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Updated: Nov 9, 2025

Author Spotlight: In Silico Creation and Impact of Carbonylated Amino Acids on Protein Structure and Function
Published on: April 26, 2024
Surface charged amino acid-based strategy for rational engineering of kinetic stability and specific activity of
Zhemin Liu1, Xiaodan Fu1, Mingxue Yuan1
1College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Abstract:
A rational workflow for engineering kinetically stable enzymes with good specific activity by surface charged amino acids engineering was proposed based on systematically analyzing the results of mutating 44 negatively charged surface amino acids of a thermophilic β-mannanase (ManAK). Computational data, combined with experimental results indicated that percentage side-chain solvent accessibility (PSSA), changes in Gibbs free energy of unfolding (∆∆Gmut) and root-mean-square fluctuations (RMSF) could be suitable for screening kinetically stable mutants. A combinational standard (∆∆Gmut < -0.5 kJ/mol and RMSF >0.68 Å) resulted a decrease in the proportion of destabilizing mutants to 12.5%. The perturbations of substrate affinity and specific activity caused by mutation were weakened as the shortest distance from Cα of mutated site to Cα of catalytic sites (DsCα-Cα) increased. Results indicated that hotspot zones contributing to the local stability and integrity of catalytic motif at elevated temperatures might be widely distributed across spatial structure of the protein, while the mutation perturbation on enzyme specific activity demonstrated a gradually weakening trend from the catalytic core to the protein surface. These findings further our understanding of the structural-functional relationships of protein and highlight a deduced workflow to engineering industrially useful enzymes.
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