title: Characterization of Four New Distinct ω-Transaminases from Pseudomonas putida NBRC 14164 for Kinetic Resolution of Racemic Amines and Amino Alcohols.
abstract: Four uncharacterized ω-transaminases (ωTAs) from Pseudomonas putida NBRC 14164 have been identified and cloned from the pool of fully sequenced genomes. The genes were functionally expressed in Escherichia coli BL21, and the enzymes were purified and characterized. Four TAs showed highly (S)-selective ωTA activity and converted (S)-α-methylbenzylamine and pyruvate to acetophenone and L-Ala. The maximum activity of cloned enzymes was in the pH range of 8.0-8.5 (Pp36420), 8.5-9.5 (Pp21050), 9.0-9.5 (PpspuC), and 9.5-10.5 (PpbauA), and the optimal temperatures were at 35 °C (Pp36420, Pp21050, and PpspuC) and 50 °C (PpbauA), respectively, with KMof 161.3 mM (Pp21050), 136.7 mM (PpbauA), 398.5 mM (Pp36420), and 130.9 mM (PpspuC) and yielding a catalytic efficiency kcat/KMof 0.015, 0.003, 0.012, and 0.023 mM-1s-1. Several racemic amines and amino alcohols were resolved by the cloned ωTAs; perfect conversions (48-50 %) were obtained by at least one enzyme, and the residual substrates were left with 97-99 % ee. Kinetic resolution of racemic phenylglycinol was done with PpspuC in a 100-mL scale. Enaniomeric excess of (S)-phenylglycinol reached 99 % with 45 % isolated yield. The high enantioselectivity and large substrate spectra of the cloned PpTAs showed an attractive potency for biotechnology application in production of chiral amines and amino alcohols.
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No.79 West Yingze Street, Taiyuan, Shanxi, 030024, China.
authors.forename: Hua-Lei
authors.initials: HL
authors.lastname: Wu
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No.79 West Yingze Street, Taiyuan, Shanxi, 030024, China. zhangjiandong@tyut.edu.cn.
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Zhang
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No.79 West Yingze Street, Taiyuan, Shanxi, 030024, China.
authors.forename: Chao-Feng
authors.initials: CF
authors.lastname: Zhang
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No.79 West Yingze Street, Taiyuan, Shanxi, 030024, China.
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Fan
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No.79 West Yingze Street, Taiyuan, Shanxi, 030024, China.
authors.forename: Hong-Hong
authors.initials: HH
authors.lastname: Chang
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No.79 West Yingze Street, Taiyuan, Shanxi, 030024, China.
authors.forename: Wen-Long
authors.initials: WL
authors.lastname: Wei
mesh_terms: D000605:Amino Alcohols / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics
chemical_list: D000605:Amino Alcohols; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000637:Transaminases
keywords: Chiral amino alcohol; Enantioselectivity; Kinetic resolution; Pseudomonas putida; Transaminase