不同搅拌策略对破伤风梭状芽孢杆菌发酵生产破伤风毒素的影响

Effects of different Clostridium tetani fermentation stirring strategies on tetanus toxin production

目的考察不同搅拌策略对破伤风梭状芽孢杆菌(Clostridium tetani,C.tetani)大规模发酵生产破伤风毒素的影响,建立稳定的C.tetani发酵工艺。方法在1500 L发酵罐中,采用不同搅拌策略发酵C.tetani。发酵过程中测定发酵液菌体浓度和毒素絮状单位(flocculation unit,Lf),并应用实时荧光定量PCR测定tent基因和tetR基因的表达量;发酵液经深层过滤、超滤、两步盐析等工艺纯化后得到精制毒素,测定精制毒素的絮状单位、体积和蛋白氮含量,并计算其产量、纯度和收率。结果不同搅拌策略对C.tetani的生长、产毒、tent基因和tetR基因的表达量以及精制毒素的产量、纯度和收率均有显著影响(P均<0.05);Stir-3组批培养结束时的平均产毒量为(110±10)Lf/mL,精制毒素的平均产量为(88±9)Lf/mL培养基,均高于其他试验组(P均<0.05);Stir-3组精制毒素的平均收率和纯度分别为79.8%±3.0%和(2798±83)Lf/mg PN,高于Stir-1和Stir-4组(P均<0.05),但与Stir-2组相比差异无统计学意义(P>0.05)。结论C.tetani大规模发酵时,可以采取两阶段搅拌控制策略(前12 h不搅拌,12 h后连续搅拌)以提高破伤风毒素的产量和纯度,不同搅拌策略可通过影响tent基因和tetR基因的表达量进而影响C.tetani的产毒量。

Objective To establish a stable process for fermentation of Clostridium tetani(C.tetani)by assessing the effects of different stirring strategies on tetanus toxin production by large-scale C.tetani fermentation.Methods Fermenters of 1500 L capacity were used for C.tetani fermentation with different stirring strategies.During the fermentation process,the C.tetani concentration and the tetanus toxin yield(flocculation unit)in the fermentation broth were measured.The tent and tetR gene expression levels were determined by real-time quantitative fluorescence PCR analysis.Refined tetanus toxin was obtained from purification by depth filtration,ultra filtration,and two-step salting-out extraction.The flocculation unit,volume and protein nitrogen content of refined tetanus toxin was measured to calculate its yield,recovery rate and purity.Results Significant differences were observed between stirring strategies regarding their effects on C.tetani growth,toxin production,tent and tetR gene expression,refined toxin yield,purity,and recovery rate(all P<0.05).The average toxin production at the end of the C.tetani fermentation process was(110+10)Lf/mL and the average refined toxin production was(88±9)Lf per milliliter of culture medium in the Stir-3 group,which were significantly higher than those in other groups(all P<0.05).The average recovery rate and purity of refined toxin in the Stir-3 group were 79.8%±3.0%and(2798±83)Lf/mg PN respectively,which were significantly higher than those in the Stir-1 and Stir-4 group(all P<0.05),but the differences between the Stir-2 and Stir-3 groups were not significant(P>0.05).Conclusion In large-scale C.tetani fermentation,a two-stage stirring control strategy(no stirring for the first 12 hours,then continuous stirring)can be adopted to improve the yield and purity of tetanus toxin.Different stirring strategies can affect the toxin production of C.tetani by affecting the expression levels of tent gene and tetR gene.