Study On The Mutation Breeding Of Protoplasts Of Cephalosporin C Producing Strain Cephalosporium Acremonium HC-4

Cephalosporin C is a kind ofβ-lactams antibiotic produced by Cephalosporium acremonium. 7-Aminocephalosporanic acid-the original nucleus of cephalosporin C, is one of the important intermediate products of P-lactam antibiotic. Its ratio in antibiotics is arising year by year. Semisynthetic antibiotic of cephalosporin is widely used in antibiotic medicine and antibacterials because it is hypoallergenic and has broad antibacterial spectrum. So, expansion of production scale and increase of production level are the major goals for the pharmaceutical companies. Strain breeding has made outstanding contributions for the fermentation industry to increase production. As an effective breeding method, the technology of protoplast mutagenesis has been widely used in Strain breeding.In this study, Cephalosporium acremonium HC-4was taken as original strain, selected the mutation strains with high productivity by protoplasts mutagenesis induced by LiCl-UV mutagenesis and microwave mutagenesis. The results were as follows:(1) The protoplasts were prepared by enzymatic hydrolysis and the regeneration rate of protoplasts was studied. The mycelia were cultured for 120 hours. The enzymatic hydrolysis conditions for preparing the protoplasts were as follows:the mixed enzyme solution included 1% snailase and 1% cellulase, the enzymolysis temperature was 30℃, the rotate speed of table concentrator was 50 r·min-1, the enzymolysis time was 3.5 h. The osmotic pressure stabilizer was KC solution. Under these conditions, the concentration of protoplasts was 4.62x107/mL. Coated the protoplasts on the regeneration medium, the regeneration rate reached 10.18%.(2) The mutagenesis parameters and mutagenesis effects of all mutative factors in the experiment were studied. It was concluded that the mutagenesis effect of LiCl-UV was more intense than that of microwave. The optimum mutagenesis conditions were that: the concentration of LiCl was 0.50% and UV irradiation time is 30 s or 60 s. Forward mutation ratios reached 50.00%.(3) Selected the mutation strains with high productivity by protoplasts mutagenesis induced by LiCl-UV mutagenesis and microwave mutagenesis. 121 mutant strains were obtained (there were 61 mutant strains of LiCl-UV mutagenesis, there were 60 mutant strains of microwave mutagenesis). The staphylococci aureus was indicator strain.45 positive mutant strains and 76 negative mutant strains were obtained by using the filter paper method in the preliminary screening. Selected 13 positive mutant strains with higher antibacterial activity and researched their genetic stability. The antibacterial activity of most positive mutant strains reduced greatly after subculturing. A strain, named ZW3-17, had steady hereditary trait. The ratios of the area of inhibition zones between ZW3-17 and HC-4 were R1:1.314209, R2:1.317152, R3:1.084906, R4:1.200840, R5:1.309286 after subculturing five times. Determined the titer of Cephalosporin C in its fermented liquor by HPLC, the titer of Cephalosporin C reached 22.923 g/L. This was increased 11.972% in compare with HC-4 and its stabilization test appeared to be stable.