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A lipase-calcium phosphate complex enzyme crystal, its preparation method and its catalytic method for synthesizing clindamycin palmitate

A technology for synthesizing clindamycin and palmitate, applied in biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of resin becoming solid waste, not meeting sustainable development, and reducing enzyme activity, and improving production. Safety, high yield, effect of reducing steps

Active Publication Date: 2020-12-22
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two main disadvantages of this immobilization method, one is that the enzyme activity decreases rapidly, and the other is that the resin will eventually become solid waste, which is not in line with sustainable development

Method used

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  • A lipase-calcium phosphate complex enzyme crystal, its preparation method and its catalytic method for synthesizing clindamycin palmitate
  • A lipase-calcium phosphate complex enzyme crystal, its preparation method and its catalytic method for synthesizing clindamycin palmitate
  • A lipase-calcium phosphate complex enzyme crystal, its preparation method and its catalytic method for synthesizing clindamycin palmitate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Preparation of clindamycin free base:

[0056] Dissolve 2.5g of clindamycin hydrochloride in 6mL of deionized water, mix well, slowly add 6mL of NaOH (1mol / L) and keep stirring, then let it stand for a while, remove the upper layer of water, the lower layer of precipitation at 45 °C in a vacuum oven for one day; the obtained white solid is clindamycin free base.

[0057] Such as figure 1 As shown, the clindamycin free base HPLC analysis spectrum that embodiment 1 prepares; Get the clindamycin free base of 1mg in 1mL methanol, mix with the organic phase membrane filter of 0.22 μ m aperture, Obtain the sample, the following sample preparation process is the same; HPLC conditions: C18 column, column temperature 25 ℃, 226nm, 0.5mL / min, mobile phase: methanol: 3% ammonium acetate = 70:30, clindamycin free The alkali retention time is 6.23min.

[0058] Such as figure 2 As shown, the LC-MS spectrum of clindamycin free base prepared in Example 1: (m / z, 425.2)....

Embodiment 2

[0059] Example 2: Preparation of calcium phosphatase crystals:

[0060] 100 μL of calcium chloride solution (200 mM) was added to 5 mL of phosphate buffered saline (PBS, 20 mM, pH 6.7) containing 0.25 mg / ml Thermomyces lanuginosus lipase (TLL), and the mixture was heated at 4°C Cultivate under the conditions for 24 hours; then centrifuge at 10,000 rpm for 5 minutes, remove the supernatant to obtain a white precipitate; finally wash the obtained precipitate three times with deionized water to obtain the calciphosphatase crystal of Thermomyces lanuginosus lipase.

Embodiment 3-1

[0063] Example 3-1: Preparation of clindamycin palmitate:

[0064] Weigh 13.25mg (0.3125mmol) of clindamycin free base and 26.48mg (0.9375mmol) of vinyl palmitate and add it to a centrifuge tube with 6.25mL of petroleum ether, and add calcium phosphate crystals (containing 150mg of hydrophobic Cottonophilus lipase) was shaken in a shaker at a speed of 200 rpm for 24 hours at 30°C; after the reaction, centrifuged, and the filter residue was washed with the washing solvent petroleum ether to obtain the supernatant; Heat and spin dry the supernatant under reduced pressure, add dichloromethane to dissolve and adjust the pH value to 1-2 with dilute hydrochloric acid, then heat and spin dry the solvent under reduced pressure, add ethanol to dissolve and add acetonitrile, stir at 0°C for 4 hours, and finally Cold filtration and drying to obtain a white solid, the white solid is clindamycin palmitate.

[0065] Such as image 3 As shown, the HPLC analysis spectrum of clindamycin pa...

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Abstract

The invention provides a lipase-calcium phosphate compound enzyme crystal, a preparation method thereof and a method for catalyzing and synthesizing clindamycin palmitate by using the lipase-calcium phosphate compound enzyme crystal, and belongs to the technical field of antibiotic drug synthesis. According to the method, clindamycin free alkali shown in the formula I and ethenyl hexadecanoate shown in the formula II serve as a substrate and react with a reaction solvent for 6-30 h at the temperature of 30-80 DEG C under the catalytic function of the lipase-calcium phosphate compound enzyme crystal, and clindamycin palmitate shown in the formula III is prepared. The lipase-calcium phosphate compound enzyme crystal prepared through the method is a calcium phosphate crystal of thermomyces lanuginosus lipase. The lipase-calcium phosphate compound enzyme crystal has the advantages that the cost of the method is low, immobilized enzyme cannot be dissolved in an organic solvent and can be recycled, the yield can reach 75.8% at most, the method is safe, reliable, low in environment pollution and suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of antibiotic drug synthesis, and relates to a lipase-calcium phosphate compound enzyme crystal, a preparation method thereof and a method for catalyzing the synthesis of clindamycin palmitate. Background technique [0002] Enzyme is a kind of protein with special three-dimensional spatial conformation produced by biological cells and having catalytic activity. As we all know, biocatalysis, as a kind of green catalysis, has attracted people's attention because of its environmental protection, energy saving and high efficiency. However, enzymes are the most commonly used in biocatalysis and are often used as catalysts. As a kind of biocatalyst, the reaction catalyzed by enzyme has the advantages of strong specificity (enzyme catalysis has regioselectivity and stereoselectivity), high catalytic efficiency, and can be operated under isothermal conditions of normal temperature and pressure. At present, the app...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/64C12N9/20
CPCC07K2299/00C12N9/20C12P7/6436C12P19/64C12Y301/01003
Inventor 王安明章鹏飞余建云
Owner HANGZHOU NORMAL UNIVERSITY
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