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Preparation method of proline endonuclease

An endoproline, proline technology, applied in biochemical equipment and methods, enzymes, peptidases and other directions, can solve the problem of no detection of proline endoprotease activity, unfavorable promotion and production, complex processes, etc. problems, to achieve the effect of wide temperature and pH adaptation range, easy control and good stability

Active Publication Date: 2017-05-17
NINGBO XINUOYA MARINE BIOTECH CO LTD
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AI Technical Summary

Problems solved by technology

At the same time, the proline endoprotease derived from Aspergillus niger has good thermal stability. When incubated at pH 5.0 and 65°C for 30 minutes, no decrease in proline endoprotease activity was detected.
[0004] At present, the production of proline endoprotease by using Aspergillus, especially Aspergillus niger, mainly adopts the method of genetic engineering. For example, Chinese patents CN200710150282.3, CN201610202929.1, and CN201310656778.3 all involve methods for producing proline endoprotease. The technical means used are to obtain the target enzyme preparation by cultivating engineering bacteria through genetic recombination. The process is complicated, and it is necessary to determine the amino acid sequence of the target enzyme preparation and design related primers, which is not conducive to industrial promotion and production.

Method used

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  • Preparation method of proline endonuclease
  • Preparation method of proline endonuclease
  • Preparation method of proline endonuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 The shake flask high-density fermentation of Aspergillus niger

[0022] Step 1: Streak the Aspergillus niger glycerin strains stored in conventional glycerol at -80°C on the wort medium plate, place it in a 30°C incubator and cultivate it for 2 days until a single colony grows; then pick a full single colony and streak Line inoculated on the wort medium plate, placed in 28°C incubator for 2 days, then took the full strain into 50mL BMGY liquid medium, 28°C, 200rpm shake flask cultured for 18h, to obtain the seed solution.

[0023] The composition of the BMGY liquid medium by weight percentage is: tryptone 20g / L, yeast extract 10g / L, 121°C, after sterilizing for 20min, adding 100mmol / L potassium phosphate at pH 6, 13.4g / L of amino nitrogen source, Biotin 4 x 10 -4 g / L (sterilized by filtration), glycerin 10g / L.

[0024] Step 2: In the aseptic environment of the ultra-clean workbench, under the protection of the flame, centrifuge the prepared seed solution ...

Embodiment 2

[0026] Embodiment 2 High-density fermentation in fermenter of Aspergillus niger

[0027] Step 1: with embodiment 1;

[0028] Step 2: In the aseptic operation room, insert the prepared seed solution into the Aspergillus niger high-density fermentation medium (same as Example 1), adjust the pH of the fermentation medium to 5.8, and cultivate at 30°C at 200-800r / min Fermentation; adjust the flow rate of 0.5 mg / L proline according to the pH and dissolved oxygen state of the fermenter, maintain the pH at 5.0-7.0, and maintain the dissolved oxygen at 35-50%, until the proline endonuclease enzyme The activity is slowly increased or no longer increased, and the fermentation is terminated. The fermentation time is 96-200h, and the fermentation bacteria concentration is 100-300g / L (calculated by the wet weight of the bacteria).

Embodiment 3

[0029] The optimal pH and stability of embodiment 3 proline endonuclease

[0030] Using proline as a substrate, determine the optimum pH and stability of proline endonuclease. Under the various pH conditions shown in Table 1, the relative activity was recorded after incubation at 37°C for 30 minutes, and the figure 1 pH curve shown. According to this curve, it can be seen that the enzyme shows the highest activity at pH 5.0, and the relative activity exceeds 60% between pH 3.5-8.0. It can be determined that its optimum pH range is 3.5-8.0. The optimum pH of Dicer is 4.5-5.5, and the method of the present invention obviously expands the optimum pH range of proline endonuclease. At the same time, from the perspective of the pH stability of the enzyme, it retains more than 80% of its initial activity between pH 3.0-7.0.

[0031] Table 1

[0032]

[0033] From the above data, it can be known that the proline endonuclease of the present invention can maintain stability in a ...

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Abstract

The invention discloses a preparation method of proline endonuclease. The preparation method comprises the following steps of preparing aspergillus niger strain liquid, culturing and fermenting, and extracting and treating fermenting liquid, wherein a BMGY liquid culture medium is used as a culture medium for preparation of the strain liquid, and culture conditions are as follows: shake-flask culture is adopted, the temperature is 28 to 35 DEG C, the speed is 200 to 250r / min, and the culture time is 12 to 18h; the prepared strain liquid is subjected to shake-flask culture, an aspergillus niger strain high-density fermenting culture medium is used as a culture medium, and culture conditions are as follows: the temperature is 28 to 35 DEG C, and the speed is 200 to 250r / min; or the prepared strain liquid is cultured in a fermenting tank, the spergillus niger strain high-density fermenting culture medium is used as a culture medium, and culture conditions are as follows: the temperature is 28 to 35 DEG C, and the speed is 200 to 800r / min; by filtering, concentrating and blending the fermenting liquid, and finely filtering or drying, liquid proline endonuclease or solid proline endonuclease is obtained. The preparation method has the advantages that the natural aspergillus niger strain is used, and proline is used as an inducing substrate, so that the proline endonuclease with high enzyme activity, wide temperature and pH (potential of hydrogen) application range and good stability is prepared under the moderate fermenting condition.

Description

technical field [0001] The invention relates to microbial fermentation engineering, in particular to a preparation method of an enzyme preparation, in particular to a preparation method of proline endonuclease. Background technique [0002] Aspergillus niger-derived prolyl endoprotease (prolyl endoprotease, PEP) is a protease that can efficiently hydrolyze the carboxy-terminal peptide bond of proline residues in proteins or polypeptides. PEP can be widely used to prevent beer haze, tea cheese formation and reduce the bitterness of protein hydrolyzate. There is no self-developed PEP enzyme product in the domestic market, and it is completely dependent on imports. Filamentous fungi have strong secretion and expression capabilities and are widely used to express homologous or heterologous proteins. As a basically safe (GRAS) microorganism, Aspergillus niger has complete and efficient protein post-translational modification, and it has the characteristics of safety, high yield...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/62
CPCC12N9/62C12Y304/21026
Inventor 田健潘叶羽施碧赢诸辉
Owner NINGBO XINUOYA MARINE BIOTECH CO LTD
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