Stereoselective Nitrile Hydrolysis by Immobilized Whole-Cell Biocatalyst

Abstract

The present work attempts to deal with the stability and reusability aspect of nitrilase from Alcaligenes faecalis for the production of (R)-(-)-mandelic acid. Four entrapment matrixes were screened to search for a suitable support, and alginate was found to have significant process advantages over its other counterparts. Thermodynamic analysis allowed us to account for decreased enantioselectivity (E) as a result of immobilization. The system was also characterized based on the Thiele modulus (phi). Efficient reusability of the biocatalyst up to 35 batches was achieved by immobilization as compared to 9 batches for free cells, and cross-linking extended it further to 40 batches. Finally, synthetic utility of the immobilized biocatalyst was demonstrated on a preparative scale to produce 640 g of (R)-(-)-mandelic acid with 97% enantiomeric excess (ee).

Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

“…Thus, the immobilization of whole cells of Alcaligenes faecalis in various entrapment matrices has been previously compared. [19] However conventional polymers used for immobilization lead to escalation of mass transfer problems resulting from dilution of enzyme activity due to the addition of a large proportion of non-catalytic mass. Development of a carrier-free immobilized preparation resulted in a highly active and reusable biocatalyst that was free from diffusion restriction problems and highly stable under deleterious conditions.…”

“…We have demonstrated earlier for whole cells of Alcaligenes faecalis that entrapment of the cells resulted in a decreased enantioselectivity of the nitrilase activity due to diffusion restrictions imposed by the matrix. [19] Preparations of the soluble nitrilase and the CLEAs were incubated with (R,S)-mandelonitrile in the temperature range from 10-50 8C and the reactions were terminated after different time intervals. For exact determinations several data points were determined at the beginning of the reactions and only data with constant E-values during this time period were used for the calculations.…”

“…We have earlier demonstrated that the entrapment of whole cells of Alcaligenes faecalis which actively produce an (R)-specific mandelonitrile converting nitrilase in polymer matrices (such as alginate) leads to a significant improvement in the handling of the catalyst but at the cost of mass-transfer limitations and dilution of enzyme activity owing to the presence of a large proportion of non-catalytic mass. [19] In addition, the diffusion restricted system was also found to result in decreased enantioselectivity of the immobilized biocatalyst. One possible strategy to circumvent the problems inevitably connected with immobilized whole cell catalysts is to work with in vitro systems.…”

Biosynthesis of Iminodiacetic Acid from Iminodiacetonitrile by Immobilized Recombinant <i>Escherichia coli</i> Harboring Nitrilase

“…After freezing dehydration, all samples were dehydrated on a lyophilizer and mounted on specimen tubs with a silver paint, gold-coated, and examined by SEM at an acceleration of 1.2 kV [Bandhyopadhyay et al, 1999;Kaul et al, 2006].…”

“…Moreover, the free cells were not strong enough against the high shearing force of the stirrer, and difficult to recover from reaction mixtures by filtration or centrifugation [Kourkoutas et al, 2004]. Therefore, it is necessary to immobilize whole cells as biocatalysts in the reactions [Andres et al, 2003;Kaul et al, 2006;Sidira et al, 2010]. The conventional approaches for cell immobilization were microcapsule, crosslinking, adsorption and entrapment [Pilkington et al, 1998].…”