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Preparing method for Aspergillus oryzae protoplast

A protoplast and Aspergillus oryzae technology, applied in the field of bioengineering, can solve the problems such as the poor enzyme production ability of Aspergillus oryzae

Inactive Publication Date: 2016-03-09
JIANGXI SCI & TECH NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In actual production, the enzyme production ability of Aspergillus oryzae is often poor

Method used

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  • Preparing method for Aspergillus oryzae protoplast
  • Preparing method for Aspergillus oryzae protoplast
  • Preparing method for Aspergillus oryzae protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] will be 10 7 Aspergillus oryzae spores were inoculated in G-P liquid medium (1% peptone, 0.5% glucose, 0.5% potassium dihydrogen phosphate, 0.1% sodium nitrate, 0.05% magnesium sulfate), and cultured with sufficient shaking at 30°C for 20 hours. Filter the culture medium with a layer of lens-cleaning paper, and wash the mycelium remaining on the lens-cleaning paper with sterilized water, take 2 g of mycelium in a sterile environment, add 10 mL of enzyme solution (1% helicase, 0.8% lyase yatalase) , the enzymatic hydrolysis time is 3h, microscopic examination counts, and collects.

[0022] Enzyme solution (helicase 1%, Yatalase lyase 0.8%) preparation: Take 1g of helicase and 0.8g of Yatalase lyase, use 0.8mol / L sodium chloride as solvent, adjust the pH to 6.0 to 100mL, mix well and set aside . 0.8mol / L sodium chloride is also used as penetrant.

[0023] It can be known from microscopic examination and counting that when the enzymatic hydrolysis is performed for 3 hou...

Embodiment 2

[0025] will be 10 7 Aspergillus oryzae spores were inoculated in potato juice medium, Chapei medium, G-P medium (peptone 1%, glucose 0.5%, potassium dihydrogen phosphate 0.5%, sodium nitrate 0.1%, magnesium sulfate 0.05%), YPD medium (glucose 2%, peptone 2%, yeast extract powder 1%), each culture medium was parallelized three times, and cultured with sufficient shaking at 30°C for 20 hours, filtered the culture medium with four layers of lens-cleaning paper, and rinsed with sterilized water The mycelium that stays on the lens-cleaning paper gets mycelium 2g in aseptic environment, puts into the enzyme liquid (helicase 1%) that makes osmotic agent with 1mol / sorbitol, the result is as follows figure 1 shown.

[0026] Depend on figure 1 It can be seen that different culture media have an influence on the growth of mycelium. When the culture medium is rich in nutrients, mycelium grows faster and the cell wall is thicker, which affects the effect of enzymatic hydrolysis. Depend ...

Embodiment 3

[0028] Take 2g of Aspergillus oryzae mycelia cultured in G-P liquid medium with sufficient shaking at 30°C for 20 hours, use 1mol / sorbitol as the penetrant, take the mycelium cultured for 20 hours, put it into the enzyme solution with 1M sorbitol as the penetrant and solvent , do three parallel, enzymatic hydrolysis for 3h.

[0029] Enzyme solution: 1M sorbitol as solvent, respectively made into 1% helicase and 0, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1 %, 1.2% Yatalase lyase mixed enzyme solution. The result is as figure 2 shown.

[0030] Depend on figure 2 It can be seen that the amount of adding Yatalase lyase in 1% helicase has an impact on the preparation of protoplasts, and when the amount of adding Yatalase lyase was 0.8%, the quality of protoplasts reached 4.2 * 10 6 individual / mL, and when the amount of Yatalase lyase added was 0.8%, the number of protoplasts was relatively small.

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Abstract

The invention discloses a preparing method for Aspergillus oryzae protoplast, and relates to the technical field of methods for protoplast preparation in bioengineering. The method includes Aspergillus oryzae culture, hypha collection, enzymolysis and the like. By the adoption of the method, the yield of the protoplast can reach 1.5*10<7> / mL, the regeneration rate can reach 90%, and a foundation is laid for Aspergillus oryzae gene modification.

Description

technical field [0001] The invention relates to a method for preparing protoplasts of Aspergillus oryzae, belonging to the technical field of bioengineering. Background technique [0002] Aspergillus oryzae is an aerobic fungus, which belongs to the subphylum Deuteromycota and the genus Aspergillus. Its mycelium is generally yellow-green, with large sporulation and multinucleation. It lacks sexual reproduction in its growth cycle. Aspergillus oryzae is a kind of strain that can produce complex enzymes. The enzymes produced include cellulase, amylase, glucoamylase, protease, etc. Therefore, Aspergillus oryzae occupies a very important position in brewing, feed, food and other industrial fields, including It is widely used in rice wine, soy sauce, commercial enzyme and medical protein production. In addition, Aspergillus oryzae has efficient protein secretion and expression ability, and its heterologous protein expression level is comparable to that of Pichia pastoris, an exc...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/06C12R1/69
CPCC12N1/14C12N1/06
Inventor 曾斌郝庆刘华
Owner JIANGXI SCI & TECH NORMAL UNIV
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