide bond predictions, molecular dynamics simulations.
- Experimental validation: site-directed mutagenesis, melting temperature assays, cosolvent resistance tests, enzyme kinetics, crystal structure analysis.
Main Results:
- Identified 218 potential stabilizing point mutations and 35 disulfide bonds computationally.
- Experimentally confirmed 29 stabilizing point mutations, primarily in two regions.
- Developed a 12-fold mutant (HheC-H12) with a 28°C higher melting temperature and enhanced cosolvent resistance.
- HheC-H12 exhibited a higher optimal catalytic temperature while retaining low-temperature activity.
- Crystal structures revealed mutations improved surface charge distribution and inter-subunit interactions.
Conclusions:
- Computational enzyme design is effective for improving HheC stability.
- The HheC-H12 mutant demonstrates significantly enhanced stability and broader applicability in organic cosolvents.
- This engineered enzyme serves as a platform for further optimization of enantioselectivity and activity.
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