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Fermentation method of antalzyme

A fermentation method and lysozyme technology, which are applied in the fields of genetic engineering and protein engineering, can solve the problems of limited sources of biological materials, different degrees of freshness, immunogenicity and toxic side effects, etc., and achieve low production costs, improved production efficiency, and improved The effect of competitiveness

Inactive Publication Date: 2009-01-28
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the lysozyme used clinically is mainly egg white lysozyme, because it is a heterologous protein, it will produce immunogenicity and toxic side effects in the human body
However, a small amount of lysozyme is extracted from human milk, placenta or urine, the source is extremely difficult, and industrial production cannot be carried out
Moreover, extracting lysozyme from biological materials has its inherent defects. First, it is limited by the source of biological materials; unstable
And natural lysozyme is expensive, so it cannot be used clinically
[0005] So far, there is no large-scale production of lysozyme in China

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 fermentation process and yield calculation

[0023] 1.1 Strain activation: Take out the strain from -80°C and revive at room temperature. Open the cryovial and repeatedly suck it several times with a pipette gun. Dilution 10 -3 times, spread the solid medium on the YPD plate, and culture it at 30°C.

[0024] 1.2 Seed culture: After 48 hours in the YPD plate solid medium, pick a 2mm diameter colony and inoculate 100ml of preferred culture based on a 1L Erlenmeyer flask, seal it with 4 layers of sterile gauze, and carry out shaking flask cultivation at a temperature of 28-30°C and a shaking speed of 300rpm. The culture was terminated after 36 h.

[0025] 1.3 Fermentation culture: put 6L of basic medium into a 15L fermenter, and autoclave at 115°C for 25min. Inoculate 1.5L of the secondary seed culture medium and cultivate for 16-24 hours to exhaust the glycerol in the culture medium. The methanol induction phase lasted for 72 hours. Repeat the incubatio...

Embodiment 2

[0028] Example 2 Effect of Methanol Concentration on Expression

[0029] The optimized medium was divided into 500ml Erlenmeyer flasks. Induction was performed after 48 h of culture. Medium A 33 , add methanol 0.25ml every 12h; culture medium B 33 , add methanol 0.5ml every 12h; medium C 33 , add methanol 1ml every 12h; Medium D 33 , add methanol 1.5ml every 12h; Medium E 33 , add methanol 2ml every 12h. After the induction started, samples were taken every 12 hours for a total of 8 times, stored at -20°C, and the relative activity of human lysozyme was detected at the end of the fermentation, and the activity growth curve was drawn.

[0030] The results showed that adding 1% methanol per 24 hours, i.e. 10ml / L methanol, had the highest activity of the protein in the culture medium; adding 0.5%, 1.5%, 2%, 2.5% methanol in the medium every 24 hours, the protein activity was not much different; The protein activity was the lowest in the medium supplemented with 3% methanol...

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Abstract

The invention belongs to the field of genetic engineering and protein engineering, more particularly relates to a fermentation method of lysozyme; the application of the lysozyme is very wide; researchers optimize the lysozyme production technology continuously so as to expect that the yield of the lysozyme is increased and the production cost is reduced simultaneously. The invention adopts the common materials in the industry such as peptone, and yeast powder, etc. to carry out production, has low cost and is applicable to production on large scale. Compared with the culture medium provided by the Invitrogen Company, the cost for producing 1g of the lysozyme is reduced from more than 6 thousand RMB to more than 2 thousand RMB; therefore, the fermentation method of lysozyme of the invention improves the competitiveness of the products and lays foundation for the expanded production of genetic engineering strains.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and protein engineering, and in particular relates to a fermentation method of lysozyme. Background technique [0002] Lysozyme is a kind of basic globulin, its full name is: 1,4-β-N-lysozyme or: mucopeptide N-acetylmuramoyl hydrolase. It can hydrolyze the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan of the bacterial cell wall, destroy the peptidoglycan scaffold, and cause the cell wall to rupture under the action of the internal osmotic pressure of the bacteria. achieve a bactericidal effect. [0003] Clinically, lysozyme has a certain effect on acute and chronic inflammation of eyes, ears, nose, throat, oral cavity, etc., especially for acute inflammation such as: acute pharyngitis, acute laryngitis, acute otitis media, etc. The effect is obvious. Used in combination with antibiotics, it also has a certain effect on severe acute inflammatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N1/16
Inventor 余龙黄鹏唐丽莎李宗林
Owner FUDAN UNIV