CN102676395A - Aspergillus usamii mutant strain and application thereof in preparation of acid protease - Google Patents
Aspergillus usamii mutant strain and application thereof in preparation of acid protease Download PDFInfo
- Publication number
- CN102676395A CN102676395A CN2012100204974A CN201210020497A CN102676395A CN 102676395 A CN102676395 A CN 102676395A CN 2012100204974 A CN2012100204974 A CN 2012100204974A CN 201210020497 A CN201210020497 A CN 201210020497A CN 102676395 A CN102676395 A CN 102676395A
- Authority
- CN
- China
- Prior art keywords
- aspergillus
- strain
- mutant strain
- enzyme
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to an aspergillus usamii mutant strain and an application thereof in preparation of acid protease, belonging to the technical field of microorganisms. The invention discloses the aspergillus usamii mutant strain (Aspergillus sp.) M1223 and a method for preparing the acid protease by utilizing the strain, the strain is collected in China General Microbiological Culture Collection Center, the collection number is CGMCC No. 5335, and the collection data is October 12, 2011. Aspergillus usamii disclosed by the invention can be used for high production of the acid protease through a liquid-state deep fermentation technology, and the enzyme can effectively degrade zein, glutenin, cytokeratin and the like under acidic conditions, and further has better application potential in foods, brewing, fur processing and other aspects.
Description
[technical field]
The present invention relates to strain Aspergillus usamii mutant strain (Aspergillus sp.) M1223 and the application in the preparation aspartic protease thereof, belong to microbial technology field.
[background technology]
Aspergillus usamii is a kind of of black mold, is widely used in the production of aspartic protease, zytase, saccharifying enzyme etc. in the industry.Proteolytic enzyme is the enzyme of peptide bond, generation amino acid and the polypeptide of one type of protein hydrolysate, according to the difference of action condition, can be divided into aspartic protease, neutral protease and Sumizyme MP.Aspartic protease is the important enzyme of zymin industry, is widely used in having bigger market potential in medicine, feed, weaving and the brewery industry.The development of microbial source aspartic protease starts from the sixties in 20th century; At present; What found ability secreting acidic proteolytic enzyme both at home and abroad mainly is various moulds; Like black mold, aspergillus oryzae, saitox aspergillus, head mold and their variant, mutant strain etc., the main bacteria seed that is applied to suitability for industrialized production is a black mold.
[summary of the invention]
The purpose of this invention is to provide bacterial strain Aspergillus usamii (Aspergillus sp.) M1223 that a plant height produces aspartic protease; With it serves as to produce bacterial strain; Utilize deep layer liquid state fermentation technology preparation aspartic protease, and tentatively inquire into its Degradation different proteins.
The bacterial strain of product aspartic protease provided by the invention is from milk-product source mill mud, to screen to obtain; Be accredited as the black mold flora through 26S rDNA; Confirm as Aspergillus usamii in conjunction with the Physiology and biochemistry evaluation; This bacterial classification obtains the mutant strain that a plant height produces acidic protein through beam-plasma mutagenesis, and called after (Aspergillus sp.) M1223 is preserved in No. 3 China Committee for Culture Collection of Microorganisms common micro-organisms centers, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City at present; Preserving number CGMCCNo.5335, preservation date are on October 12nd, 2011.
Correlative study to this bacterial strain obtains as drawing a conclusion:
1. substratum
Screening culture medium (g/L): casein food grade 3, potassium hydrogenphosphate 1, sal epsom 0.5, Repone K 0.5, ferrous sulfate 0.01, sucrose 30, agar 20g, PH nature.
Seed culture medium (g/L): SODIUMNITRATE 3, potassium hydrogenphosphate 1, sal epsom 0.5, Repone K 0.5, ferrous sulfate 0.01, sucrose 30, PH nature.
Fermention medium (g/L): soybean cake powder 30～100, Semen Maydis powder 5～15, Sodium phosphate, dibasic 2～5, ammonium chloride 10～15, calcium chloride 2～5, PH nature.
2. culture condition
Seed culture condition:, shake in the bottle 105～6 spore/ml of inoculum size, 200r/min, 30 ℃ of fermentation culture 2～3 days with spore inoculating at the 250ml that contains the 30ml seed culture medium.
Shake-flask culture condition: shake in the bottle inoculum size 5% (V/V), 200r/min, 30 ℃ of fermentation culture 3～4 days at the 500ml that contains the 50ml fermention medium.
5L jar fermentation condition: in containing the 5L fermentor tank of 3L fermention medium, inoculum size 5% (V/V), 500r/min, air flow 0.8～1vvm, 30 ℃ of fermentation culture 3～4 days.
