Identification and characterization of a novel thermostable transaminase (TATP) from Thermorudis peleae

Abstractω-Transaminases (ω-TAs) are significant candidate enzymes in the biosynthesis of chiral amines for the pharmaceutical and chemical industries. Novel thermostable ω-TAs for potential use in these industries can be discovered using homologous sequence alignment. In this study, we used well-studied B3-TA from hot spring metagenomes, the Sbv333-TA from Streptomyces sp., and TATR from Thermomicrobium roseum as starting templates to BLAST search, and found a yet unidentified thermostable ω-TA (TATP) from Thermorudis peleae. We cloned TATP into vector pET28a, leading to pET28a-TATP, and overexpressed it in Escherichia coli. The enzyme showed the highest activity at pH 8.8 and 73.6 °C, with remarkable thermostability and tolerance toward organic solvents methanol and ethanol. Substrate specificity analysis showed that TATP enzyme is active toward a broad range of substrates including glyoxylate, pyruvate, 2-phenylpropionaldehyde, 2-oxobutyrate, propionaldehyde, acetaldehyde, ethyl acetoacetate, etc. Especially, TATP enzyme presented relatively good activity toward pyruvate and glyoxylate. In addition, the active sites of TATP were analyzed via the approaches of protein sequence alignment, three-dimensional structure simulation, and coenzyme pyridoxamine phosphate docking, which provided a guideline for further enzyme engineering and suggested a potential industrial thermostable ω-TA for chiral amines synthesis.Keywords: ω-TransaminasesThermorudis peleaethermal stabilitybiocatalysis Disclosure statementNo potential conflict of interest was reported by the author(s).Additional informationFundingThis work is supported by the National Key R&D Program of China [2021YFC2104200, 2022YFF0710101], Youth Innovation Promotion Association Fellowship Program, CAS [2022327], Cooperative research project between the Chinese Academy of Sciences and the Office of the Iranian Vice President, 154232KYSB20200029; Innovation and Entrepreneurship Team of Jiangsu Province [JSSCTD202145], Youth Leading Talent Program of Gusu [ZXL2022477]; China Postdoctoral Science Foundation [grant no. 2022M722337].