Institute of Biochemistry, Protein Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17489, Greifswald, Germany.
Institut of Food Chemistry and Food Technology, Justus-Liebig-University of Gießen, Heinrich-Buff-Ring 17, 35392, Gießen, Germany.
Appl Microbiol Biotechnol. 2022 Sep;106(17):5563-5574. doi: 10.1007/s00253-022-12071-1. Epub 2022 Aug 6.
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.
胺转氨酶（ATA）将酮转化为具有光学活性的胺，用于制备活性药物成分和构建块。由于对序列-功能关系的广泛了解，可以在蛋白质数据库中鉴定新型 ATA。然而，从氨基酸序列预测热稳定性和操作稳定性是一个持续存在的挑战，也是鉴定用于工业应用的高效 ATA 生物催化剂的重要步骤。在这项研究中，我们进行了数据库挖掘，并对β-丙氨酸：丙酮酸转氨酶簇（3N5M）的选定假定酶进行了表征（一个亚家族，迄今为止只有少数成员被描述过，其四聚体结构被认为对操作稳定性有积极影响）。四种假定的转氨酶（TA-1：Bilophilia wadsworthia、TA-5：Halomonas elongata、TA-9：Burkholderia cepacia 和 TA-10：Burkholderia multivorans）以可溶性形式在大肠杆菌中作为四聚体获得。在比较这些四聚体与已知的二聚体转氨酶时，我们发现确实可以在这个蛋白质亚家族中鉴定到具有高操作稳定性的新型 ATA，但我们也发现了与假设的相关性的例外，即四聚体组装会导致稳定性增加。从 Burkholderia multivorans 中发现的 ATA 具有广泛的底物特异性，包括异丙胺接受能力，在丙酮酸转化 1-苯乙胺时非常活跃（6 U/mg），在储存和操作条件下高达 70°C 时具有热稳定性。此外，在 30°C 下孵育 16 小时期间，可使用 50%（v/v）的异丙醇或 DMSO 作为共溶剂，而不会对酶产生不稳定的影响。关键点：• 数据库挖掘在β-丙氨酸：丙酮酸转氨酶亚家族中鉴定出一种耐热胺转氨酶。• 四聚体转氨酶在 30°C 操作条件下可耐受 50% DMSO 和异丙醇。• 四聚体结构不一定与更高的操作稳定性相关。
