Benzoylformic acid was synthesized from the hydrolysis of benzoyl cyanide by a newly isolated Rhodococcus sp. CCZU10-1. In this study, an aqueous–toluene biphasic system was developed for highly efficient production of benzoylformic acid from the hydrolysis of benzoyl cyanide. In the aqueous–toluene biphasic system, the phase volume ratio, buffer pH and reaction temperature were optimized. Using fed-batch method, a total of 932mM benzoylformic acid accumulated in the reaction mixture after the 10th feed. Moreover, enzymatic hydrolysis of benzoyl cyanide using calcium alginate entrapped resting cells was carried out in the aqueous–toluene biphasic system, and efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 14.26g benzoylformic acid g−1 dry cell weight (DCW) cell after 20 cycles of repeated use.
