Applications of acid protease in preparation of pathogenic bacterium inhibition preparation

A technology of acid protease and pathogenic bacteria, applied in the field of bioengineering, can solve problems such as the lack of clear research on properties, and achieve the effect of controlling decay

Active Publication Date: 2017-06-13
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, no one has isolated and purified the P6281 protease of Trichoderma harzianum, and its properties have not been clearly studied, and there is no direct evidence that the protease inhibits fungal growth, and no one has ever directly applied it to antibacterial

Method used

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  • Applications of acid protease in preparation of pathogenic bacterium inhibition preparation
  • Applications of acid protease in preparation of pathogenic bacterium inhibition preparation
  • Applications of acid protease in preparation of pathogenic bacterium inhibition preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 acid protease P6281 anti-pathogenic fungus effect experiment

[0050] Botrytis cinerea, Rhizoctonia solani, Aspergillus flavus, Aspergillus fumigatus and Mucor circinosa involved in this embodiment are respectively Botrytis cinerea GIM3.47, Rhizoctonia solani GIM3.512, Aspergillus fumigatus GIM3.20 Mucor circinerii GIM3.79 were purchased from Guangdong Microbial Culture Collection Center; Aspergillus flavus GIM3.493 was purchased from Institute of Microbiology, Chinese Academy of Sciences.

[0051] (1) Plant pathogenic bacteria Botrytis cinerea and Rhizoctonia solani preserved at -80°C, animal and plant pathogenic bacteria Aspergillus flavus, Aspergillus fumigatus, and conditional pathogenic bacteria Mucor circinii were respectively inoculated on PDA plates, and cultivated at 25°C for 5 sky;

[0052] (2) Pick the mycelia with a diameter of 4mm with an inoculation loop and inoculate them into the PDA medium (experimental group) containing 10% of the medium vo...

Embodiment 2

[0057] Application of embodiment 2 acid protease P6281 in fruit storage

[0058] Rinse the PDA plate of Botrytis cinerea cultured for 5 days with sterile water to obtain a bacterial suspension, and mix an equal volume of 20 μL of pure enzyme solution (containing 2 μg of P6281 protease) and bacterial suspension (concentration of about 10 5 each / mL) injection and inoculation to apples, oranges and cucumbers which were surface-sterilized with 1% sodium hypochlorite solution, as the experimental group; Cultivate at room temperature to observe the development of Botrytis cinerea.

[0059] The results are attached Figure 6-8 As shown, the left side of each figure is the experimental group of P6281+ Botrytis cinerea suspension, and the right side is the control group. Figure 6 In order to observe the development of botrytis cinerea for 4 days, record the development radius of botrytis cinerea lesions, the average radius of the lesion circle in the control group was 0.76cm, and no...

Embodiment 3

[0060] Example 3 Heterologous expression and purification of Trichoderma harzianum acid protease P6281

[0061] (1) Trichoderma harzianum culture:

[0062] Inoculate Trichoderma harzianum GIM 3.442 (purchased from Guangdong Microbial Culture Collection Center) to PDA solid medium (200 mL of potato flour filtrate, 4 g of glucose, 0.75 g of magnesium sulfate, 0.75 g of potassium dihydrogen phosphate, and 4 g of agar powder) Incubate at 30°C for 2 days.

[0063] (2) Trichoderma harzianum total RNA extraction:

[0064] (1) Take about 100 mg of mycelia with tweezers after high-pressure steam sterilization, put it into a mortar pre-cooled with liquid nitrogen, add a small amount of liquid nitrogen, grind quickly with a mortar, then add a small amount of liquid nitrogen, continue grinding, repeat 3 times until all the mycelium completely turns into white powder.

[0065] (2) Add 2mL RNAiso Plus (purchased from Dalian Bao Biological Engineering Co., Ltd.) to the mortar, try to cove...

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Abstract

The present invention discloses applications of an acid protease in preparation of a pathogenic bacterium inhibition preparation. According to the present invention, on the basis of the successful recombination expression purification of the acid protease P6281 of Trichoderma harzianum, the inhibition effect of the acid protease P6281 on animal and plant pathogenic fungi is first verified, and the acid protease P6281 is firstly applied in the fresh keeping of fruits and vegetables, the biological prevention and control, and the storage and mildew prevention of crops; the used protease is derived from Trichoderma harzianum, is non-toxic and safe, and cannot cause environment pollution; the problems that the direct application of the Trichoderma harzianum preparation is unsuitable for the growth of fruits and vegetables, the direct application of the Trichoderma harzianum preparation can cause the rotting of fruits and vegetables, and the like are overcome; and the acid protease can be combined with the commonly-used fruit and vegetable fresh-keeping technology, such that the gray mold of fruits and vegetables during the storage can be controlled, and the postharvest decay can be effectively controlled.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the application of an acidic protease in preparing preparations for inhibiting pathogenic bacteria. Background technique [0002] Botrytis cinerea is a necrotrophic pathogenic fungus with a wide host range, which can cause more than 200 kinds of fruits, vegetables and flowers Botrytis cinerea. It is widely distributed in the air, and its harm has become more and more serious in recent years, and the crop yield reduction rate can reach 60%. Botrytis cinerea can not only infect field crops, but also cause huge losses in the post-harvest stage of plants. Among all storage diseases, the losses caused by it are the most serious. [0003] Rhizoctonia solani is a soil-borne pathogenic fungus with a wide range of hosts, which can cause diseases of rice, wheat, cotton, and various vegetables and fruits. At present, it is widely distributed and has become a worldwide d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N63/02A01P3/00A23B7/154A23L3/3463
CPCA23B7/154A23L3/3463A01N63/10
Inventor 罗晓春邓俊劲李志伟黄伟谦
Owner SOUTH CHINA UNIV OF TECH
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