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Method for converting echinocandin B through actinoplanes utahensis

A technology of swimming actinomycetes and echinocandin, applied in the direction of microorganism-based methods, biochemical equipment and methods, immobilized on or in inorganic carriers, etc., can solve the cumbersome enzyme purification process, high production cost, Poor stability and other problems, to achieve the effect of simple process, low cost and good stability

Pending Publication Date: 2018-03-09
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The early conversion method was to directly add the echinocandin B substrate to the amidohydrolase fermentation system for conversion. This method has disadvantages such as poor stability and low conversion rate.
Later, some researchers expressed the amidohydrolase gene in Streptomyces, prepared it as a free enzyme or an immobilized enzyme, and then established a stable transformation system for transformation. However, the preparation and immobilization of the free enzyme required tedious enzyme purification processes. higher production costs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1. Cell culture and immobilization

[0018] Inoculate actinoplanes utahensis on the plate medium and culture at 25°C for 10 days; inoculate the cultured bacteria on the plate into the seed medium and cultivate at 25°C for 12 hours; inoculate the seed solution into the fermentation medium , the volume percentage of the inoculum was 10%, and cultured at 28°C for 24h. Collect 50g of bacteria by centrifugation, suspend in 50mL of phosphate buffer with pH 5.0, add 100mL of 2% sodium alginate solution by mass volume fraction and mix well, and then inject 0.1mol / L of

[0019] CaCl 2 In the solution, immobilize at 25°C for 40min. Afterwards, immobilized beads of about 2 mm were obtained by filtration, washed with phosphate buffer solution of pH 5.0 and prepared for column loading.

[0020] 2. Column packing

[0021] The prepared cell-immobilized beads were uniformly suspended in pH 5.0 phosphate buffer, and added to a glass column with a diameter of 5 cm and a height of 20 ...

Embodiment 2

[0031] 1. Cell culture and immobilization

[0032] Inoculate actinoplanes utahensis on the plate medium and culture at 25°C for 10 days; inoculate the cultured bacteria on the plate into the seed medium and cultivate at 25°C for 12 hours; inoculate the seed liquid into the fermentation medium , the volume percentage of the inoculum was 10%, and cultured at 28°C for 24h. Collect 40 g of bacteria by centrifugation and suspend in 40 mL of phosphate buffer with a pH of 6.0, add to 100 mL of 1% sodium alginate aqueous solution and mix well, then inject 0.2 mol / L CaCl steadily with a syringe 2 In aqueous solution, immobilize at 30°C for 25min. Afterwards, small beads of about 2 mm were obtained by filtration, washed with a phosphate buffer solution of pH 6.0, and prepared for column loading.

[0033] 2. Column packing

[0034] The prepared cell-immobilized beads were evenly suspended in phosphate buffer solution with pH 6.0, and added to a glass column with a diameter of 5 cm and...

Embodiment 3

[0044] .Cell culture and immobilization

[0045] Inoculate actinoplanes utahensis on the plate medium and culture at 25°C for 10 days; inoculate the cultured bacteria on the plate into the seed medium and cultivate at 25°C for 12 hours; inoculate the seed solution into the fermentation medium , the volume percentage of the inoculum was 10%, and cultured at 28°C for 24h. Collect 20 g of bacteria by centrifugation and suspend in 20 mL of acetate buffer with a pH of 6.5, add to 50 mL of 3% sodium alginate aqueous solution and mix well, then inject 0.2 mol / L CaCl steadily with a syringe 2In aqueous solution, immobilize at 20°C for 40min. Afterwards, small beads of about 2 mm were obtained by filtration, washed with acetate buffer solution with a pH of 6.5, and prepared for column loading.

[0046] 2. Column packing

[0047] The prepared cell-immobilized beads were uniformly suspended in acetate buffer solution with a pH of 6.5, and added to a glass column with a diameter of 5 c...

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PUM

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Abstract

The invention discloses a method for converting echinocandin B through actinoplanes utahensis. The method comprises the following steps: (1) culturing actinoplanes utahensis so as to obtain a bacterial liquid, centrifuging to collect bacteria, suspending the bacteria in a buffer saline solution, adding a sodium alginate solution, uniformly mixing, stably injecting into a CaCl2 solution, performingimmobilization, and putting the immobilized beads into a column for later use; (2) dissolving echinocandin B into an organic solvent, putting into the buffer saline solution till the concentration ofthe echinocandin B is 0.1-5g / L, further putting the echinocandin B into the column obtained in the step 1), and performing conversion. The method has the remarkable advantages of being simple in process and low in cost, and the prepared immobilized cells have the advantages of good stability, high conversion rates and multiple times of repeated use.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a method for transforming echinocandin B by using Actinomycetes uthaides, in particular to a method for efficiently transforming echinocandins B by immobilizing Actinomyces uthaides cells method. Background technique [0002] In the 1970s, it was discovered that echinocandins (Echinocandins) can non-competitively inhibit the activity of fungal cell wall β-1,3-glucan synthase, and exhibit broad antibacterial spectrum and strong activity. It is an important drug of choice for the treatment of fungal infections in immunosuppressed patients and immunonormal patients, mainly acting on Candidas and Aspergillus. There are three main types of echinocandins, among which echinocandin B (Echinocandin B) is the most important type. After removing the side chain of echinocandin B, a semi-synthetic precursor nucleus is obtained, which undergoes a series of chemical modifications Derivatives w...

Claims

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Application Information

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IPC IPC(8): C12P21/04C12N11/10C12N11/14C12R1/04
CPCC12N11/10C12N11/14C12P21/02
Inventor 张贵民薛国希
Owner LUNAN PHARMA GROUP CORPORATION
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