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Freeze-drying protective agent of glycosaminoglycan synthetase and application of freeze-drying protective agent

A technology of freeze-drying protective agent and glycosaminoglycan, which is applied in the field of freeze-dried preparation of glycosaminoglycan synthetase and its preparation, can solve the problems such as no effect found, achieve simple formula, facilitate transportation and long-term storage, good The effect of resolubility

Pending Publication Date: 2022-04-05
BLOOMAGE BIOTECHNOLOGY CORP LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this lyoprotectant is a protective agent formulation for microorganisms and has not been used in enzyme preparations
[0009] From the analysis of the prior art, it can be seen that up to now, a kind of effective lyoprotectant suitable for glycosaminoglycan synthetase has not been found.

Method used

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  • Freeze-drying protective agent of glycosaminoglycan synthetase and application of freeze-drying protective agent
  • Freeze-drying protective agent of glycosaminoglycan synthetase and application of freeze-drying protective agent
  • Freeze-drying protective agent of glycosaminoglycan synthetase and application of freeze-drying protective agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Prepare the purified enzyme solution of heparin synthase I for the synthesis of heparin and heparan sulfate, the steps are as follows:

[0034] 1) Construction of engineering strains

[0035] For the specific construction process of strain BL21(DE3)-GaKfiA, please refer to the literature "The second member of the bacterial UDP-N-acetyl-D-glucosamine: heparosan alpha-1,4-N-acetyl-D-glucosaminyltransferase superfamily: GaKfiA from Gallibacterium anatis".

[0036] 2) Strain recovery and fermentation culture

[0037] Take out the cryopreservation tube of BL21(DE3)-GaKfiA strain in the -80℃ refrigerator and inoculate it into LB solid medium (containing 100 μg / mL kanamycin) for activation, pick a single colony in LB (containing 100 μg / mL kanamycin) ) seed medium (37°C, 220r / min), transfer the seed solution to 1L LB (containing 100μg / mL kanamycin) liquid medium for expansion and culture, and when the OD is about 0.6, add the final The concentration was 0.5mM IPTG, induced at ...

Embodiment 2

[0041] Prepare heparin synthase II purified enzyme solution for synthesizing heparin and heparan sulfate, the steps are as follows:

[0042] 1) Construction of engineering strains

[0043] For the construction method of strain BL21(DE3)-BtHS1, refer to patent CN113564139A.

[0044] 2) Strain recovery and fermentation culture

[0045] Take out the cryopreservation tube of BL21(DE3)-BtHS1 strain in the -80℃ refrigerator and inoculate it into LB solid medium (containing kanamycin 100 μg / mL) for activation, pick a single colony in LB (containing kanamycin 100 μg / mL) ) seed medium (37°C, 220r / min), transfer the seed solution to 1L LB liquid medium (containing 100μg / mL kanamycin) for expansion, and cultivate until the OD is about 0.6, then add the final The concentration was 0.5mM IPTG, induced at 22°C and 220r / min for 16-20h, the bacteria were collected by centrifugation, and placed in a -20°C refrigerator for later use.

[0046] 3) Purification of Heparin Synthase II

[0047] ...

Embodiment 3-9

[0049] A preparation method of a freeze-dried preparation of heparin synthase I, comprising the following steps:

[0050] 1. Dissolve ectoine, trehalose and hydroxypropyl-β-cyclodextrin with a certain amount of purified water to obtain a lyoprotectant solution, which is filtered through a sterile filter;

[0051] 2. Mix the lyoprotectant solution with the purified enzyme solution of heparin synthase I to obtain the total raw material solution, so that the concentration of heparin synthase I in the total raw material solution is 0.05wt.%, so that ectoine, trehalose and hydroxyl The final concentration of propyl-β-cyclodextrin in the total raw material solution meets the requirements in Table 1, and water makes up 100wt.%.

[0052] Table 1

[0053]

[0054]

[0055] Note: "wt.%" refers to the grams of ingredients contained in 100ml of liquid.

[0056] 3. Divide the total raw material solution according to the amount of 1ml / sterile vial, and then place it in a -80°C refri...

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Abstract

The invention discloses a freeze-drying protective agent of glycosaminoglycan synthetase and application of the freeze-drying protective agent. The freeze-drying protective agent is prepared from trehalose, hydroxypropyl-beta-cyclodextrin and ectoine. The formula of the freeze-drying protective agent is different from the formula of the traditional freeze-drying protective agent, the components of the formula are simple, the freeze-dried glycosaminoglycan synthase is loose in structure, regular in shape and appearance and good in solubility, and particularly, the enzyme activity of the freeze-dried glycosaminoglycan synthase is slightly different from that of the freeze-dried glycosaminoglycan synthase before freeze-drying. And the enzyme activity can be kept above 90% after being stored at 4 DEG C and 25 DEG C for 90 days, which indicates that the freeze-drying protective agent can effectively reduce the activity loss of the glycosaminoglycan synthase in the freeze-drying process, and is beneficial to the storage and transportation of the glycosaminoglycan synthase.

Description

technical field [0001] The invention relates to a freeze-drying protectant for glycosaminoglycan synthase, and also relates to a freeze-dried preparation of glycosaminoglycan synthase and a preparation method thereof, belonging to the technical field of biological products. Background technique [0002] Glycosaminoglycans are a family of heteropolysaccharides with complex structures, which can be divided into hyaluronic acid (HA), heparan sulfate (HS) and heparin (Heparin) according to their disaccharide repeating units, glycosidic bonds, and sulfation sites. ), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS). Glycosaminoglycans exist in cells and play key roles in cell adhesion, chemokine signaling, biochemical cascades, and pathogen recognition, and are widely used in clinical drugs and health products. The three traditional sources of glycosaminoglycans are animal tissue extraction, chemical synthesis, and enzymatic synthesis. Due to the struct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10
Inventor 赵岩郭学平生举正邓建群杜冉冉陆震孙元军孟庆营金艳
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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