龟裂链霉菌高产土霉素菌株的诱变筛选及其发酵条件

Screening of High Yielding Oxytetracycline Producing Strain Streptomyces rimosus and Optimization of Its Fermentation Condition

对出发菌株186-6冻存的孢子悬液进行了活化及复性,先后经过6轮筛选,获得1株高产菌株,命名为P-11.与186-6相比,高产菌株的发酵效价提高了20.75%.对高产菌株进行5次斜面传代,对其遗传特性进行考察,与原始效价相比,高产菌株的效价分别为99.8%,100.2%,98.5%,97.4%,96.6%,说明高产菌株的遗传稳定性良好.对高产菌株特性考察的结果表明:菌种纯度为98.4%,磷耐受性比原始菌株186#有很大提高.代谢参数研究表明,其前期代谢较186#菌株慢.对原发酵培养基进行优化,得到新配方:淀粉120g/L,黄豆饼粉15.5g/L,棉籽饼粉5g/L,CaCO315g/L,(NH4)2SO414.5g/L,NaCl 4.7g/L,玉米浆10g/L,KH2PO40.1g/L,消沫剂0.1g/L,CoCl 0.01g/L,淀粉酶0.12g/L.与原配方相比,土霉素发酵效价提高了25%左右.不同氨基酸对土霉素效价的影响结果表明:对提高土霉素效价起积极作用的3种氨基酸是Thr,Ser和Phe.

After activation and regeneration of the spore suspension of Streptomyces rimosus 186-6, a target strain named P-11 was obtained through compound mutagenesis of microwave and LiC1, random breeding, NTG-induced mutagenesis, breeding of resistance to its metabolic products, NaNO2-induced mutagenesis,and screening of phosphate resistance. The fermentation titer of strain P-11 was higher than that of strain 186-6 by 20. 75%. Strain P-11 was subcultured on slant for 5 times to study its hereditary capacity. Compared to the original titer, the fermentation titers of each generation of strain P-11 were 99.8% ,100.2 %,98.5 % ,97.4 % and 96.6 % respectively,which showed that strain P-11 had good genetic stability. Study on the features of strain P-11 indicated that the purity of strain P-11 was 98.4 % and its phosphorus tolerability was improved. The metabolic parameters of strain P-11 declared that its early metabolism was lower than that of strain 186-6. To optimize the fermentation medium,response surface methodology （RMS） was used, and a new recipe, which contains starch 120 g/L,soybeans meal 15.5 g/L,cottonseed meal 5 g/L,CaCO3 15 g/L,（NH4）2SO4 14.5 g/L,NaCl 4.7 g/L,corn syrup 10 g/L,KH2PO4 0.1 g/ L,defrother 0.1 g/L,CoC1 0.01 g/L,and amylase 0.12 g/L,was obtained. Compared to the original recipe, the new recipe increased the titer of oxytetracycline by about 25 %. The results of the effects of amino acid on the titer of oxytetracycline indicated that Thr, Ser and Phe played an active role in improving the titer of oxytetracycline.