Strain improvement of Arthrobacter simplex by protoplast fusion

Abstract

An L-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium, Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20 μg ml-1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20-40% polyethylene glycol 6000. The fusion frequency was about 1.5-1.7 x 10-3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20-60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement of Arthrobacter strains for enzyme production.