Improving the catalytic thermostability of Bacillus altitudinis W3 ω-transaminase by proline substitutions

Abstract

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.

Fig. 1

Multiple sequence alignment of ω-TAs from Bacillus altitudinis W3 and Geobacillus thermodenitrificans subsp. Thermodenitrificans DSM465, Sphaerobacter Thermophilus DSM 20745, Parageobacillus thermoglucosidasius DSM2542 and Geobacillus thermoleovorans ARTRW1 using DNAMAN software. Five sites at the wild-type marked by black frames prepared for mutant. The red Arabic numbers represent the positions of amino acids in wild-type enzymes

Fig. 2

Thermostability of WT ω-BPTA and mutant enzymes. a Thermal inactivation of WT ω-BPTA, mutant D192P, T237P and D192P/T237P at different temperatures over 15 min (T₅₀¹⁵). b Thermal inactivation half-life (t_1/2) of WT ω-BPTA, mutant D192P, T237P and D192P/T237P at 40 °C. c The effect of catalytic temperature on the WT ω-BPTA, mutant D192P, T237P and D192P/T237P

Fig. 3

Hydrogen bonds in wild-type BPTA, mutant D192P, T237P and D192P/T237P. a Wild-type ω-BPTA: the O atom of Thr237 forms one hydrogen bond each with the N atoms of His239 (3.4 Å), Asp240 (3.2 Å), and Val241 (3.3 Å). b D192P: a new hydrogen bond has been formed between the O atom of Pro192 and the N atom of Gly189 (2.8 Å). c T237P: the O atom of Pro237 forms one hydrogen bond each with the N atoms of His239 (3.4 Å), Asp240 (3.2 Å), and Val241 (3.3 Å). d D192P/T237P: a new hydrogen bond has been formed between the O atom of Pro192 and the N atom of Gly189 (2.8 Å) and the O atom of Pro237 forms one hydrogen bond each with the N atoms of His239 (3.4 Å), Asp240 (3.2 Å), and Val241 (3.3 Å)

Fig. 4

Hydrophobic interaction in mutant D192P, T237P and D192P/T237P. a D192P: two new hydrophobic interactions have been formed between Asp192 and Leu249 (5.1 Å), Ala254 (6.1 Å). b T237P: a new hydrophobic interaction has been formed between Phe236 and Pro237 (4.8 Å). c D192P/T237P: three new hydrophobic interactions have been formed between Asp192 and Leu249 (5.1 Å), Ala254 (6.1 Å) and between Phe236 and Pro237 (4.8 Å)