Semi-rational evolution of ω-transaminase from <i>Aspergillus terreus</i> for enhancing the thermostability.

Tingting Cai; Jiaren Cao; Shuai Qiu; Changjiang Lyu; Fangfang Fan; Sheng HU; Weirui Zhao; Lehe Mei; Jun Huang

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Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability.

Author: Tingting CAI ¹ ; Jiaren CAO ¹ ; Shuai QIU ¹ ; Changjiang LYU ¹ ; Fangfang FAN ¹ ; Sheng HU ² ; Weirui ZHAO ² ; Lehe MEI ² ; Jun HUANG ¹
Author Information

1. School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, Zhejiang, China.
2. School of Biological and Chemical Engineering, Ningbo Tech University, Ningbo 315100, Zhejiang, China.
Publication Type:Journal Article
Keywords: chiral amines; molecular dynamic simulation; semi-rational design; thermostability; α-helix; ω-transaminases
MeSH: Transaminases/chemistry*; Molecular Docking Simulation; Amines/chemistry*; Pyruvic Acid/metabolism*; Enzyme Stability
From: Chinese Journal of Biotechnology 2023;39(6):2126-2140
CountryChina
Language:Chinese
Abstract: ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t_1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T¹⁰₅₀) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.