Citrate synthase

An enzyme that catalyzes the condensation of oxaloacetic acid and acetyl-CoA to yield citric acid and coenzyme A.

Method 1. Dilute alkali-enzymatic extraction method to extract 250kg of 20g/L (2%) NaOH solution, place it in a soaking tank, add 40kg of white and dry cartilage, and slowly stir once every half an hour at outdoor temperature, and wait for the leaching solution When the relative density reaches 5°Be (20°C), the material is discharged, filtered with gauze, and the bone residue is soaked in an appropriate amount of distilled water for 20 minutes, filtered, and the two filtrates are combined, the total volume is 200L, and the extract is obtained. Pig larynx (nasal) cartilage [NaOh]→[room temperature] leaching solution Enzymatically extract the leaching solution and place it in the digestion tank, slowly add an equal volume of hydrochloric acid under stirring and adjust the pH to 8.8-9, heat on a circulating water bath, and the internal temperature reaches 50℃ Add 1300g of pancreatin equivalent to 1:25 times (high-power pancreatin is preferred), continue to increase the temperature, and control the digestion temperature at 53-54°C for a total of 7 hours. During the hydrolysis process, the increase of amino acids causes the pH to drop, so 100g/L (10%) NaOH solution is needed to adjust the pH to 8.8-9. Check the hydrolysis end point. Take a little reaction solution and filter it in a colorimetric tube. Add 1-2 drops of 100 g/L (10%) trichloroacetic acid to 10ml of the filtrate. If it is slightly turbid, it means that the digestion is good, otherwise increase pancreatin as appropriate. Extraction solution [HCl, pancreatin]→[pH8.8-9, 53-54℃, 7h] Enzymatic hydrolysate, keep the temperature in the adsorption tank at 53-54℃, adjust pH 6.8-7 with 1:1 hydrochloric acid, add activity 7kg of white clay, 200g of activated carbon, stir, adjust pH 6.8-7 with 100 g/L (10%) NaOH solution, stir and absorb for 1 hour, then adjust pH 5.4 with 1:2 hydrochloric acid, stop heating, let stand for a while, filter , Get a clear filtrate. Enzymatic hydrolysis solution [activated white clay, activated carbon]→[pH6.8-7, 1h] Filtrate precipitation and drying The above clear filtrate is quickly adjusted to pH6 with 100 g/L (10%) NaOH solution, and the volume of the clear solution is 10 g/ L (1%) of sodium chloride, dissolve and filter until clear. The filtrate is slowly added with 90% ethanol with 75% alcohol content under stirring. Stir once every 30 minutes for about 4-6 times to make the fine particles increase and settle. Let it stand for more than 8 hours and absorb the supernatant. The precipitate is fully dehydrated and washed twice with absolute ethanol, drained, dried at 60-65°C or vacuum dried. The filtrate [NaCl, 90%]→[pH6, 8h or more] precipitate [absolute ethanol]→[60-65℃] dry preparation according to the formula contains chondroitin sulfate 20 g/L (2%), sodium chloride 8.5 g/L (0.85%), weigh 107% of labeled cartilage sulfate powder (calculated as pure product), sprinkle into water for injection, swell, stir to dissolve, add sodium chloride, adjust pH to about 5.5, heat Bring to a boil, filter while it is hot with a Buchner funnel, add 3-5 g/L (0.3%-0.5%) activated carbon to the filtrate and quickly heat it to a boil, keep it for 15 minutes, wrap the filter paper with an sand stick, and vacuum filter when the liquid is boiled , The filtrate is cooled to room temperature, and water for injection is added to the full amount, filtered with a No. 3 vertical melting funnel until clear, filled with 2ml per tube, sterilized to obtain chondroitin sulfate injection. Dry product [NaCl, Water for Injection]→[pH5.5] Chondroitin Sulfate Injection Method 2. Dilute alkali-concentrated salt extraction method for leaching and extraction Put the clean and clean broken cartilage in the extraction tank, and the concentration is 3-3.5 The mol/L sodium chloride solution (high salt concentration is suitable) completely immerses the bone residue. Adjust pH 12-13 with 50% sodium hydroxide solution, stir and extract for 10-15 hours, filter, and re-extract the filter residue as above. During the extraction process, pH 12-13 should be adjusted at any time, and the two extraction solutions should be combined. Pig larynx (nasal) cartilage [NaOH, NaCl]→[pH12-13, 10-15h]→Extract solution hydrochloride hydrolysis The extract solution is adjusted to pH7-8 with 2mol/L hydrochloric acid, and the temperature is rapidly raised to 80-90℃, and kept for 20min After cooling, filter to clarify the filtrate and discard the residue to obtain the salt solution. Extraction solution [HCl]→[pH7-8, 80-90℃] Salt hydrolysis solution removes acidic protein, adjusts the salt solution solution to pH 2-3, stirs for 10 minutes, stands for storage and then filters until clear, adjust pH 6.5, add 2 Use deionized water to make the sodium chloride concentration of the solution about 1mol/L. Salt hydrolyzate [water, NaCl]→[pH2-3, pH6.5] The filtrate is precipitated and dried. The above solution is added with 95% ethanol to make the volume fraction of alcohol reach 50%-60% (measured with an ethanol meter) and stored until clear. The supernatant is removed by siphoning, the precipitation is drained, dehydrated and dried to obtain the finished product of chondroitin sulfate. Filtrate [ethanol]→chondroitin sulfate finished product method 3. Enzymatic hydrolysis—resin extraction method to mince whale cartilage, add 1mol/L sodium hydroxide solution to soak, hydrolyze at 40℃ for 2h or add pH7.5 water soak, with protease Incubate and hydrolyze at 55°C for 20h, then add hydrochloric acid to neutralize it, filter, and adjust the sodium chloride concentration in the filtrate to 0.5mol/L. Then, the solution was passed through an Amberlite IRA-933 ion exchange resin column, after the adsorption was completed, washed with 0.5 mol/L sodium chloride solution, and then eluted with 1.8 mol/L sodium chloride solution at a flow rate of 2L/h, and the eluent Desalination, ethanol precipitation, centrifugation, collection of precipitation, vacuum drying, and then the finished product. Whale cartilage slurry [1mol/L NaOH, protease, HCl]→[40℃, 2h; 55℃, 20h; pH7] filtrate [0.5 mol/L NaCl, IRA-933 resin column]→ eluent [ethanol] product application The chondroitin sulfate sample prepared by this process was found to be a single zone by paper electrophoresis, the antigen test result was negative, and the sulfur content was 7.2%. Its advantage is that the combination of enzyme hydrolysis or dilute alkali hydrolysis and resin exchange technology ensures that the chondroitin sulfate molecule does not degrade and solves the problem of purity of the finished product. The method is simple and the yield is high, which is a more practical preparation method.

It is used for certain neurological headaches, neuralgia, arthralgia, migraine, arteriosclerosis, etc.; it can also be used for the adjuvant treatment of hearing impairment and hepatitis caused by streptomycin.