In comparison with its counterpart from N. meningitides, all conserved motifs were found in the N-termini of E. coli CMP-NeuAc synthetase. E. coli CMP-NeuAc synthetase seems to have redundant C-termini with a less effect on its activity. To explain this speculation, a series of recombinant DNAs with deletion from 3'-end of CMP-NeuAc synthetase were produced by PCR, ligated into expression vector pET-15b and expressed in BL21(DE3)pLysS. After induction with IPTG, we found that the recombinant enzyme with deletion of 189 amino acids from C0termini retained its activity. This result demonstrates that the 229 amino acids of N-termini was the minimal functional domain of E. coli CMP-NeuAc synthetase. The deletions altered the optimum pH and thermostability of active truncated enzymes, indicating that the truncated C-terminal amino acids of E. coli CMP-NeuAc synthetase could affect the conformation of the enzymatic catalytic domain and therefore affect its catalytic activity and thermostability, although it is not involved in enzymatic activity directly.
Amino Acid Sequence ; Cytidine Monophosphate N-Acetylneuraminic Acid ; metabolism ; Enzyme Stability ; Escherichia coli ; enzymology ; Hydrogen-Ion Concentration ; Molecular Sequence Data ; Mutation ; N-Acylneuraminate Cytidylyltransferase ; chemistry ; physiology ; Structure-Activity Relationship

Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5'-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.
Cytidine ; pharmacology ; Cytidine Monophosphate ; pharmacology ; Enterobacter aerogenes ; enzymology ; Enzyme Induction ; Pentosyltransferases ; biosynthesis ; Vidarabine ; biosynthesis

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.
Cytidine Monophosphate ; metabolism ; Escherichia coli ; genetics ; Industrial Microbiology ; methods ; Phosphotransferases (Phosphate Group Acceptor) ; metabolism ; Uridine Kinase

With PVA as the carrier, the frozen beer yeast cells were immobilized for production of CTP from CMP. we explored the optimal condition of the immobilization from the aspects of the type, concentration of the PVA, and the immobilizing methods of cells In all 8 continuous batch of fermentation under the reactional condition of the immobilized cells, the conversion rate of CTP were maintained about 85% - 95%. Moreever, the storage stability of immobilized cells were investigated, and the products was also isolated and identifided by HPLC.
Beer ; microbiology ; Bioreactors ; microbiology ; Cells, Immobilized ; cytology ; metabolism ; Chromatography, High Pressure Liquid ; Cytidine Monophosphate ; metabolism ; Cytidine Triphosphate ; metabolism ; Fermentation ; Industrial Microbiology ; methods ; Polyvinyl Alcohol ; Saccharomyces cerevisiae ; cytology ; metabolism

BACKGROUND: The aim of this study was to evaluate the effect of continuous infusion of ondansetron compared with bolus injection on the incidence of postoperative nausea and vomiting (PONV) in intravenous, patient-controlled analgesia (PCA). METHODS: Sixty three women undergoing laparoscopic-assisted vaginal hysterectomy were randomly allocated according to the method of ondansetron administration: bolus injection of ondansetron (8 mg) after the operation (Bolus group, n = 21); continuous infusion after ondansetron (8 mg) mixed to PCA (PCA 8 mix group, n = 22); and continuous infusion after ondansetron (16 mg) mixed to PCA (PCA 16 mix group, n = 20). The PONV were measured at 1 hr, 6 hr, 24 hr and 48 hr after operation and pain scores (visual analog scale, VAS) were checked. RESULTS: The incidence of PONV during 48 hr in the Bolus group (23.8%) and PCA 16 mix group (20.0%) were significantly lower than PCA 8 mix group (54.5%) (P < 0.05). The three groups showed similar VAS pain scores. CONCLUSIONS: Our results suggest that continuous infusion of ondansetron 16 mg is as effective as a bolus injection of ondansetron (8 mg) at preventing PONV in high-risk patients.
Analgesia, Patient-Controlled ; Anesthesia ; Arabinonucleotides ; Cytidine Monophosphate ; Female ; Humans ; Hysterectomy, Vaginal ; Incidence ; Nausea ; Ondansetron ; Passive Cutaneous Anaphylaxis ; Piperidines ; Postoperative Nausea and Vomiting ; Propofol ; Vomiting