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Preparation method for cephalosporins acremonium protoplasts

A technology of cephalosporins and protoplasts, which is applied in the field of preparation of cephalosporins protoplasts, can solve the problems of soft texture, difficult quantitative control, and large differences in spore suspension batches, etc., and achieve high regeneration rate and high activity Good results

Inactive Publication Date: 2017-03-22
SHAANXI EYOUNG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cephalosporium acremonium seldom produces spores, the texture of the thallus is soft, and the prepared spore suspension varies greatly between batches, so it is difficult to quantitatively control the inoculation
Moreover, due to the different mycelium differentiation state of the bacteria of the same age, the quality of the prepared protoplasts is significantly different.
It can be seen that "bacterial age" cannot accurately express the hyphae state

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] A preparation method of cephalosporin protoplast, is characterized in that comprising the following steps:

[0018] (1) Dissolve cellulase, helicase and lysozyme in osmotic pressure buffer, filter and sterilize through a 0.22 micron microporous membrane, and store at 4°C until use;

[0019] (2) Wash the spores of Cephalosporium acremonium with physiological saline, inoculate them in 50ml of CSL medium in 250ml shake flasks, and culture at 28°C for 4 days with shaking at 220r / min;

[0020] (3) Then transfer to YPS medium 50ml250ml shake flask, shake 220r / min at 28°C and culture for 18h;

[0021] (4) Take 50ml of the bacteria solution and centrifuge at 6600xg10min to collect the bacteria; accurately weigh the wet weight of 1g of mycelia, wash with sterile water 3 times, collect the bacteria by centrifugation, resuspend in 10mmol / LDTT solution 10ml, 30-50℃ Shake at 250r / min and incubate for 1h under the conditions for pretreatment; take the pretreatment solution and centr...

Embodiment 2

[0026] A preparation method of cephalosporin protoplast, is characterized in that comprising the following steps:

[0027] (1) Dissolve cellulase, helicase and lysozyme in osmotic pressure buffer, filter and sterilize through a 0.22 micron microporous membrane, and store at 4°C until use;

[0028] (2) Wash the spores of Cephalosporium acremonium with physiological saline, inoculate them in 50ml of CSL medium in 250ml shake flasks, and culture at 28°C for 4 days with shaking at 220r / min;

[0029] (3) Then transfer to YPS medium 50ml250ml shake flask, shake 220r / min at 28°C and culture for 18h;

[0030] (4) Take 50ml of the bacteria solution and centrifuge at 6600xg10min to collect the bacteria; accurately weigh the wet weight of 1g of mycelia, wash with sterile water 3 times, collect the bacteria by centrifugation, resuspend in 10mmol / LDTT solution 10ml, 30-50℃ Shake at 250r / min and incubate for 1h under the conditions for pretreatment; take the pretreatment solution and centr...

Embodiment 3

[0035] A preparation method of cephalosporin protoplast, is characterized in that comprising the following steps:

[0036] (1) Dissolve cellulase, helicase and lysozyme in osmotic pressure buffer, filter and sterilize through a 0.22 micron microporous membrane, and store at 4°C until use;

[0037] (2) Wash the spores of Cephalosporium acremonium with physiological saline, inoculate them in 50ml of CSL medium in 250ml shake flasks, and culture at 28°C for 4 days with shaking at 220r / min;

[0038] (3) Then transfer to YPS medium 50ml250ml shake flask, shake 220r / min at 28°C and culture for 18h;

[0039] (4) Take 50ml of the bacteria solution and centrifuge at 6600xg10min to collect the bacteria; accurately weigh the wet weight of 1g of mycelia, wash with sterile water 3 times, collect the bacteria by centrifugation, resuspend in 10mmol / LDTT solution 10ml, 30-50℃ Shake at 250r / min and incubate for 1h under the conditions for pretreatment; take the pretreatment solution and centr...

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PUM

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Abstract

A preparation method for cephalosporins acremonium protoplasts comprises the following steps that cellulase, helicase and lywallzyme are dissolved in an osmotic pressure buffer agent, and are filtered and degermed through a 0.22 micron millipore membrane, and a product is preserved at 4 DEG C for standby use; and spores of cephalosporins acremonium are washed out through normal saline, inoculated in a 50-250 ml shake flask containing a CSL culture medium, vibrated at the speed of 220r / min at 28 DEG C, and cultivated for four days. Through the preparation method for the cephalosporins acremonium protoplasts, a large number of protoplasts can be effectively obtained, activity is very good, and the regeneration rate is very high.

Description

technical field [0001] The invention relates to a method for preparing protoplasts of Cephalosporium cephalosporins. Background technique [0002] Cephalosporium acremonium - major production of lactam antibiotics. However, because the cell wall structure of filamentous fungi is quite complex, it is difficult to transform foreign genes into Cephalosporium acremonium to construct a transformation system. The use of protoplasts to complete the transformation has been proven to be an effective means. During the preparation and regeneration of protoplasts, the conditions of enzymatic hydrolysis, the state of mycelium and the type of regeneration medium are important factors. In this study, the appropriate enzyme types, compounding ratio and enzymatic hydrolysis time were investigated, and the optimal enzymatic hydrolysis conditions for the preparation of Cephalosporium acremonium protoplasts were screened. At the same time, the results of this experiment suggest that the myce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12R1/75
CPCC12N1/14
Inventor 张俊
Owner SHAANXI EYOUNG TECH
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