Characterization of an enantioselective amidase with potential application to asymmetric hydrolysis of (R, S)-2, 2-dimethylcyclopropane carboxamide

Abstract

Amidase is a promising synthesis tool for chiral amides and related derivatives. In the present study, the biochemical properties of the Delftia tsuruhatensis CCTCC M 205114 enantioselective amidase were determined for its potential application in chiral amides synthesis. D. tsuruhatensis CCTCC M 205114 amidase was purified 105.2 fold with total activity recovery of 4.26%. The enzyme is a monomer with a subunit of approximately 50 kDa by analytical gel filtration HPLC and SDS-PAGE. It had a broad substrate spectrum and displayed high enantioselectivity against R-2, 2-dimethylcyclopropane carboxamide and R-mandelic amide. The amidase was applied to enantioselective hydrolysis of R-2, 2-dimethylcyclopropane carboxamide from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate S-2, 2-dimethylcyclopropane carboxamide. This enzyme did not require metal ions for the hydrolysis reaction. Its optimal pH and temperature were 8.0 and 35°C, respectively. The K m and V max of the amidase for R-2, 2-dimethylcyclopropane carboxamide were 2.54 mM and 8.37 lmol min -1 mg protein -1 , respectively. After 60 min of the reaction, R-2, 2-dimethylcyclopropane carboxamide was completely hydrolyzed, generating S-2, 2-dimethylcyclopropane carboxamide with a yield of 45.9% and an e.e. of above 99%. Therefore, this amidase can serve as a promising producer for S-2, 2-dimethylcyclopropane carboxamide and other amides.

Soluble and functional expression of a recombinant enantioselective amidase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization

“…The recombinant amidase exhibited excellent activity toward most of the substrates (Table 4). These results are similar to those reported for amidases from Comamonas acidovorans KPO-2771-4 [42], R. erythropolis AJ270 [25], and D. tsuruhatensis CCTCCM 205114 [10]. In contrast, amidases from P. yayanosii CH1 [9], Helicobacter pylori [43], and Rhodococcus rhodochrous M8 prefer aliphatic amides [44], while an amidase from Pseudomonas sp.…”

“…Some amidases have very strict chemo-, regio-, and enantioselectivities, allowing them to be used to produce optically pure amides, carboxylic acids, and related derivatives. They have been used to produce (S)-2,2-dimethylcyclopropanecarboxamide [2,[10][11][12], piperazine-2-carboxylic acid [13], phenylalanine [14], S-naproxen [15], and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide [16]. These chemicals and their derivatives have important applications in the fine chemical and pharmaceutical industries.…”

Simultaneous purification of nitrile hydratase and amidase of Alcaligenes sp. MTCC 10674

“…Purified amidase was obtained in fraction number 14, 15 and 16 (Fig. 3 ), and 9.2-fold purification was achieved (Table 2 ), previously amidase has been purified from Pseudonocordia thermophila and Delftia tsuruhatensis CCTCCM 205114 up to 48 and 105-fold, respectively (Egorova et al 2004 ; Wang et al 2011 ). The purified amidase consists of two subunits of 52 and 49 kDa (Fig.…”

Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid

“…Similarly, the enzyme exhibited none or negligible activity against short-chain aliphatic amides, such as acetamide (Entry 11, Fig 9 ) and simple aromatic amides, such as benzamide (Entry 10, Fig 9 ). In sharp contrast, its homologs in Delftia tsuruhatensis CTCC M 205114 [ 38 ], Geobacillus subterraneus RL-2a [ 39 ], Paracoccus sp. M-1 [ 40 ], Pseudonocardia thermophila [ 41 ] exhibited excellent activity towards these substrates.…”