Bioconversion of Benzoic Acid to cis, cis-Muconic Acid by Corynebacterium pseudodiphtheriticum

Abstract

The culture conditions for bioconversion of benzoic acid to cis, cis-muconic acid by Corynebacterium pseudodiphtheriticum M2128 were investigated. Using optimized medium and fedbatch fermentation conditions in a 5-L fermentor, cis, cis-muconic acid was produced from benzoate to a final concentration of 3.05 g/L after 96 h-cultivation. The molar conversion was 47%. Catechol 1,2-dioxygenase of Strain M2128 was purified 145-fold as measured by specific activity from a cell-free extract by ammonium sulfate fractionation, DEAD-Sepharose CL-6B, Sephadex G-150 and Phenyl-Toyopearl column chromatography, The overall yield of the purified enzyme was 37%. The purified enzyme gave an apparent single protein band on SDS-PAGE. The molecular weight of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sephadex G-150 was 38,000 and 75,000, respectively, indication that catechol 1,2-dioxygenase from Strain ＜2128 is a dimmer. The purified enzyme was stable over the pH range 7.0 to 11.0 at 30℃ for 2 h. The optimal pH and temperature were 7.0 and 45℃, respectively. The product of the purified enzyme, when it acted on catechol, was identified as cis, cis-muconic acid using absorption spectra, TLC and HPLC.