Process selection and characterisation for the biocatalytic hydration of poorly water soluble aromatic dinitriles

The biotransformation of poorly water soluble aromatic dinitriles is of industrial and scientific interest. Although processes do exist for the transformation of water soluble nitriles, such as acrylonitrile, no description of a process suitable for the large scale bio trans formation of poorly water soluble nitriles appears in the literature. In this work we illustrate a systematic design procedure for optimising the production of 3-cyanobenzamide from 1,3-dicyanobenzene (1,3-DCB). The regio-selective nitrile hydratase (NHase) of the well characterised Rhodococcus R312 strain was initially selected as catalyst. Isolation of the NHase at process scale however was not feasible due to the rigid cell wall of the bacteria and the poor stability of the isolated enzyme. The whole cell form of the biocatalyst was thus used even though the activity of the associated amidase could overmetabolise the amide product into the corresponding acid. To overcome productivity limitations imposed by the characteristically low aqueous solubility of this class of substrate (similar to 0.34 g l(-1) in the case of 1,3-DCB) the use of an aqueous-organic two-phase bioreactor was investigated. After screening a wide range of solvents to act as a substrate reservoir toluene was selected as the organic phase due to the most favourable combination of Log P value (2.9) and 1,3-DCB saturation concentration (similar to 30 g l(-1)). The effects of phase volume ratio (0.05-0.3), wet weight biomass concentration (1.25-200 g(ww) l(-1)) and substrate concentration in the organic phase (5-25 g l(-1)) were then combined in a process map to define a suitable operating window where the maximum space-time yield of amide formation could be obtained. Compared to a sin.-le-phase transformation, the two-phase process yielded 12 times as much of the amide product of which less than 8% w/w was lost due to over-metabolism.