PROTOPLAST FORMATION AND REGENERATION IN PAH-DEGRADING BACTERIAL STRAINS Pseudomonas AND Sphingomonas

摘要

To improve the formation and regeneration frequency of protoplasts in Gram-negative bacteria, the effects of some factors on protoplast isolation and regeneration from polycyclic aromatic hydrocarbons (PAH)-degrading bacterial strains Pseudomonas sp. GP3A (GenBank EU233280) and Sphingomonas sp. GY2B (GenBank DQ139343) were investigated. Pretreatment with penicillin and EDTA enhanced the protoplast formation in both the bacterial strains. The optimal condition for GP3A and GY2B protoplast formation was in 5 mg/ml lysozyme at 37 oC for 100 min and 80 min in a shaking culture, respectively. The double culture had a higher regeneration frequency than the mixed or single culture. The regeneration medium containing 20 mM CaCl2, 20 mM MgCl2, and 50 mM L-serine that was cultured at 30 oC showed higher regeneration frequency for GY2B. For GP3A, regeneration medium supplemented with 30 mM CaCl2, 20 mM MgCl2, and 100mM L-serine incubated at 30 oC could improve the regeneration frequency. GP3A and GY2B protoplast has been fused and regenerated on selective agar.