Shifting the pH Optima of (R)-Selective Transaminases by Protein Engineering

Abstract

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme.

Creating Polyploid Escherichia Coli and Its Application in Efficient L‐Threonine Production

“…pH tolerance is important for maintaining normal bacterial physiological functions. [ 22 ] The doubling time of polyploid E. coli TH‐103Z at pH 5 was 6.5% higher than that at pH 7, but the doubling time of haploid E. coli TH increased by 35.9%. TH‐103Z grew better than did TH at pH 5 (Figure 4d ).…”

Expanded Substrate Specificity in D-Amino Acid Transaminases: A Case Study of Transaminase from Blastococcus saxobsidens

“…Considering the protonation states of both the internal aldimine of BlasaTA and the amino groups of substrates (D-amino acids and (R)-amines), the assistance of the α-carboxylate group in the deprotonation of D-amino acids may be a marked advantage when the Michaelis complex is forming [36]. Recently, a proton transfer system exploring the conserved histidine residue was suggested for (R)-ATAs [49]; however, no similar system was observed in BlasaTA or its homologue, CpuTA. At the same time, CpuTA is characterized by similar values of activity towards D-amino acids and primary aromatic (R)-amines [25].…”

Evaluating Multienzyme Cascade Routes for Pharmaceutically Relevant Molecules

“…[19,20] Here, the recombinant enzymes can be engineered to remove, insert, or mutate specific amino acids in the enzyme structure. Various reports have demonstrated drastic changes in the enzyme properties upon protein engineering, including a complete switch in stereoselectivity, [21,22] expanded substrate scope, [23][24][25][26] enhanced activity [27] and thermostability, [28] tolerance to a broad range of pH [29] and different co-solvents, [30,31] and high substrate loading. [32] Computer-assisted technologies have eased the process to engineer the enzymes for desired functions.…”