Method for preparing purine nucleoside phosphorylase by solid state fermentation

A technology of purine nucleoside phosphorylase and solid-state fermentation, which is applied in the field of preparing purine nucleoside phosphorylase by solid-state fermentation, and can solve the problems of improving the activity of purine nucleoside phosphorylase products, demanding equipment and operating techniques, and taking a long time. , to achieve the effect of less time-consuming, high production efficiency and less energy consumption

Active Publication Date: 2010-02-03
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to overcome the existing method for preparing purine nucleoside phosphorylase by liquid fermentation of Bacillus subtilis, which has strict requirements on equipment and operation technology, high energy consumption and long time consumption, and the ob...

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  • Method for preparing purine nucleoside phosphorylase by solid state fermentation
  • Method for preparing purine nucleoside phosphorylase by solid state fermentation
  • Method for preparing purine nucleoside phosphorylase by solid state fermentation

Examples

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Embodiment 1

[0140] This example illustrates the method for preparing purine nucleoside phosphorylase by solid-state fermentation of the present invention.

[0141] 1. Preparation of Bacillus subtilis seed liquid:

[0142] Under aseptic conditions, use a pipette to rinse the purchased ACCC 10741 slant with 5 ml of sterile normal saline, absorb the resulting washing solution, add it to 45 ml of sterile normal saline and shake to make a bacterial suspension. Take 5 milliliters of the bacterium suspension and insert it into 50 milliliters of liquid seed culture medium, shake the shaker at 37 ° C and 150 rpm for 6 hours, and then obtain a turbid first-stage seed liquid, which is obtained by cell counting. The cell density was 1.2 x 10 5 cells / ml.

[0143]Get the first-stage seed liquid, and inoculate it into two 1000-milliliter culture bottles containing 300 ml of seed culture medium with an inoculum amount of 20 milliliters per bottle. After cultivating on a shaking table for 10 hours, one...

Embodiment 2

[0174] Cultivate ACCC 10741 according to the method of Example 1, the difference is that the bacterial suspension obtained by washing the strains preserved on the slant with sterile physiological saline is heated to 68° C. in a water bath within 1 minute and incubated for 15 minutes, and cooled within 2 minutes. to 37°C; then cultured on a shaker at 37°C and 150 rpm for 5 hours to obtain a turbid first-stage seed solution, and the cell density obtained by cell counting was 2.9×10 6 cells / ml. Obtain 22 grams of purine nucleoside phosphorylase dry powder. The specific activity of the obtained purine nucleoside phosphorylase is 1.00KU / gram. The yield of this embodiment is 10KU / kg solid fermentation medium liquid part.

Embodiment 3

[0176] This example illustrates the method for preparing purine nucleoside phosphorylase by solid-state fermentation of the present invention.

[0177] 1. Preparation of Bacillus subtilis seed liquid:

[0178] Under aseptic conditions, use a pipette to rinse the purchased ACCC 10741 slant with 5 ml of sterile normal saline, absorb the resulting washing solution, add it to 45 ml of sterile normal saline and shake to make a bacterial suspension. Take 5 milliliters of the bacterium suspension and insert it into 50 milliliters of liquid seed culture medium, shake it at 35 DEG C and 180 rpm for 8 hours, and then obtain a turbid first-stage seed liquid, which is obtained by cell counting. The cell density was 1.1 x 10 5 cells / ml.

[0179] Get the first-stage seed liquid, and inoculate it into two 1000-milliliter culture bottles containing 300 ml of seed culture medium with an inoculum volume of 20 milliliters per bottle. After cultivating on a shaking table for 10 hours, one of t...

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Abstract

The invention provides a method for preparing purine nucleoside phosphorylase by solid state fermentation of bacillus subtilis, wherein, the mould-free corn cobs after sterilization with the particlediameter of 20-40 meshes and the water content of 30% are adopted as the solid matrix of the solid state fermentation culture medium. The inventor of the invention overcomes the prejudice that 'purinenucleoside phosphorylase products are more easily separated from liquid state fermentation of bacillus subtilis, compared with solid state fermentation of bacillus subtilis' which is held by the prior art; by utilizing the solid state fermentation method of the invention, efficient separation of purine nucleoside phosphorylase products from bacillus subtilis can be realized. The method of the invention has the advantages of less energy consumption, less time consumption and higher activity of the obtained purine nucleoside phosphorylase products.

Description

technical field [0001] The invention relates to a method for preparing enzyme by solid-state fermentation, more specifically, the invention relates to a method for preparing purine nucleoside phosphorylase by solid-state fermentation. Background technique [0002] Purine Nucleoside Phosphorylase (PNP, enzymatic classification number EC 2.4.2.1) catalyzes the following reaction: [0003] [0004] Among them, inosine is an important product of the reaction catalyzed by serum adenosine deaminase and 5'-nucleotidase, so purine nucleoside phosphorylase can be used to measure serum adenosine deaminase and 5'-nucleotidase associated with hepatobiliary diseases - Clinical biochemical diagnostic kit for nucleotidase activity. [0005] The existing purine nucleoside phosphorylase is generally obtained by liquid fermentation of Bacillus subtilis. For example, CN101289646 discloses a method for obtaining purine nucleoside phosphorylase by liquid fermentation of Bacillus subtilis (A...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12R1/125
Inventor (请求不公开姓名)
Owner BEIJING LEADMAN BIOCHEM
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