662.8±65.0 °C at 760 mmHg
~222 °C (dec.)
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provides coniferyl ferulate(CAS#:63-37-6) MSDS, density, melting point, boiling point, structure, formula, molecular weight etc. Articles of coniferyl ferulate are included as well.>> amp version: coniferyl ferulate
Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5′-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.
5′-cytidine monophosphate; immobilized enzyme; polyphosphate kinase; uridine-cytidine kinase.
[Concomitant use of immobilized uridine-cytidine kinase and polyphosphate kinase for 5'-cytidine monophosphate production]
Sijia Wu 1, Jie Li 1, Chenlong Hu 1, Junyu Tian 1, Tong Zhang 1, Ning Chen 1, Xiaoguang Fan 1
2020 May 25
Isothermal titration calorimetry (ITC) can benefit from operating in miniaturized devices as they enable quantitative, low-cost measurements with reduced analysis time and reagents consumption. However, most of the existing devices that offer ITC capabilities either do not yet allow proper control of reaction conditions or are limited by issues such as evaporation or surface adsorption caused inaccurate solution concentration information and unintended changes in biomolecular properties because of aggregation. In this paper, we present a microdevice that combines 3D-printed microfluidic structures with a polymer-based MEMS thermoelectric sensor to enable quantitative ITC measurements of biomolecular interactions. Benefitting from the geometric flexibility of 3D-printing, the microfluidic design features calorimetric chambers in a differential cantilever configuration that improves the thermal insulation and reduces the thermal mass of the implementing device. Also, 3D-printing microfluidic structures use non-permeable materials to avoid potential adsorption. Finally, the robustness of the polymeric MEMS sensor chip allows the device to be assembled reversibly and leak-free, and hence reusable. We demonstrate the utility of the device by quantitative ITC characterization of a biomolecular binding system, ribonuclease A (RNase A) bind with cytidine 2′-monophosphate (2’CMP) down to a practically useful sample concentration of 0.2 mM. The thermodynamic parameters of the binding system, including the stoichiometry, equilibrium binding constant, and enthalpy change are obtained and found to agree with values previously reported in the literature.
3D-printed microfluidic structures; Isothermal titration calorimeter; MEMS thermoelectric sensor; Polymer substrate.
Isothermal titration calorimetry in a 3D-printed microdevice
Yuan Jia 1 2, Chao Su 2 3, Maogang He 4, Kun Liu 5, Hao Sun 6, Qiao Lin 7
2019 Nov 11
The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.
LC-MS/MS; cytidine-5′-monophospho-N-acetylneuraminic acid; human leukocytes; hydrophilic interaction chromatography.
Quantitation of cytidine-5'-monophospho-N-acetylneuraminic acid in human leukocytes using LC-MS/MS: method development and validation
Meng Fang 1, Xin Xu 2, Michael Zhang 1, Yifan Shi 1, Guodong Gu 1, Wanjing Liu 1, Bradley Class 3, Carla Ciccone 3, William A Gahl 3, Marjan Huizing 3, Nuria Carrillo 3, Amy Q Wang 2