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Aspergillus niger of high yield oxidation-resistant low temperature glucose oxidase

A technology of glucose oxidase and Aspergillus niger, which is applied in the field of bioengineering and can solve problems such as increased production costs

Active Publication Date: 2017-12-19
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, almost all glucose oxidase depends on imports, and the production cost also increases accordingly.

Method used

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  • Aspergillus niger of high yield oxidation-resistant low temperature glucose oxidase
  • Aspergillus niger of high yield oxidation-resistant low temperature glucose oxidase
  • Aspergillus niger of high yield oxidation-resistant low temperature glucose oxidase

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Experimental program
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Effect test

Embodiment 1

[0036] The mutagenesis selection of embodiment 1 bacterial strain

[0037] Preparation of spore suspension: elute the spores on the slant of the original strain with an appropriate amount of sterile physiological saline, place them in a pre-sterilized triangular flask with glass beads, vibrate on a shaker for 20 minutes, and filter them out with sterilized absorbent cotton. Mycelium to dispersed single spore suspension, counted with a hemocytometer. diluted to 10 8 individual / mL spore suspension.

[0038] Microwave mutagenesis: put a test tube containing 5 mL of spore suspension in a beaker containing ice, use a microwave oven with a frequency of 2450 MHz and an output power of 700 W, irradiate the test tubes one by one at different times, and the dilution gradient is 10 -1 ~10 -6 . Take 10 of each dose -4 ~10 -6 Spread 0.2 mL of spore suspension at 3 dilutions on the plate medium, incubate at 30°C for 2-3 days, count the number of colonies, and draw the lethality curve...

Embodiment 2

[0046] Example 2 Production of glucose oxidase by liquid fermentation of bacterial strain CH870 and its extraction

[0047] (1) Seed tank cultivation:

[0048] Tank pressure 0.05-0.08MPa, cultivation temperature 30°C, ventilation volume 30m 3 / h, the stirring speed is 180r / min, and the pH is controlled to 7.0; cultivate until the thalli are darkly stained, strong, and free of bacteria, and the seed liquid is obtained after the cultivation is completed;

[0049] Seed tank medium: glucose 20%, peptone 25%, (NH 4 ) 2 SO 4 5%, K 2 HPO 4 1%, MgSO 4 ·7H 2 O0.5%, pH 7.0;

[0050] (2) Fermentation tank culture

[0051] Inoculate the seed liquid into the fermentation medium with 5% inoculation amount, the tank pressure is 0.05-0.08Mpa, the culture temperature is 30°C, the stirring speed is 300r / min, the pH is controlled at 6.5-7.0; the ventilation rate: 0-40h is 30m 3 / h, 40-58h is 45m 3 / h, 58h - 40m for the tank 3 / h; when the pH rises to 7.0, start feeding and control ...

Embodiment 3

[0067] Embodiment 3 glucose oxidase enzymatic assay method

[0068] Substrate system: pipette 2 mL of 0.07 g / L o-dianisidine solution and 1 mL of 5% glucose solution in a graduated test tube with a 1 mL pipette gun. Use a 0.5mL pipette to pipette 0.1mL of 0.1g / L horseradish peroxidase solution into the same graduated test tube. The substrate system was placed in a constant temperature water bath at 30 °C for 10 min.

[0069] Determination of enzyme activity: Use a pipette gun to draw 0.1mL of the diluted enzyme solution and shake it evenly in the substrate. Using a blank tube as a control, quickly measure the absorbance at 460nm with a visible spectrophotometer. Read the initial absorbance value as A 0 And timing, record the absorbance value A every 1min n , a total of 5min was measured.

[0070] From the measured absorbance value, calculate the enzyme activity of the enzyme solution according to the following formula: X1 (U / mL)

[0071] ΔA n+1 =A n+1 -A n (n=0,1,2,3,4...

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Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to aspergillus niger of producing low temperature glucose oxidase and a production method of the glucose oxidase. The aspergillus niger is aspergillus niger CH870, and has a collection number of CGMCC (China General Microbiological Culture Collection Center) No. 14138. Enzyme activity of fermentation liquid of the glucose oxidase produced by fermentation of the strain reaches above 2300U / ml; the produced glucose oxidase is stable in a pH (potential of hydrogen) scope of 2.0-8.0; the most suitable reaction temperature is 20 DEG C; the glucose oxidase is good in stability and has oxidation resistance; the residual enzyme activity is 100% under the condition of 30mmol hydrogen peroxide; the residual enzyme activity is 90.8% under the condition of 200mmol hydrogen peroxide; and the glucose oxidase is wide in application.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, in particular to a low-temperature glucose oxidase with improved oxidation resistance and a production method thereof. Background technique: [0002] Glucose oxidase (GOD) can specifically oxidize β-D-glucose into gluconic acid and hydrogen peroxide. Glucose oxidase has a wide range of applications in the fields of food, medicine and biology. [0003] Because glucose oxidase can catalyze glucose to consume oxygen and produce gluconic acid and hydrogen peroxide, it is widely used in the food industry, mainly in the following four aspects: removal of residual glucose in food, deoxygenation, sterilization, determination of food glucose content. [0004] Glucose oxidase, as a new type of green enzyme preparation, was listed as one of the 12 permitted feed additives by the state in 1999. The mechanism of action of glucose oxidase to promote growth is: the glucose oxidase added in animal feed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/04C12R1/685
CPCC12N9/0006C12Y101/03004C12N1/145C12R2001/685
Inventor 王兴吉郭庆文贾仁洁张杰刘文龙
Owner SHANDONG LONGKETE ENZYME PREPARATION
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