FLUORESCEIN DIPHOSPHATE, TETRAAMMONIUM SALT

Method one: Enzyme conversion method Preparation of enzyme liquid The yeast residue that has been used for multi-generation fermentation; Suspension and an appropriate amount of distilled water, repeated freezing and thawing 3 times at -20°C to obtain the enzyme liquid. Yeast residue (containing FDP synthase) [-20°C] → Enzyme solution conversion, impurity removal The above enzyme solution was added with substrate (8% sucrose, 4% NaH2PO4, 0.29% MgCl2, pH6.5) and reacted at 30°C 6h, boil for 5min, centrifuge to remove contaminants to obtain the conversion solution. Enzyme liquid [substrate] → [30℃, 6h] conversion liquid [boiling] → [30min] conversion liquid adsorption, elution conversion liquid was passed through DEAE-C anion exchange column chromatography, washed with distilled water to pH 7.0, Then carry out separation and elution, collect the eluent and add CaCl2 to generate its calcium salt precipitate, and filter to obtain FDP calcium salt. Conversion supernatant [DEAE-C exchange column, water] → [pH7.0] eluent [CaCl2] → FDP calcium salt conversion, sodium salt FDP calcium salt suspended in water, using 732 [H+] resin to convert it FDPH4, adjust the pH to 5.3-5.8 with 2mol/L NaOH to form crude FDPNa3. By ultrafiltration, sterilization, depyrogenation, lyophilization, you can get FDPNa3 boutique. FDP calcium salt [732 [H+] resin] → FDPH4 [2mol/L NaOH] → [pH5.3-5.8] FDPNa3 crude product [filtration] → [sterilization, mycoplasma] super filtrate → [lyophilized] FDPNa3 boutique Method 2: Culture of FDP-producing bacteria by immobilized cell preparation method Saccharomyces cerevisiae was inoculated on wort slant medium and cultured at 26°C for 24 hours, transferred to seed medium, cultivated to logarithmic growth period, and transferred to fermentation medium. It was fermented and cultured at 28°C for 24h, stood still for one week, centrifuged, and collected bacterial cells. FDP producing bacteria seed [medium]→[26℃, 24h] Preparation of FDP producing bacterial cell immobilized cells Take the activated bacterial cells and suspend them with equal volume of normal saline, preheat to 40℃, and heat with 4 times the amount of normal saline Dissolve carrageenan (carrageenan dosage is 3.2g/L), the two are mixed and stirred at 45 ℃ for 10min, poured into a molding vessel, cooled at 4-10 ℃ for 30min, add the same amount of 22.2g/L KCl solution and soak and harden for 4h, Cut into 3mm×3mm×3mm pieces and serve. FDP-producing cells [Carrageenan, KCl]→[4h] Immobilized cells The activation of immobilized cells uses a surfactant containing a substrate, soaked at 35°C for 24h, and washed with 0.3mol/L KCl solution Soak in physiological saline and set aside. Immobilized cells [0.3mol/L KCl, NaCl]→[35℃, 24h] Activated immobilized cells Transform, remove impurities The activated immobilized cells are used to fill the column reactor. The above method is followed by passing 30℃ substrate solution ( Containing 8% sucrose, 4% NaH2PO4, 0.3% ATP, 0.29% MgCl2) collect the conversion solution, remove the impurities and get the conversion solution. Activate immobilized cells [substrate] → [30°C] transforming solution [depuration protein] → absorb, elute, and form calcium salts in the transformed clear solution. Pass the transformed clear solution through the treated DEAE-C anion exchange column and wash it. After elution, collect the eluent and add an appropriate amount of CaCl2 to generate FDP calcium salt precipitate. Conversion supernatant [DEAE-C column] → eluent [CaCl2] → FDP calcium salt to acid, sodium salt FDP calcium salt was suspended in sterile water, converted to FDPH4 with 732 [H+] resin, using 2mol /L NaOH to adjust the pH to 5.3-5.8 into sodium salt, decolorization of activated carbon, ultrafiltration, lyophilization, to obtain FDPNa3 boutique. FDP calcium salt [732 [H+] resin] → FDPH4 [2mol/L NaOH] → [pH5.3-5.8] FDPNa3 [ultrafiltration] → ultrafiltrate [lyophilized] → FDPNa3 boutique.

Fluorogenic alkaline phosphatase probe.

Molecular Weight:560.4
Hydrogen Bond Donor Count:4
Hydrogen Bond Acceptor Count:11
Rotatable Bond Count:2
Exact Mass:560.10733165
Monoisotopic Mass:560.10733165
Topological Polar Surface Area:184
Heavy Atom Count:37
Complexity:797
Covalently-Bonded Unit Count:5
Compound Is Canonicalized:Yes