The enantioselective hydrolysis of mandelonitrile with whole cells of a recombinant Escherichia coli expressing nitrilase activity was severely inhibited by the substrate at high concentrations (>300mM), which resulted in a low yield of the target product (R)-(-)-mandelic acid. To relieve the substrate inhibition and to enhance the (R)-(-)-mandelic acid productivity, eight water-organic solvent biphasic systems were attempted in this work. Toluene was found to be the most suitable solvent as the organic phase among the solvents tested. Various parameters were systematically examined and optimized in shake flasks. The phase volume ratio, buffer pH and reaction temperature were shown to be sensitive parameters affecting both the yield and the enantiopurity of product in the biphasic system. Under the optimized conditions, significant enhancement of substrate tolerance from 200mM to 500mM and average productivity from 179.6gl(-1)d(-1) to 352.6gl(-1)d(-1) were achieved. Subsequently, the biocatalytic hydrolysis of mandelonitrile was successfully carried out in a stirred reactor (2-l scale) by repeated use of the calcium alginate entrapped cells for 5 batches, affording 110.7g (R)-(-)-mandelic acid in 98.0% ee (enantiomeric excess) and a specific production of 13.8g (mandelic acid) g(-1) (cell), respectively.
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