Journal
PROTEIN SCIENCE
Volume 23, Issue 1, Pages 110-116Publisher
WILEY
DOI: 10.1002/pro.2388
Keywords
designed protein stabilization; electrostatic stabilization; lipase stabilization; surface charge-charge interaction; Lipk107
Categories
Funding
- National Natural Science Foundation of China [20976053/B060804, 21002029/B020706]
- National High Technology Research and Development Program of China [2012AA020403]
- 973 Program [2012CB721003]
- National Major Science and Technology Projects of China [2012ZX09304009]
- National University of Singapore (Suzhou) Research Institute [R-2012-N-007]
- State Key Laboratory of Bioreactor Engineering
- State Key Laboratory of Materials-Oriented Chemical Engineering [KL10-09]
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We report a suite of enzyme redesign protocol based on the surface charge-charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with approximate to 10 degrees C higher half-inactivation temperature than wild-type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14-fold increase in the half-inactivation time at 50 degrees C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.
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