title: An enzymatic assay for high-throughput screening of cytidine-producing microbial strains.
abstract: Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH3, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058-10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more).
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors: Huina
authors: H
authors: Dong
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors: Yongfei
authors: Y
authors: Liu
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; The Light Industry Technology and Engineering, School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China.
authors: Xin
authors: X
authors: Zu
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors: Ning
authors: N
authors: Li
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors: Feiran
authors: F
authors: Li
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; National Engineering Laboratory for Industrial Enzymes, Tianjin, 300308, China.
authors: Dawei
authors: D
authors: Zhang
mesh_terms: D001419:Bacteria / Q000378:metabolism*; D003562:Cytidine / Q000032:analysis*; D003564:Cytidine Deaminase / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D005285:Fermentation; D057166:High-Throughput Screening Assays / Q000379:methods*; D007215:Indophenol / Q000737:chemistry; D015203:Reproducibility of Results; D014529:Uridine / Q000737:chemistry
chemical_list: D007215:Indophenol; D003562:Cytidine; D003564:Cytidine Deaminase; D014529:Uridine