Method for preparing nuclease P1 by ferment process

A nuclease and fermentation technology, applied in the biological field, can solve the problems of high production cost, long fermentation cycle, and low enzyme activity unit, and achieve the effect of reducing equipment cost and simplifying the process

Inactive Publication Date: 2007-09-19
北京燕京中科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the original production method, whether solid or liquid culture is used, the unit of enzyme activit

Method used

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  • Method for preparing nuclease P1 by ferment process
  • Method for preparing nuclease P1 by ferment process
  • Method for preparing nuclease P1 by ferment process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 prepares bran spores

[0042] 1. Activation of strains

[0043] Take 1 ring of bacteria and insert it into the potato slant medium, culture it at 28-30°C for 3 days as the first generation, and so on, transfer it to the fourth generation slant medium, and store it routinely for later use.

[0044] 2. Expand training

[0045] Fully wash the fresh fourth-generation slant with sterile water to obtain a spore suspension, take 0.1-2.0 ml and insert it into the prepared bran medium, and cultivate it at 28-30° C. for 3 days. Bran spore count reaches 10 9 pcs / g.

Embodiment 2

[0046] Embodiment 2 (2 tons of fermentation tanks)

[0047]1. Medium formula: phosphate 15Kg, magnesium sulfate 3Kg, calcium carbonate 3Kg, zinc sulfate 1.2Kg, industrial peptone 15Kg, industrial glucose 9Kg, make up to 1500L with water.

[0048] 2, inoculum size: 900 grams of bran spores (embodiment 1);

[0049] 3. Cultivation temperature: 29~30℃;

[0050] 4. Cultivation speed: 150rpm~250rpm, when the dissolved oxygen drops below 40%, adjust the speed;

[0051] 7. Ventilation volume: 1.5m 3 / min;

[0052] 8. Tank pressure: 0.05~0.06Mpa;

[0053] 9. Initial pH value: 6.0

[0054] 10. Supplementary culture medium:

[0055] The fermentation culture is about 24.1 hours, and the dry material required for the 250L fermenter culture medium is added to the medium for the first time;

[0056] Fermentation culture is about 29.5 hours, and the medium is supplemented with the dry material required for the 250L fermenter medium for the second time;

[0057] The fermentation cultur...

Embodiment 3

[0067] Embodiment 3 (2 tons of fermentation tanks)

[0068] 1. Medium formula: phosphate 3Kg, magnesium sulfate 1.3Kg, calcium carbonate 1.3Kg, zinc sulfate 1.5Kg, industrial peptone 30Kg, industrial glucose 6Kg, make up to 1500L with water.

[0069] 2. Inoculation amount: 840 grams of bran spores;

[0070] 3. Cultivation temperature: 29~30℃;

[0071] 4. Cultivation speed: 150rpm~270rpm, when the dissolved oxygen drops below 40%, adjust the speed;

[0072] 7. Ventilation volume: 1.5m 3 / min;

[0073] 8. Tank pressure: 0.05~0.06Mpa;

[0074] 9. Initial pH value: 5.9

[0075] 10. Supplementary culture medium:

[0076] Ferment and cultivate for about 23 hours, start the medium and add the dry material required for the 250L fermenter medium for the first time;

[0077] Fermentation and cultivation for about 28 hours, the second supplement of the medium with the dry material required for the medium of the 250L fermenter;

[0078] Ferment and cultivate for about 33 hours, an...

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Abstract

The invention provides a fermentation method for preparing nuclease P1, which using directly penicillium citrinum sporogon as seed, fermenting by means of fed-batch sugar supplement to improve activity of the nuclease P1, the mehod without first and second order seed culture shorts production cycle greatly and raises efficiency. The invetion also uses obtained enzyme for preparing 5'-mononucleotide with 85-90% rate of enzymatichydrolysis and 15 mg/ml of producing rate of nucleotide. The invention also provides a penicillium citrinum 3.2788-Y01 of nuclease P1, which fungus stroing number is CGMCC NO.1943, the stem can raise one times active unit of the nuclease P1.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a production and preparation of nuclease P 1 Methods. Background technique [0002] Nucleic acid degradation products—nucleotides and their derivatives are widely used in food, medicine, agriculture, cosmetics, and biochemical reagents (Chen Taosheng, Modern Industrial Microbiology Volume 1, Shanghai Science and Technology Press, 1979, 153-164 ), while nuclease P 1 It is the main enzyme for enzymatically hydrolyzing RNA and DNA into 5'-nucleotides. [0003] In the 1960s, in order to develop the monosodium glutamate market, Japan began to obtain flavor enhancers—flavored nucleotides (5’-GMP and 5’-IMP, etc.) from degraded RNA. In 1957, Japanese scholar Guo Zhongming and others screened a strain of Penicillium citrinum (Agric Biol Chem 25, 693 (1961)), which can secrete an extracellular enzyme with a phosphodiesterase activity. Function, enzymatically digests RNA to generate 5'NMP....

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N1/14C12R1/80
Inventor 洪娜陈慎黎高沃
Owner 北京燕京中科生物技术有限公司
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