Expression and characterization of a novel ω-transaminase from Burkholderia phytofirmans PsJN / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 912-926, 2016.
Artículo
en Chino
| WPRIM
| ID: wpr-242287
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.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Proteínas Bacterianas
/
Clonación Molecular
/
Burkholderia
/
Escherichia coli
/
Genética
/
Transaminasas
/
Metabolismo
Idioma:
Chino
Revista:
Chinese Journal of Biotechnology
Año:
2016
Tipo del documento:
Artículo
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