A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase.

Jun Huang,Hong-Peng Wang,Le-He Mei,Chang-Jiang Lv,Jia-Qi Mei,Wei-Rui Zhao,Wan-Li Zhu,Sheng Hu

doi:10.3390/molecules24071194

Abstract

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

Highlights

Chiral amines are indispensable building blocks for numerous biologically active compounds and active pharmaceutical ingredients [1,2,3]
Mutual Information Server to Infer Coevolution (MISTIC) provided an integrated view of AT-amine transaminases (ATAs) in regards to the mutual information (MI) between residues (Figure S1A, in the Supplementary Materials), sequence conservation, cumulative MI (cMI), and proximity MI (pMI)
Residues with high cMI scores were rich in shared MI; residues with high pMI scores were mostly located in proximity to functionally important residues (Figure S1B)

Summary

Introduction

Chiral amines are indispensable building blocks for numerous biologically active compounds and active pharmaceutical ingredients [1,2,3]. Amine transaminases (ATAs) have become the most prominent biocatalysts for the generation of optically pure chiral amines because of their high stereoselectivity and environmentally benign reaction conditions [2,5,6,7]. In industrial applications, biocatalysts, such as ATAs, are often required to enhance the rate of a reaction and the solubility of the reactant while simultaneously diminishing the risk of microbial contamination. The development of enzymes with a higher thermostability would expand the applicability of ATAs in industrial processes. To enhance the thermal stability of an (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), rational strategies, such as the combination of the B-factor profile and folding free energy calculations (∆∆Gfold ), the introduction of disulfide bridges, and consensus mutagenesis, were

Methods

Results

Discussion

Conclusion