Expression and characterization of a novel ω-transaminase from Burkholderia phytofirmans PsJN.
- Author:
Yuncheng DU
1
;
Wenyue DONG
1
;
Jinju JIANG
1
;
Qijia CHEN
1
;
Jinhui FENG
1
;
Qiaqing WU
1
;
Dunming ZHU
1
Author Information
- Publication Type:Journal Article
- Keywords: Burkholderia phytofirmans PsJN; aromatic β-amino acids; chiral resolution; ω-transaminase
- MeSH: Bacterial Proteins; biosynthesis; genetics; Burkholderia; enzymology; Cloning, Molecular; Escherichia coli; genetics; metabolism; Transaminases; biosynthesis; genetics
- From: Chinese Journal of Biotechnology 2016;32(7):912-926
- CountryChina
- Language:Chinese
- Abstract: Production of chiral amines and unnatural amino-acid using ω-transaminase can be achieved by kinetic resolution and asymmetric synthesis, thus ω-transaminase is of great importance in the synthesis of pharmaceutical intermediates. By genomic data mining, a putative ω-transaminase gene hbp was found in Burkholderia phytofirmans PsJN. The gene was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme (HBP) was purified by Ni-NTA column and its catalytic properties and substrate profile were studied. HBP showed high relative activity (33.80 U/mg) and enantioselectivity toward β-phenylalanine (β-Phe). The optimal reaction temperature and pH were 40 ℃ and 8.0-8.5, respectively. We also established a simpler and more effective method to detect the deamination reaction of β-Phe by UV absorption method using microplate reader, and demonstrated the thermodynamic property of this reaction. The substrate profiling showed that HBP was specific to β-Phe and its derivatives as the amino donor. HBP catalyzed the resolution of rac-β-Phe and its derivatives, the products (R)-amino acids were obtained with about 50% conversions and 99% ee.
