胞苷生产菌的选育及发酵

Screening and fermentation of cytidine-producing Bacillus subtilis mutants

目的选育胞苷脱氨酶缺失枯草芽孢杆菌,提高胞苷发酵单位,并对间歇补料分批发酵方式进行了初步研究。方法(1)以胞苷脱氨酶缺失枯草芽孢杆菌CDS36为突发菌株,对其进行紫外诱变,经6-杂氮尿嘧啶(6AU)、5-氟胞苷(5FCR)等进行定向抗性筛选;(2)采用间歇补料分批发酵,葡萄糖初始浓度10.0%,48h后补加10.0%,72h补加5.0%的补料方式培养,并测定发酵单位。结果(1)通过紫外诱变和抗性筛选得到了6-杂氮尿嘧啶、5-氟胞苷抗性的突变株CDS36-800-17,抗6-杂氮尿嘧啶和5-氟胞苷的临界浓度分别为1000mg/L和800mg/L;(2)通过间歇补料分批发酵方式发现培养96h,胞苷发酵单位可达4.86g/L,培养120h可达5.58g/L,比未经补料分批发酵时提高了20.0%。结论(1)筛选获得胞苷发酵单位较高的突变株CDS36-800-17,并具有较好的遗传稳定性,可稳定发酵;(2)间歇补料分批发酵方式可提高胞苷产量。

Objective To derive a mutant which can increase the yield of cytidine from Bacillus subtilis which deficient in eytidine deaminase and observe the process of eytidine production of intermittent fed-batch fermentation. Methods （ 1 ） Inducing Bacillus subtilis CDS36 （deficient in eytidine deaminase） by ultraviolet, the mutant was selected directionally and stepwisely on the agar plates containing 6AU and 5FCR. （2）Intermittent fed-batch fermentation： 10% glucose was fed 48 hours later and 5% glucose was fed again 72 hours later, when the initial glucose concentration was 10%, the yield of cytidine was detected . Results （1）a mutant Bacillus subtilis CDS36-800-17 resisatnat to 6AU and 5FCR was otained. The critical resistance concentrations of 6AU and 5FCR were 1000mg/L and 800mg/L ,respectively; 2）The yield of eytidine was 5.58g/L and that of eytidine was 4.86g/L 96 and 120 hours after fermentation, Cytidine production was increased 20% through feed fermentation than through batch fermentation. Conclusion Cytidine overproducing mutant, Bacillus subtilis CDS36-800-17 with stable heredity and fermentation has been obtained,.Intermittent fed-batch fermentation methods can enhance the yoel of cytidine.