Highly Efficient Chemoenzymatic Synthesis of l-Phosphinothricin from N-Phenylacetyl-d,l-phosphinothricin by a Robust Immobilized Amidase.

A chemoenzymatic strategy was developed for the highly efficient synthesis of l-phosphinothricin employing a robust immobilized amidase. An enzymatic hydrolysis of 500 mM N-phenylacetyl-d,l-phosphinothricin resulted in 49.9% conversion and 99.9% ee of l-phosphinothricin within 6 h. To further evaluate the bioprocess for l-phosphinothricin production, the biotransformation was performed for 100 batches under a stirred tank reactor with an average productivity of 8.21 g L-1 h-1. Moreover, unreacted N-phenylacetyl-d-phosphinothricin was racemized and subjected to the enzymatic hydrolysis, giving l-phosphinothricin with a 22.3% yield. A total yield of 69.4% was achieved after one recycle of N-phenylacetyl-d-phosphinothricin. Significantly, this chemoenzymatic approach shows great potential in the industrial production of l-phosphinothricin.

Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor.

To develop a highly efficient method for aprepitant chiral intermediate (S)-4-fluorophenylglycine, a continuous reaction system was established in packed bed bioreactor using amidase covalently immobilized on epoxy resin as biocatalyst. The epoxy resin was firstly modified by metal-chelate method and functional groups (Cu2+-IDA) generated were able to rapidly adsorb amidases, which were further covalently bound onto the modified resin with 90.1% immobilization yield and 80.2% activity recovery. The immobilized amidase exhibited excellent thermal stability with the longest half-life of 1456.8 h at 40 °C ever reported. (S)-4-fluorophenylglycine was continuously produced using the reaction system with 49.9% conversion, 99.9% ee, and an outstanding space-time yield of 5.29 kg L-1 d-1. Moreover, the efficient reaction system exhibited a high operational stability and retained 86.3% catalytic activity after 25-day continuous operation. This efficient continuous bioprocess presents great industrial potential for large-scale production of (S)-4-fluorophenylglycine.

Efficient chemoenzymatic synthesis of (S)-α-amino-4-fluorobenzeneacetic acid using immobilized penicillin amidase.

An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of racemic N-phenylacetyl-4-fluorophenylglycine. The optimum temperature, pH and agitation rate of the reaction were determined to be 40 °C, 9.5 and 300 rpm, respectively. Kinetic resolution of 80 g L-1N-phenylacetyl-4-fluorophenylglycine by immobilized amidase 20 g L-1 resulted in 49.9% conversion and >99.9% e.e. within 3 h. The unreacted N-phenylacetyl-4-fluorophenylglycine can be easily racemized and then recycled as substrate. The production of (S)-α-amino-4-fluorobenzeneacetic acid was further amplified in 1 L reaction system, affording excellent conversion (49.9%) and enantioselectivity (99.9%). This chemoenzymatic approach was demonstrated to be promising for industrial production of (S)-α-amino-4-fluorobenzeneacetic acid.

Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli.

BACKGROUND: Ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is a versatile chiral precursor for many pharmaceuticals. Although several biosynthesis strategies have been documented to convert ethyl 4-chloro-3-oxobutanoate (COBE) to (R)-CHBE, the catalytic efficiency and stereoselectivity are still too low to be scaled up for industrial applications. Due to the increasing demand of (R)-CHBE, it is essential to explore more robust biocatalyst capable of preparing (R)-CHBE efficiently. RESULTS: A stereoselective carbonyl reductase toolbox was constructed and employed into the asymmetric reduction of COBE to (R)-CHBE. A robust enzyme designed as BgADH3 from Burkholderia gladioli CCTCC M 2012379 exhibited excellent activity and enantioselectivity, and was further characterized and investigated in the asymmetric synthesis of (R)-CHBE. An economical and satisfactory enzyme-coupled cofactor recycling system was created using recombinant Escherichia coli cells co-expressing BgADH3 and glucose dehydrogenase genes to regenerate NADPH in situ. In an aqueous/octanol biphasic system, as much as 1200 mmol COBE was completely converted by using substrate fed-batch strategy to afford (R)-CHBE with 99.9 % ee at a space-time yield per gram of biomass of 4.47 mmol∙L-1∙h-1∙g DCW-1. CONCLUSIONS: These data demonstrate the promising of BgADH3 in practical synthesis of (R)-CHBE as a valuable chiral synthon. This study allows for the further application of BgADH3 in the biosynthesis of chiral alcohols, and establishes a preparative scale process for producing (R)-CHBE with excellent enantiopurity.

Chemoenzymatic synthesis of (S)-duloxetine using carbonyl reductase from Rhodosporidium toruloides.

A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000mM) in a space-time yield per gram of biomass (DCW) of 22.9mmolL(-1)h(-1)gDCW(-1) at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee.

Asymmetric synthesis of optically active methyl-2-benzamido-methyl-3-hydroxy-butyrate by robust short-chain alcohol dehydrogenases from Burkholderia gladioli.

Three short-chain alcohol dehydrogenases from Burkholderia gladioli were discovered for their great potential in the dynamic kinetic asymmetric transformation of methyl 2-benzamido-methyl-3-oxobutanoate, and their screening against varied organic solvents and substrates. This is the first report of recombinant enzymes capable of achieving this reaction with the highest enantio- and diastereo-selectivity.