Improving the thermostability of glutamate decarboxylase by consensus mutagenesis

Conference Dates

September 15-19, 2019


Glutamate decarboxylase (GAD) is a crucial enzyme for the preparation of gamma-aminobutyric acid (GABA) by biological methods. In this study, the Consensus Finder (http://cbs-kazlab.oit.umn.edu/) was used, and 8 sites were screened with the most prevalent amino acid (over 60% threshold) among the homologous family members, and stabilized variants of GAD were performed by site-directed mutagenesis. The results showed that the mutants T383K and A163S showed higher thermostability than that of GAD wild-type. Compared to GAD wild type, the A163S and T383K mutants displayed the shift in thermostability, with 1.74-fold and 1.45-fold increase in half-life (t1/2) at 55 °C, 2.9 °C and 1.8 °C increase in T50 10. By the differential scanning fluorimetry (DSF) analysis, the best mutant, T383K, showed a melting temperature (Tm) of 41.9 °C, which corresponded to a 1.3 °C increase relative to the wild-type GAD. Molecular dynamics simulation indicated that mutations of A163S and T383K lowered the overall root mean square deviation (RMSD) for the overall residues at 310K and consequently increased the protein rigidity.

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