棒状链霉菌原生质体再生和克拉维酸产量变化的研究

Study on the protoplast regeneration and the changing of clavulanic acid production of Streptomyces clavuligerus

在棒状链霉菌菌种选育过程中，采用原生质体形成与再生，结合物理和化学诱变处理，得到产克拉维酸稳定性好、产量高的菌株。试验结果表明，改进后的方法原生质体形成率达到１０￣６／ｍｌ数量级。原生质体再生后的菌株，对物理及化学诱变剂的敏感性增加，与再生前对照菌株相比，同剂量死亡率增加了１％～３５％。对未经过原生质体形成与再生的菌株用ＥＭＳ、ＵＶ、ＮＴＧ诱变处理所得到的１７１３只斜面和经再生后的菌株用同样诱变剂处理所得到的１２３０只斜面进行发酵试验。经效价测定表明，产量突变株的频率分布向正向移动，正变菌株出现频率较未再生突变株高２４．０６％，且出现了１．４６％较对照高一倍以上的在未再生诱变株中没有出现过的高产株，菌株稳定性提高，小菌落突变率由３１％降低到８％，而抗生素产量提高到原来的３２６％，斜面的保藏时间明显延长，传代后Ｆ＿３效价保持在９０％以上。

In the strain-improvement program of Streptomyces clavuligerus,protoplast regeneration combined with physical and chemical mutagenization was adopted to select high-yielding strains.Results showed,with improved method,protoplast regeneration rate attained the order of 106/ml.Compared with strains in control,the strains of protoplast-regenerated were more sensitive to physical and chemical mutagenic agent,death-rate of same dose increased 1%～35%,1713 strains without protoplast regeneration and 1230 protolast regenerated strains were mutagenized by EMS,UV and NTG respectively,then carried fermentation test.The titer-test showed,the curve of normal distribution has a positive move, rate of positive mutants is 24.06%higher than mutants without protoplast regeneration,the production of 1.46% strains was increased by more than 1 fold than the strains in control,the stability of strains was improved,small colony mutation rate decreased from 31%to 8%,antibiotics production was 326%higher than that of original strain,the storage time of strains was highly prolonged,and the titer of F3 kept over 90%.