CN109161541A - A kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield - Google Patents

A kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield Download PDF

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CN109161541A
CN109161541A CN201811010797.8A CN201811010797A CN109161541A CN 109161541 A CN109161541 A CN 109161541A CN 201811010797 A CN201811010797 A CN 201811010797A CN 109161541 A CN109161541 A CN 109161541A
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long handle
medium
low energy
handle trichoderma
ion implantation
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徐双贵
胡培
李垒
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Anhui Xin Kang Feed Co Ltd
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Anhui Xin Kang Feed Co Ltd
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi

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Abstract

The invention discloses a kind of methods of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield, are related to technical field of bioengineering.It include: that the long handle trichoderma strain of original acid protease is seeded on solid fermentation plating medium；The long handle trichoderma strain on plating medium is taken to be isolated and purified using isolation by dilution method；It is taken with oese and takes long handle reesei spores on slant medium, be added in sterile water and be diluted；Spore suspension is taken to be spread evenly across in empty sterile petri dish, the target chamber for being then placed in ion implantation apparatus carries out ion implanting；It chooses in the good strain inoculated to seed culture medium of upgrowth situation；The strain inoculated of above-mentioned selection is cultivated into fermentation liquid culture medium.The present invention is repeatedly cultivated by different nutrient mediums and separating-purifying, while also using Low energy N+ ions, improves the yield of the acid protease of long handle trichoderma, while obtaining acid protease also to have high temperature resistance.

