论文标题:产氨短杆菌Corynebacterium ammoniagenes转化乳清酸（OA）生成尿苷酸（UMP）的研究
Research of Conversion of Orotic Acid to Uridine-5-monophosphate by Corynebacterium Ammoniagenes
论文作者 李慧
论文导师 钱秀萍,论文学位 硕士,论文专业 微生物学
论文单位 上海师范大学,点击次数 732,论文页数 81页File Size1022k
2005-05-01论文网 http://www.lw23.com/lunwen_85342/ 产氨短杆菌;乳清酸;尿苷酸;菌种选育;发酵转化;细胞转化
Corynebacterium ammoniagenes; Orotic acid; Uridine-5-monophosphate; strain breeding; fermentable conversion; enzymatic conversion
尿嘧啶核苷酸(Uridine 5’monophosphate,UMP)是一种重要的药物、药物前体和食品添加剂。本论文的目的在于研究以乳清酸为底物,利用微生物转化生成UMP。本研究采用HPLC 法和液质联用法确证了产氨短杆菌Corynebacterium ammoniagenesR248 能将底物乳清酸转化生成UMP。研究了发酵转化和细胞转化反应体系中几种因素对R248 菌株转化UMP 的影响,结果表明,乳清酸1g/L、生物素50μg/L、pH 5.0、25℃转化培养4 天后, UMP 生成能力为420mg/L。乳清酸20g/L、细胞量20%、pH 7.2、250ml 三角瓶装液量5ml、32℃反应26 小时后,R248 的细胞转化能力为991mg/L。采用链霉素抗性、卡那霉素抗性和产物结构类似物抗性等作为筛选手段,对产氨短杆菌R248 进行离子束和紫外诱变,选育出一株UMP 生成能力比出发菌株R248 提高7 倍突变株M14。对高活性突变株M14 的细胞转化和发酵转化条件进行优化。结果表明,发酵转化中突变株M14 转化能力为0.98g/L;突变株M14 在乳清酸20g/L、细胞量30%、pH 8.06、葡萄糖50g/L、磷酸盐15g/L、21×180mm 试管装液量5ml、32℃细胞转化6 小时后UMP 的转化能力为2.1g/L。产氨短杆菌R248 发酵转化和细胞转化生成UMP,高活性突变菌株M14 的选育,以及突变株M14 转化条件的优化,对进一步研究产氨短杆菌Corynebacterium ammoniagenes 转化乳清酸生成UMP 具有重要意义。
Uridine-5-monophosphate (UMP) is an important medicine, pharmaceutical precursor andfood addition. A special aim of our research was to indicate the possibility that UMP could beproduced from orotic acid.The analysis of HPLC and MS-HPLC were employed to corroborate the conversion oforotic acid to UMP by Corynebacterium ammoniagenes. The results revealed that the yield ofUMP was 420mg/L when Corynebacterium ammoniagenes R248 was incubated at 25℃, pH5.0 in a medium containing 1g/L orotic acid,50μg/L biotin for 4 days,and 991mg/L of UMPwas accumulated in the enzymatic conversion reaction which consisted of 20g/L orotic acid,20%Corynebacterium ammoniagenes R248 cells.Five ml of the reaction mixture was poured intoa 250-ml flask and incubated at 32℃,pH 7.2 for 26 hours.The screening methods of streptomycin, kanamycin and pyrimidine analogue 5-FC wereused when the strain was treated with UV and N+.The mutant M14 of Corynebacteriumammoniagenes was obtained whose UMP yield was 7-fold higher than the parent strain R248.The conditions of fermentable and enzymatic conversion of M14 were optimized.In thefermentable conversion, 0.98g/L UMP was accumulated.The optimum enzymatic reactioncontained 20g/L orotic acid,30% wet cells,50g/L glucose, 15g/L KH2PO4.Five ml of thereaction mixture was poured into a 21×180mm tube and incubated at 32℃for 6 hours. The pHof the reaction mixture was maintained at 8.06. Ultimately,the yield of UMP was 2.1g/L.The fermentable and enzymatic conversion to accumulate UMP by Corynebacteriumammoniagenes R248, screening mutant with high capacity and optimizing the convertconditions are essential to research the conversion of orotic acid to UMP by Corynebacteriumammoniagenes further.
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