Characterization of proteins from the 3N5M family reveals an operationally stable amine transaminase

Abstract

Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the β-alanine:pyruvate transaminase cluster (3N5M) — a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1: Bilophilia wadsworthia, TA-5: Halomonas elongata, TA-9: Burkholderia cepacia, and TA-10: Burkholderia multivorans) were obtained in a soluble form as tetramers in E. coli. During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from Burkholderia multivorans features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C. Key points • Database mining identified a thermostable amine transaminase in the β-alanine:pyruvate transaminase subfamily. • The tetrameric transaminase tolerates 50% DMSO and isopropanol under operating conditions at 30 °C. • A tetrameric structure is not necessarily associated with a higher operational stability Graphical abstract

Comparison of Four Immobilization Methods for Different Transaminases

“…ATA-Vfl is a dimeric, (S)selective wildtype transaminase from Vibrio fluvialis, and it has been frequently examined and used in several studies [60][61][62][63][64]. ATA-Bmu is an (S)-selective wildtype transaminase from Burkholderia multivorans, and was recently discovered by database mining as a member of the β-alanine:pyruvate transaminase family [65]. Enzymes of this family exhibit a tetrameric structure associated with high operational stability.…”

Activity Levels of Amine Transaminases Correlate with Active Site Hydrophobicity

“…The operating stabilities of the ATAs from this study are better than those reported for Vibrio fluvialis ATA [27]. They are in the same range as a wild-type ATA identified in a metagenomic library probably originating from Pseudomonas sp., TA-5LH9 [28], but less stable than the engineered variant of TA-5LH9 created by Bo ¨rner and co-workers [29] and our recently characterized wild-type ATA from Bulkholderia multivorans [30], which both are tetrameric enzymes.…”

Identification, characterization and application of M16AT, a new organic solvent–tolerant (R)–enantio–selective type IV amine transaminase from Mycobacterium sp. ACS1612

“…The optimal reaction temperature for the enzyme was determined to be 55 °C, as shown in Figure 3B. Ideally, enzymes should exhibit high thermostability under “operational stability” (when substrates and conditions required for catalysis are present), similar to the higher thermal stability they should possess under storage conditions (referred to as “resting stability” because the catalyst is in a more stable resting state) [27] . To assess the thermal stability of M16AT under operational conditions, its stability under storage conditions was first tested.…”

“…Ideally, enzymes should exhibit high thermostability under "operational stability" (when substrates and conditions required for catalysis are present), similar to the higher thermal stability they should possess under storage conditions (referred to as "resting stability" because the catalyst is in a more stable resting state). [27] To assess the thermal stability of M16AT under operational conditions, its stability under storage conditions was first tested. Figure 3C shows that after treatment at 55 °C for 30 minutes under storage conditions, the residual activity of M16AT was approximately 45 %.…”