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Enzyme Microb Technol. 2019 Jan;120:52-60. doi: 10.1016/j.enzmictec.2018.10.003. Epub 2018 Oct 6.

Enzymatic synthesis of sitagliptin intermediate using a novel ω-transaminase.

Author information

1
Department of Systems Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea. Electronic address: Kim-rlarjsgml11@naver.com.
2
Department of Systems Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea. Electronic address: hw5827@naver.com.
3
Department of Systems Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea. Electronic address: taresh.khobragade@yahoo.com.au.
4
Department of Systems Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea. Electronic address: mahi1709@gmail.com.
5
Department of Systems Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea. Electronic address: Sihyong21@naver.com.
6
Department of Systems Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea. Electronic address: Sanghany3@naver.com.
7
Department of Systems Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea. Electronic address: wonyumi12@gmail.com.
8
CKD Bio Research Institute, Ansan-si, Gyeonggi-do 15604, Republic of Korea. Electronic address: inschoi6@ckdbio.com.
9
Department of Systems Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea. Electronic address: hyungdon@konkuk.ac.kr.

Abstract

Enantiopure β-amino acids are essential precursors of various pharmaceuticals, agrochemicals and other industrially important chemicals. In this study, we selected sixteen potential ω-Transaminases (ω-TAs) by BLAST and phylogenetic tree analysis. These ω-TAs were cloned, purified and tested for their reactivity for the synthesis of model β-amino acid (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid [3-ATfBA], a key precursor for sitagliptin. In an enzymatic cascade, lipase converted β-ketoester substrate to β-keto acid, which was subsequently aminated by the selected ω-TA to its corresponding β-amino acid. A potent enzyme from Ilumatobacter coccineus (ω-TAIC) was identified for the production of 3-ATfBA. The pH dependency of the product inhibition suggested that lowering the reaction pH to 7.0 can circumvent the inhibition of ω-TAIC by 3-ATfBA and about 92.3% conversion of 100 mM β-keto ester substrate could be achieved. The applicability of this enzymatic system was further evaluated at the scale of 140 mM, wherein 3-ATfBA was generated with excellent conversion (81.9%) and enantioselectivity (99% ee). Furthermore, ω-TAIC was successfully used for the synthesis of various β-amino acids from their corresponding β-keto ester substrates.

KEYWORDS:

Ilumatobacter coccineus ω-transaminase; Lipase; Phylogenetic tree; Product inhibition; Sitagliptin precursor; β-Amino acids

PMID:
30396399
DOI:
10.1016/j.enzmictec.2018.10.003
[Indexed for MEDLINE]

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