Abstract
Immobilized cells of a recombinant Escherichia coli expressing nitrilase from Pseudomonas putida were used to catalyze the hydrolysis of mandelonitrile (2-hydroxy-2-phenylacetonitrile) to (R)-(−)-mandelic acid. The cells had been immobilized by entrapment in an alginate matrix. Conditions for the hydrolysis reaction were optimized in shake flasks and in a packed bed reactor. In shake flasks the best conditions for the reaction were a temperature of 40 °C, pH 8, biocatalyst bead diameter of 4.3 mm, sodium alginate concentration in the gel matrix of 2 % (w/v, g/100 mL), a cell dry mass concentration in the bead matrix of 20 mg/mL, an initial substrate concentration of 50 mM and a reaction time of 60 min. Under these conditions, the conversion of mandelonitrile was nearly 95 %. In the packed bed reactor, a feed flow rate of 20 mL/h at a substrate concentration of 200 mM proved to be the best at 40 °C, pH 8, using 4.3 mm beads (2 % w/v sodium alginate in the gel matrix, 20 mg dry cell concentration per mL of gel matrix). This feed flow rate corresponded to a residence time of 0.975 h in the packed bed.
Highlights
The conventional harsh chemical methods are extensively substituted by environment-friendly biocatalysts for the synthesis of pharmaceutically important intermediates (Kumar et al 2010)
Conditions for the hydrolysis reaction were optimized in shake flasks and in a packed bed reactor
This study reports the use of immobilized whole cells of the recombinant Escherichia coli BL21 (DE3) for producing (R)-(-)-mandelic acid from mandelonitrile in shake flask and packed bed reactor
Summary
The conventional harsh chemical methods are extensively substituted by environment-friendly biocatalysts for the synthesis of pharmaceutically important intermediates (Kumar et al 2010). Nitrilase-mediated production of (R)-(-)mandelic acid has significant advantage over chemical methods of production; it offers excellent enatioselectivity and above all possibility of carrying out dynamic kinetic resolution which provides 100 % theoretical yield of the product (Yamamoto et al 1991; Banerjee et al 2006; Singh et al 2005; Xue et al 2011) (Fig. 1). This study reports the use of immobilized whole cells of the recombinant Escherichia coli BL21 (DE3) for producing (R)-(-)-mandelic acid from mandelonitrile in shake flask and packed bed reactor. P. putida was reported as an ideal candidate for stereo-selective hydrolysis of mandelonitrile, considering its higher growth rate, higher reaction rate and higher stability as compared with other nitrilase-producing microorganisms. P. putida nitrilase attains comparable specific activity within a shorter period of time and the fermentation time was reported to be lower for biocatalyst generation (Kaul et al 2004)
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