As a green biocatalyst, transaminase with high thermostability can be better employed to synthesize many pharmaceutical intermediates in industry. To improve the thermostability of (R)-selective amine transaminase from Bacillus altitudinis W3, related mutation sites were determined by multiple amino acid sequence alignment between wild-type ω-transaminase and four potential thermophilic ω-transaminases, followed by replacement of the related amino acid residues with proline by site-directed mutagenesis. Three stabilized mutants (D192P, T237P, and D192P/T237P) showing the highest stability were obtained and used for further analysis. Comparison with the wild-type enzyme revealed that the double mutant D192P/T237P exhibited the largest shift in thermostability, with a 2.5-fold improvement of t 1/2 at 40 °C, and a 6.3 °C increase in T 50 15, and a 5 °C higher optimal catalytic temperature. Additionally, this mutant exhibited an increase in catalytic efficiency (k cat/K m) relative to the wild-type enzyme. Modeling analysis indicated that the improved thermostability of the mutants could be associated with newly formed hydrophobic interactions and hydrogen bonds. This study shown that proline substitutions guided by sequence alignment to improve the thermostability of (R)-selective amine transaminase was effective and this method can also be used to engineering other enzymes.
Keywords: Amino transferase; Enzyme thermostability; Hydrogen bonds; Hydrophobic interactions; Proline substitutions; Protein engineering.
© King Abdulaziz City for Science and Technology 2020.