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Enzymatic synthesis of chiral amines with omega-transaminase

IP.com Disclosure Number: IPCOM000193291D
Publication Date: 2010-Feb-17
Document File: 5 page(s) / 290K

Publishing Venue

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Abstract

Enantiomerically pure chiral amines are useful intermediates for the synthesis of pharmaceutical compounds. Therefore, methods for the preparation of enantiomerically pure chiral amines are desirable. Enzymatic methods based on kinetic resolution have been developed, but these suffer from a theoretical yield of 50%. omega-Transaminases present an attractive option as they catalyze the asymmetric synthesis of chiral amines from the corresponding prochiral ketones. Certain characteristics of omega-transaminases, such as limited substrate scope and product inhibition, may limit their utility in industrial applications. In an effort to identify omega-transaminases with improved properties for industrial use, mutants of Vibrio fluvialis omega-transaminase were prepared and evaluated for chiral amine synthesis. This investigation has resulted in the identification of V. fluvialis omega-transaminase mutants with improved properties for a range of ketone substrates.

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Enzymatic synthesis of chiral amines with omega-transaminase

Background

Enantiomerically pure chiral amines are useful intermediates for the synthesis of pharmaceutical compounds.  Therefore, methods for the preparation of enantiomerically pure chiral amines are desirable.  Enzymatic methods based on kinetic resolution have been developed, but these suffer from a theoretical yield of 50%.  omega-Transaminases present an attractive option as they catalyze the asymmetric synthesis of chiral amines from the corresponding prochiral ketones.  Certain characteristics of omega-transaminases, such as limited substrate scope and product inhibition, may limit their utility in industrial applications.  In an effort to identify omega-transaminases with improved properties for industrial use, mutants of Vibrio fluvialis omega-transaminase were prepared and evaluated for chiral amine synthesis.  This investigation has resulted in the identification of V. fluvialis omega-transaminase mutants with improved properties for a range of ketone substrates. 

Methods

General screening procedure:

Screening reactions were carried out at 1 ml scale under the following conditions: 100 mM – 200 mM ketone substrate, 100 mM – 200 mM (S)-1-phenylethylamine, 2 mM pyridoxal phosphate, 40 g/l – 80 g/l E. coli cells, 100 mM potassium phosphate (pH 7.0), 30°C, 800 rpm (Thermomixer-R).   The production of amine products was determined by HPLC after derivatization with Marfey’s reagent.  For HPLC analysis, reaction samples were added to 9 volumes of acetonitrile and mixed for 2 minutes at 750 rpm.  The diluted reaction sample (50 microliters) was treated with 1M aqueous sodium bicarbonate (5 microliters) and Marfey’s reagent (N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide, 200 microliters of a 5 g/l solution in acetonitrile) for 1 h at 40°C.  The derivatization reaction was quenched with 5 microliters of 1M HCl, diluted with 50 microliters of acetonitrile and analyzed by HPLC (Gemini C18 (3 microliters, 4.6 x 150 mm), 1 ml/min 55:45 1% triethylamine in water adjusted to pH 3 with phosphoric acid:acetonitrile, detection at 340 nm.    

Preparation of transaminase variants:

The V. fluvialis aminotranferase wild type gene was synthesized by DNA 2.0 and cloned into the pET28b expression vector (Novagen/EMD Biosciences, San Diego, CA). The vector was transformed into the chemically competent BL21(DE3) Gold E. coli strain (Stratagene,

La Jolla

,

CA

) through the heat shock protocol described by the manufacturer and plated on LB/Kanamycin (50ug/mL) plates. Liquid cultures were grown by placing a single colony in 3mL LB media at 37°C, 210 rpm, overnight. This culture was then diluted 1/50 in 25 or 50 mL Overnight Express TB media (reconstituted as directed, Novagen/EMD Biosciences,

San Diego

,

CA

) in a 125mL or 250mL, respectively, baffled vented flask and put at 30°C, 210 rpm, for 24 hours. The cells were then pelleted and frozen at -20°C for use in the scre...