Autodisplay of Nitrilase from Alcaligenes faecalis in E-coli Yields a Whole Cell Biocatalyst for the Synthesis of Enantiomerically Pure (R)-Mandelic Acid

In the present study, a whole cell biocatalyst for the synthesis of (R)-mandelic acid from mandelonitrile was constructed. For this purpose, nitrilase from Alcaligenes faecalis subsp. faecalis ATCC 8750 was displayed on the surface of Escherichia coli by using Autodisplay. Localization of the nitrilase in the cell envelope of E. coli was monitored by SDS-PAGE and surface exposure was verified by its accessibility to externally added protease. The whole cell biocatalyst converted up to 2.6 mm of (R)-mandelic acid under optimum conditions at pH 7.5 and 45 degrees C within 24 h (1 mL culture, OD578 = 10). By using chiral HPLC, the ee value of the product was determined to be > 99%. The surface displayed nitrilase showed an apparent Km value of 3.6 mm and an apparent V-max value of 1 nmol min(-1) mL(-1) when a bacterial suspension of OD578 3 was used. Substrate inhibition by benzaldehyde was similar to that of the free enzyme. The whole-cell biocatalyst retained 55% of its initial (R)-mandelic acid production after 5 cycles of repeated use, and could be stored at -70 degrees C for 180 d without a substantial loss of activity. In addition the whole cell biocatalyst converted 9.3 mm phenylacetonitrile within 16 h.