Biocatalyst preparation from Pseudomonas putida SM-6 for conversion of dl-lactate to pyruvate

The production of pyruvate using enzymatic methods is a valuable process for its cheap substrate, high conversion ratio, simple composition of reaction, and convenience of recovery. Cell-free extracts from Pseudomonas putida SM-6 that contain both lactate oxidase (LOD) and catalase were employed as catalysts for biocatalysis. In order to obtain an efficient and cost-effective biocatalyst, the cultivation of P. putida SM-6 both in shake flask and at bioreactor level was investigated. On enzyme production, the suitability of glucose instead of lactate as a carbon source was studied. Maximum LOD production occurred when a mixture of lactate and glucose was used as a carbon source. Lactate was assimilated first and the increase of nitrogen concentration had a positive effect on cell growth and glucose assimilation. The optimum cultivation temperature was 29-31 °C, and the neutral or basic initial medium pH was in favor of both cell growth and LOD production. The interrelation between various process parameters was evaluated for the cultivation of P. putida SM-6 in submerged culture. Using a fed-batch cultivation strategy in a 2-l bioreactor, an increase in cell mass was achieved but without a concomitant increase in LOD production compared with cultivation in shake flask. Bioconversion of lactate to pyruvate was carried out using cell-free extract acquired by cell disruption of P. putida SM-6. Ethylenediaminetetraacetic acid (EDTA) was added into the biocatalytic system to prohibit the degradation of pyruvate by other enzymes in the cell-free extract. As a result, on a preparative scale, cell-free extract which contained 3.04 mg/ml crude enzyme (10.0 U/ml LOD, 877 U/ml catalase) produced pyruvate of 423 mM from dl-lactate of 660 mM in a reaction period of 22 h. The molar conversion yield of pyruvate on the basis of the amount of lactate consumed was 0.80 g/g.