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An enzymatic assay for high-throughput screening of cytidine-producing microbial strains.

Dong H, Liu Y, Zu X, Li N, Li F, Zhang D - PLoS ONE (2015)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: 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.

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).

No MeSH data available.


Related in: MedlinePlus

(A). Effect of different cytidine concentrations on CDA assay in H2O.Cytidine standard curves were conducted under standard reaction conditions in section 2.5 in H2O (B), LB medium (C), M9 medium (D) and 10-fold diluted LB medium (E), respectively. Each plot represents the average of three samples. Absorbance was measured using a microplate reader.
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pone.0121612.g003: (A). Effect of different cytidine concentrations on CDA assay in H2O.Cytidine standard curves were conducted under standard reaction conditions in section 2.5 in H2O (B), LB medium (C), M9 medium (D) and 10-fold diluted LB medium (E), respectively. Each plot represents the average of three samples. Absorbance was measured using a microplate reader.

Mentions: The cytidine assay was developed by coupling CDA with the indophenol method and conducted according to the method in section 2.5. The resulting indophenol had a maximum absorption at 630 nm. The addition of cytidine resulted in a proportional color development, which was translated to a linear standard curve (Fig. 3B-E). The upper range of the assay (in water) was also been explored (Fig. 3A). The increase of absorbance begins to fall when the concentration of cytidine is higher than 10 mM. The linear range and detection limit in H2O, LB medium, M9 medium and 10-fold diluted LB medium were listed in Table 1. The regular M9 medium contains (NH4)2SO4, which has significant influence on the reaction. Therefore, we replaced it with urea. The modified M9 medium enabled highly sensitive detection of analytes, even though containing a low concentration of cytidine. LB medium has negative influence on the assay, but the influence decreased as the medium was diluted (Fig. 3E). This result demonstrated that an accurate concentration of cytidine can be determined specifically using this colorimetric assay. Therefore, the fermentation samples in LB medium should be diluted before CDA reaction. The dilution step is necessary when using complex medium.


An enzymatic assay for high-throughput screening of cytidine-producing microbial strains.

Dong H, Liu Y, Zu X, Li N, Li F, Zhang D - PLoS ONE (2015)

(A). Effect of different cytidine concentrations on CDA assay in H2O.Cytidine standard curves were conducted under standard reaction conditions in section 2.5 in H2O (B), LB medium (C), M9 medium (D) and 10-fold diluted LB medium (E), respectively. Each plot represents the average of three samples. Absorbance was measured using a microplate reader.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376533&req=5

pone.0121612.g003: (A). Effect of different cytidine concentrations on CDA assay in H2O.Cytidine standard curves were conducted under standard reaction conditions in section 2.5 in H2O (B), LB medium (C), M9 medium (D) and 10-fold diluted LB medium (E), respectively. Each plot represents the average of three samples. Absorbance was measured using a microplate reader.
Mentions: The cytidine assay was developed by coupling CDA with the indophenol method and conducted according to the method in section 2.5. The resulting indophenol had a maximum absorption at 630 nm. The addition of cytidine resulted in a proportional color development, which was translated to a linear standard curve (Fig. 3B-E). The upper range of the assay (in water) was also been explored (Fig. 3A). The increase of absorbance begins to fall when the concentration of cytidine is higher than 10 mM. The linear range and detection limit in H2O, LB medium, M9 medium and 10-fold diluted LB medium were listed in Table 1. The regular M9 medium contains (NH4)2SO4, which has significant influence on the reaction. Therefore, we replaced it with urea. The modified M9 medium enabled highly sensitive detection of analytes, even though containing a low concentration of cytidine. LB medium has negative influence on the assay, but the influence decreased as the medium was diluted (Fig. 3E). This result demonstrated that an accurate concentration of cytidine can be determined specifically using this colorimetric assay. Therefore, the fermentation samples in LB medium should be diluted before CDA reaction. The dilution step is necessary when using complex medium.

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: 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.

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).

No MeSH data available.


Related in: MedlinePlus