Characterization of ELP-fused ω-Transaminase and Its Application for the Biosynthesis of β-Amino Acid
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Optically pure amines, β-amino acids and γ-amino acids are the valuable precursors to produce biologically active compounds. The ω-TAs are the class of enzymes which are widely used to produce such compounds. In this work (S)-ω-transaminase from the thermophilic eubacterium Sphaerobacter thermophilus (St-TA) was fused with Elastin-like polypeptides (ELPs) through the cloning process and expressed in E. coli cells. The characterization of this fusion complex was performed with respect to thermostability and effect of DMSO. Where in case of St-TA-ELP-V60, major difference in the transition temperature (Tt) was observed, wherein a Tt of 38 and 70°C was observed at the increasing concentration of DMSO from 5 to 25% (v/v). Interestingly, these fusion proteins the activity was preserved even after the aggregation of fusion complex at Tt. The substrate specificity and product inhibition analysis showed that ω-TA-ELPs had comparable results as that of wild type ω-TA. Moreover, the fused ω-TA could be efficiently reused for up to 20 batches of transamination reaction. Furthermore, the applicability of the fusion protein for the production of a sitagliptin precursor (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid (3-ATfBA) was evaluated, wherein 3-ATfBA was synthesized with good conversion (65%).
Keywords(S)-ω-transaminase elastin-like polypeptides chiral amines bio-catalysis thermostability
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- 5.Ahsan, M. M., S. Sung, H. Jeon, M. D. Patil, T. Chung, and H. Yun (2017) Biosynthesis of medium-to long-chain a,ω-diols from free fatty acids using CYP153A monooxygenase, carboxylic acid reductase, and E. coli endogenous aldehyde reductases. Catalysts 8: 4.Google Scholar
- 15.Iwasaki, A., K. Matsumoto, J. Hasegawa, and Y. Yasohara (2012) A novel transaminase, (R)-amine: pyruvate aminotransferase, from Arthrobacter sp. KNK168 (FERM BP-5228): purification, characterization, and gene cloning. Appl. Microbiol. Biotechnol. 93: 1563–1573.Google Scholar
- 30.Ramisetti, A., J. M. R. Boggu, P. Shekhar, and B. V. Subba Reddy (2016) Stereoselective synthesis of dipeptidyl peptidase-4 (DPP-4) inhibitor, (R)-sitagliptin. Chem. Select. 1: 5445–5447.Google Scholar
- 39.Akhani, R. C., A. T. Patel, M. J. Patel, S. R. Dedania, J. S. Patel, and D. H. Patel (2018) Column chromatography free purification of recombinant a-amylase from bacillus licheniformis by tagging with hydrophobic elastin like polypeptide. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 88: 1249–1255.CrossRefGoogle Scholar
- 40.Christensen, T., K. Trabbic-Carlson, W. Liu, and A. Chilkoti (2007) Purification of recombinant proteins from E. coli at low expression levels by inverse transition cycling. Anal. Biochem. 360: 166–168.Google Scholar