Description

A kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield
Technical field
The invention belongs to technical field of bioengineering, high more particularly to a kind of low energy N+ ion implantation mutagenesis long handle trichoderma The method for producing protease.
Background technique
Acid protease is one kind of hydrolase, can be under the conditions of slightly sour, by inscribe and circumscribed effect by dynamic plant Object protein is hydrolyzed to small peptide and amino acid, is widely used in food, medicine, leather industry, fur softening and depilation processing.In recent years Come, it is same in animal body to supplement since acid protease has the protein that can fast and effeciently assist animal to decompose in feed The deficiency of source enzyme promotes animal especially absorption of the young animal to a variety of nutriments, promotes its growth, enhances body disease-resistant Ability.With the development of intensive Production of Livestock and Poultry, environmental pollution is exacerbated.Wherein, nitrogen is the main matter for polluting environment.Thus The utilization efficiency of protide nutriment in feed is improved, reducing its discharge is just particularly important.Further, since China's albumen Matter feed resource there is a serious shortage of, need to seek unconventional protein resource, and animal to the utilization efficiency of unconventional protein feedstuff very It is low.Therefore, acid protease is shown huge potential by as a kind of novel biological feed additive in feed industry Value, the increasingly great attention by feed, cultivation and Animal nutrition circle.
And existing long handle trichoderma its acid protease low output, therefore the proteinase production for how improving long handle trichoderma is It is solved the problems, such as needed for of the invention.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield, lead to It crosses different nutrient mediums repeatedly to be cultivated and separating-purifying, while also using Low energy N+ ions, improve long handle The yield of the acid protease of trichoderma, while obtaining acid protease also there is high temperature resistance.
In order to solve the above technical problems, the present invention is achieved by the following technical solutions:
The present invention is a kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield, is included the following steps:
The long handle trichoderma strain of original acid protease is seeded on solid fermentation plating medium by step 1,28 DEG C of trainings It supports 3-5 days, continuous activation 3 times spare；
Step 2 takes the long handle trichoderma strain on plating medium to be isolated and purified using isolation by dilution method；And take purifying Long handle trichoderma strain afterwards is inoculated on slant medium and carries out shaking flask culture 2-4 days；
Step 3 is taken with oese and takes long handle reesei spores on slant medium, is added in sterile water and is diluted growth Handle reesei spores number concentration is the spore suspension of 4000-6000/ml；
Step 4 takes spore suspension to be spread evenly across in empty sterile petri dish, is dried up with sterile wind, is then placed in ion The target chamber of implanter carries out ion implanting, with sterile water elution after injection, is coated on activation plate；
Step 5 is chosen in the good strain inoculated to seed culture medium of upgrowth situation, in seed culture after culture 3-5 days Primary surface pours into one layer of milk；Choose the biggish bacterial strain of the ratio between the transparent loop diameter generated and colony diameter；
The strain inoculated of above-mentioned selection is carried out culture 8-15 days into fermentation liquid culture medium by step 6, and fermented and cultured terminates The activity of enzyme is measured afterwards；Choosing the high bacterial strain of enzymatic activity and being seeded to culture obtained strains on seed culture medium again is that high yield is resistance to The long handle trichoderma of high-temperature acidic protease.
Further, the solid fermentation plating medium includes wheat bran, dregs of beans and cornstarch；The wheat bran, dregs of beans Weight ratio with cornstarch three is 80-100:8-10:1-3；Also add in the solid fermentation plating medium of the per kilogram Added with ammonium sulfate 15-20g, the pH value of the solid fermentation plating medium is between 6-7.
Further, the slant medium includes: 15-25 parts of potato leachate, agar 1-3 by weight part Part, 1-3 parts of glucose；
The preparation method of the slant medium include: take potato clean peeling, be cut into ding shape and add boiling is rotten, uses yarn Filtrate is obtained by filtration in cloth；It is uniform that agar, glucose heating stirring are added into filtrate, appropriate steam is added after being cooled to 40-45 DEG C Distilled water constant volume sterilizes after bottling.
Further, the revolving speed of the shaking flask culture is set as 150-300rpm/min；The environment temperature of the shaking flask culture Degree is set as 27-29 DEG C.
Further, the seed culture medium are as follows: yeast extract 3-7g, tryptone 3-7g, glucose 8-12g, KH2PO415-20g、MgSO43-5g simultaneously adds deionized water to be settled to 1000ml；The PH of the seed culture medium is 5.5-6.
Further, the fermentation liquid culture medium is beancake powder 3.65%, corn flour 0.625%, fish meal 0.625%, sulphur Sour ammonium 1.0%, CaCl20.5%, Na2HPO40.2%, the PH of soya-bean cake hydrolyzate 6%, the fermentation liquid culture medium is 5-6.
The invention has the following advantages:
The present invention is repeatedly cultivated by different nutrient mediums and separating-purifying, while also using Low energy nitrogen ions Injection, improves the yield of the acid protease of long handle trichoderma, while obtaining acid protease also to have high temperature resistance；This hair The long handle trichoderma of bright acquisition produces acidic protein vigor and improves 35.5% than opportunistic pathogen strain, and passes through multiple secondary culture fermentation test, Producing enzyme vigor and its high temperature resistance are highly stable；, clearly by low energy N+ ion implantation mutagenesis, the strain enzyme-producing of acquisition is imitated for this It is big that rate promotes amplitude.
Certainly, it implements any of the products of the present invention and does not necessarily require achieving all the advantages described above at the same time.
Specific embodiment
A kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield, includes the following steps:
The long handle trichoderma strain of original acid protease is seeded on solid fermentation plating medium by step 1,28 DEG C of trainings It supports 3-5 days, continuous activation 3 times spare；
Step 2 takes the long handle trichoderma strain on plating medium to be isolated and purified using isolation by dilution method；And take purifying Long handle trichoderma strain afterwards is inoculated on slant medium and carries out shaking flask culture 2-4 days；
Step 3 is taken with oese and takes long handle reesei spores on slant medium, is added in sterile water and is diluted growth Handle reesei spores number concentration is the spore suspension of 4000-6000/ml；
Step 4 takes spore suspension to be spread evenly across in empty sterile petri dish, is dried up with sterile wind, is then placed in ion The target chamber of implanter carries out ion implanting, with sterile water elution after injection, is coated on activation plate；
Step 5 is chosen in the good strain inoculated to seed culture medium of upgrowth situation, in seed culture after culture 3-5 days Primary surface pours into one layer of milk；Choose the biggish bacterial strain of the ratio between the transparent loop diameter generated and colony diameter；
The strain inoculated of above-mentioned selection is carried out culture 8-15 days into fermentation liquid culture medium by step 6, and fermented and cultured terminates The activity of enzyme is measured afterwards；Choosing the high bacterial strain of enzymatic activity and being seeded to culture obtained strains on seed culture medium again is that high yield is resistance to The long handle trichoderma of high-temperature acidic protease.
Wherein, solid fermentation plating medium includes wheat bran, dregs of beans and cornstarch；Wheat bran, dregs of beans and cornstarch three The weight ratio of person is 90:9:2；Ammonium sulfate 15g, solid fermentation plate are also added in the solid fermentation plating medium of per kilogram The pH value of culture medium is between 6-7.
Wherein, slant medium includes: 20 parts of potato leachate, 2 parts of agar, 2 parts of glucose by weight part；
The preparation method of slant medium include: take potato to clean peeling, be cut into ding shape and add boiling it is rotten, with gauze mistake Filter obtains filtrate；It is uniform that agar, glucose heating stirring are added into filtrate, appropriate distilled water is added after being cooled to 40-45 DEG C Constant volume sterilizes after bottling.
Wherein, the revolving speed of shaking flask culture is set as 150-300rpm/min；The environment temperature of shaking flask culture is set as 27-29 ℃。
Wherein, seed culture medium are as follows: yeast extract 5g, tryptone 5g, glucose 8-12g, KH2PO418g、MgSO44g And deionized water is added to be settled to 1000ml；The PH of seed culture medium is 5.5-6.
Wherein, fermentation liquid culture medium is beancake powder 3.65%, corn flour 0.625%, fish meal 0.625%, ammonium sulfate 1.0%, CaCl20.5%, Na2HPO40.2%, soya-bean cake hydrolyzate 6%, the PH for the liquid culture medium that ferments is 5-6.
In the description of this specification, the description of reference term " one embodiment ", " example ", " specific example " etc. means Particular features, structures, materials, or characteristics described in conjunction with this embodiment or example are contained at least one implementation of the invention In example or example.In the present specification, schematic expression of the above terms may not refer to the same embodiment or example. Moreover, particular features, structures, materials, or characteristics described can be in any one or more of the embodiments or examples to close Suitable mode combines.Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.Preferred embodiment is not There is the details that detailed descriptionthe is all, does not limit the invention to the specific embodiments described.Obviously, according to this specification Content can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is in order to preferably explain this The principle and practical application of invention, so that skilled artisan be enable to better understand and utilize the present invention.This hair It is bright to be limited only by the claims and their full scope and equivalents.

