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In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were screened. Among them, Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed between S. rimosus (N-cetylation function) and S. aureofaciens (p-hydroxylation function) and also between S. lividans and S. globisponts. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixed S. rmosus (pro- his-) and S. aureofaciens (ilv-) protoplasts with 40% (w/v) polyethylene glycol 3350 (PEG) for 3min gave 8.3 X 10-7 of fusion frequency. Treatment of mixed S. lividans (pant-) and S. globisponts (leu-) protoplasts with 50% (w/v) PEG for 3 min at 30oC gave 1.2 X 10-6 of frequency. Among the fused strains, up to 40-50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts of S. aureofaciens (2% frequency) lost their p-hydroxylation function.
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