Lipase-calcium phosphate compound enzyme crystal, preparation method thereof and method for catalyzing and synthesizing clindamycin palmitate by using lipase-calcium phosphate compound enzyme crystal

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

Active Publication Date: 2018-07-31
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...

Method used

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  • Lipase-calcium phosphate compound enzyme crystal, preparation method thereof and method for catalyzing and synthesizing clindamycin palmitate by using lipase-calcium phosphate compound enzyme crystal
  • Lipase-calcium phosphate compound enzyme crystal, preparation method thereof and method for catalyzing and synthesizing clindamycin palmitate by using lipase-calcium phosphate compound enzyme crystal
  • Lipase-calcium phosphate compound enzyme crystal, preparation method thereof and method for catalyzing and synthesizing clindamycin palmitate by using lipase-calcium phosphate compound enzyme crystal

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 stir constantly, then let it stand for a while, remove the upper layer of water, and the lower layer of precipitation is 45 Dry in a vacuum drying oven at ℃ for one day; the white solid obtained is clindamycin free base.

[0057] Such as figure 1 As shown, the Clindamycin free base HPLC analysis profile prepared in Example 1; Take 1 mg of Clindamycin free base into 1 mL of methanol, mix well and filter with a 0.22 μm organic phase filter membrane. 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, ...

Embodiment 2

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

[0060] 5mL of phosphate buffer (PBS, 20mM, pH 6.7) containing 0.25mg / ml Thermomyces lanuginosa lipase (TLL) was added with 100μL of calcium chloride solution (200mM), and the mixture was kept at 4℃ Cultivate for 24 hours under conditions; then centrifuge at 10000 rpm for 5 minutes to remove the supernatant to obtain a white precipitate; finally, the obtained precipitate is washed three times with deionized water to obtain calcineurin crystals of Thermomyces lanuginosa 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 into a centrifuge tube with 6.25mL of petroleum ether, and add calcineurin crystals (containing 150mg of Cotton-like Thermomyces spores lipase), shake in a shaker at 200 rpm for 24 hours at 30°C; after the reaction, centrifuge, and wash the filter residue with the washing solvent petroleum ether to obtain the supernatant; The supernatant was heated under reduced pressure and spin-dried, dissolved in dichloromethane and adjusted to pH 1-2 with dilute hydrochloric acid, then heated under reduced pressure to spin-dry the solvent, dissolved in ethanol and added acetonitrile, stirred at 0°C for 4h, and finally Cold filtered and dried to obtain a white solid, which is clindamycin palmitate.

[0065] Such as image 3 As shown, the HPLC analysis pattern of clindamycin palmitate prepared in Example 3-1; HPLC conditions: C18 colu...

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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 complex enzyme crystal, a preparation method thereof and a method for catalyzing the synthesis of clindamycin palmitate. Background technique [0002] Enzymes are a type of protein with a special three-dimensional conformation that is produced by biological cells and has catalytic activity. As we all know, because of its green environmental protection and energy saving and high efficiency, biocatalysis has attracted people's attention as a kind of green catalysis. However, enzymes are the most commonly used in biocatalysis and are often used as catalysts. As a 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 normal temperature and pressure and other temperature conditions. At present, the ...

Claims

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Application Information

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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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