Claims (6)

1. a kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield, which comprises the steps of:
The long handle trichoderma strain of original acid protease is seeded on solid fermentation plating medium by step 1,28 DEG C of culture 3- 5 days, continuous activation 3 times spare；
Step 2 takes the long handle trichoderma strain on plating medium to be isolated and purified using isolation by dilution method；And it takes after purification Long handle trichoderma strain is inoculated on slant medium and carries out shaking flask culture 2-4 days；
Step 3 is taken with oese and takes long handle reesei spores on slant medium, is added in sterile water and is diluted into long handle wood Mould spore number concentration is the spore suspension of 4000-6000/ml；
Step 4 takes spore suspension to be spread evenly across in empty sterile petri dish, is dried up with sterile wind, is then placed in ion implanting The target chamber of machine carries out ion implanting, with sterile water elution after injection, is coated on activation plate；
Step 5 is chosen in the good strain inoculated to seed culture medium of upgrowth situation, in seed culture base table after culture 3-5 days Pour into one layer of milk in face；Choose the biggish bacterial strain of the ratio between the transparent loop diameter generated and colony diameter；
The strain inoculated of above-mentioned selection is carried out culture 8-15 days into fermentation liquid culture medium by step 6, is surveyed after fermented and cultured Measure the activity of enzyme；Choosing the high bacterial strain of enzymatic activity and being seeded to culture obtained strains on seed culture medium again is high-yield thermostable The long handle trichoderma of acid protease.
2. a kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield according to claim 1, special Sign is that the solid fermentation plating medium includes wheat bran, dregs of beans and cornstarch；The wheat bran, dregs of beans and cornstarch The weight ratio of three is 80-100:8-10:1-3；Ammonium sulfate is also added in the solid fermentation plating medium of the per kilogram 15-20g, the pH value of the solid fermentation plating medium is between 6-7.
3. a kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield according to claim 1, special Sign is that the slant medium includes: 15-25 parts of potato leachate, 1-3 parts of agar, glucose 1-3 by weight part Part；
The preparation method of the slant medium include: take potato to clean peeling, be cut into ding shape and add boiling it is rotten, with gauze mistake Filter obtains filtrate；It is uniform that agar, glucose heating stirring are added into filtrate, appropriate distilled water is added after being cooled to 40-45 DEG C Constant volume sterilizes after bottling.
4. a kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield according to claim 1, special Sign is that the revolving speed of the shaking flask culture is set as 150-300rpm/min；The environment temperature of the shaking flask culture is set as 27- 29℃。
5. a kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield according to claim 1, special Sign is, the seed culture medium are as follows: yeast extract 3-7g, tryptone 3-7g, glucose 8-12g, KH2PO415-20g、 MgSO43-5g simultaneously adds deionized water to be settled to 1000ml；The PH of the seed culture medium is 5.5-6.
6. a kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield according to claim 1, special Sign is, the fermentation liquid culture medium is beancake powder 3.65%, corn flour 0.625%, fish meal 0.625%, ammonium sulfate 1.0%, CaCl20.5%, Na2HPO40.2%, the PH of soya-bean cake hydrolyzate 6%, the fermentation liquid culture medium is 5-6.
CN201811010797.8A 2018-08-31 2018-08-31 A kind of method of low energy N+ ion implantation mutagenesis long handle trichoderma high proteinase yield Withdrawn CN109161541A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114437939A (en) * 2022-01-20 2022-05-06 中国林业科学研究院森林生态环境与自然保护研究所 Liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114437939A (en) * 2022-01-20 2022-05-06 中国林业科学研究院森林生态环境与自然保护研究所 Liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia

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