Peroxidase production from hairy root cultures of red beet (Beta vulgaris)

Abstract

The genetically transformed roots of red beet have been shown, for the first time, to produce very high levels of peroxidase (POD; EC 1.11.1.7) accounting for 1.21 x 10⁶ Units L^-1. Of the ten clones established using different strains of Agrobacterium rhizogenes, one was that from the strain LMG-150, three each from A 2/83, A 20/83 and A4. All the clones showed true integration of T-DNA when tested by PCR and Southern hybridization methods. Each clone differed significantly from the others in growth, hormone dependency and POD production where LMG-150 produced highest biomass (140 g FW L^-1) as well as POD (ranging from 8000-9000 U g^-1 FW and 1.18 x 10⁶ U L^-1 with a specific activity of 600 U mg^-1 protein) on hormone-free medium, both in shake-flask as well as in bioreactor with a further enhancement to 1.21 x 10⁶ U L^-1 upon the addition of extra calcium chloride (5 mM). PAGE with active staining showed 4 distinct bands of R_m 0.06, 0.16, 0.25, 0.38 and 0.46 in the biomass and bands at R_m 0.06, 0.16, 0.25 and one extra band of R_m 0.575 in the spent medium where isozymes of R_m 0.38 and 0.46 were totally absent. The pH optima and other properties were grossly comparable with the standard horse-radish POD (HRP) with better thermal stability than HRP and therefore, the present source appears to offer a cheaper and additional alternative for the commercial production of POD.