Enzymatic production of 2-amino-2,3-dimethylbutyramide by cyanide-resistant nitrile hydratase

Abstract

A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l(-1)) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity were further improved to 50 g l(-1), 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application.

High-Level Expression of Nitrile Hydratase in Escherichia coli for 2-Amino-2,3-Dimethylbutyramide Synthesis

“…The NHase library, containing 24 recombinant NHases from different sources, was reserved in this lab (Table S1). The original nitrile converting strains were obtained from Whole cells of Rhodococcus boritolerans CCTCCM 208108 harboring NHase were exploited for biosynthesis of ADBA [10][11][12]. In a biphasic system (n-hexane and water), a yield of 91% was reached after 19 h of reaction at 10 • C in fed-batch mode [10].…”

“…The original nitrile converting strains were obtained from Whole cells of Rhodococcus boritolerans CCTCCM 208108 harboring NHase were exploited for biosynthesis of ADBA [10][11][12]. In a biphasic system (n-hexane and water), a yield of 91% was reached after 19 h of reaction at 10 • C in fed-batch mode [10]. However, in the original strains, NHases are often accompanied by amidases and nitrilases which could transform the amide products into carboxylic acids [13].…”

“…After incubation at 35 • C for 6 min, the reaction was terminated by the addition of 10 µL hydrochloric acid (6.0 M). After centrifugation at 10,625× g for 10 min, the supernatant was analyzed by GC to determine the ADBA formation [10,11]. One unit of NHase activity (U) was defined as the amount of whole cells or free enzymes needed to produce 1 µmol of ADBA per minute at 35 • C and pH 7.0.…”

“…It is believed that metal ions at the active site improve the hydration process and aid in the enzyme folding [7]. Whole cells of Rhodococcus boritolerans CCTCCM 208108 harboring NHase were exploited for biosynthesis of ADBA [10][11][12]. In a biphasic system (n-hexane and water), a yield of 91% was reached after 19 h of reaction at 10 °C in fed-batch mode [10].…”

A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases

“…Nitrile metabolizing enzymes are of outstanding synthetic importance because of their ability to afford amides, carboxylic acids, amines and cyanohydrins, compounds used as building blocks in the pharma and chemical industries ( van Pelt 2011 , Chen 2009 , Lin 2012 , Yamada 2001 ). They can also be potent biocatalysts for detoxification of anthropogenic toxic nitrile pollutants that are widespread throughout the world ( Vesela 2012 , Vesela 2010 ).…”