3. thick enzymatic property:
(1) be the effect substrate with the casein, this enzyme optimum temperature is 40 ℃, and the righttest action pH is 3.0.
(2) under the righttest action condition, enzymic activity reaches 5600U/ml.
(3) this enzyme molecular weight is about 45KD.
Aspergillus usamii disclosed by the invention (Aspergillus sp.) M1223 is through liquid submerged fermentation technology high yield aspartic protease; This enzyme can be under acidic conditions effective degrading maize prolamine, glutenin, cytokeratin etc., at food, brewage, aspect such as fur processing has application potential preferably.
[embodiment]
Embodiment 1: the screening of bacterium producing multi enzyme preparation and mutagenic and breeding
Primary dcreening operation: soil sampling 1g is dissolved in the 50ml sterilized water around milk-product source mill; Shake up and process soil supension; Soil supension diluted get 200 μ l behind the different multiples and coat in the solid plate of above-mentioned screening culture medium; Cultivate after 2～3 days, the separation of ruling of the single bacterium colonies of several strains that picking hydrolysis circle/diameter is big, and be stored in the seed slant medium.
Multiple sieve: a few strain bacterium that primary dcreening operation is obtained shake the multiple sieve of bottle, and the shake-flask culture base is above-mentioned fermention medium, and at 30 ℃, 200r/min cultivates that to survey in the fermented liquid enzyme after 3～4 days alive, obtains producing the highest strain bacterium called after M12 of enzyme.
Beam-plasma mutagenesis: with the above-mentioned bacterial strains is starting strain, carries out the N of 8 dosage
+Ion beam mutagenesis, mutagenesis dosage are respectively (20,40,60,80,100,150,200,300) * 10
14, through dull and stereotyped primary dcreening operation, shake the multiple sieve of bottle and obtain producing the highest plant mutant strain of enzyme, called after M1223.
Embodiment 2: strain identification
The fresh bacterium liquid in vegetative period of taking the logarithm, centrifugal collection mycelia is pressed genome extraction agent box and extracts genomic dna.And obtain about 550bp product with fungi 26S rDNA universal primer amplification; Serve the sea and give birth to the order-checking of worker's biotechnology Engineering Co., Ltd; Get 26S rDNA sequence, in GenBank, carry out the BLAST comparison and confirm that it belongs to black mold crowd (Aspergillus niger), this bacterium is at slope Kou Shi substratum (NaNO
215g/L, K
2HPO
41g/L, KCl 0.5g/L, MgSO
40.5g/L, FeSO
40.01g/L, sucrose 30g/L, agar 20g/L) and go up growth, explain to have the effect of assimilation nitrous acid, it is black that microscopically is observed conidial head, determines that it is black mold crowd's Aspergillus usamii (Aspergillus sp.) M1223 according to black mold crowd key.This bacterium is preserved in Chinese microorganism strain preservation center at present, and preserving number CGMCC No.5335, preservation date are on October 12nd, 2011.
Embodiment 3: the mensuration of protease activity
The mensuration that the proteolytic enzyme enzyme is lived adopts the industrial protease preparation measuring method of GB (QB1805.3-93).
The live definition of unit of enzyme: the 1ml liquid enzymes, under 40 ℃, the condition of pH3.0, the PM caseinhydrolysate produces 1 microgram tyrosine, is 1 enzyme activity unit, representes with U/ml.
Embodiment 4: the aspartic protease zymologic property
(1) temperature is to the influence of enzymic activity: the same reaction system, under 30～100 ℃, measure this proteolytic enzyme enzyme activity respectively, and must this enzyme optimum temperature be 40 ℃.
(2) pH is to the influence of enzymic activity: the same reaction system, and under pH2-9, survey this enzyme enzyme respectively and live, must the righttest action pH of this enzyme be 3.0.
(3) mensuration of protease molecule amount size: the crude enzyme liquid that fermentation is obtained passes through SDS-PAGE electrophoretic analysis molecular weight of albumen, and this enzyme molecular weight is about 45KD.
The fermentation test of embodiment 5M1223 bacterial strain 5L jar
Spore is cultivated: in the eggplant bottle that contains 50ml solid seed culture medium,, place 30 ℃ of constant incubators to cultivate 3～5 days with spore or mycelia streak inoculation, and ripe to spore.With saline water spore is washed, process the spore suspension of 107～8/ml.
Seed culture: shake in the bottle at the 250ml that contains the 30ml seed culture medium, insert above-mentioned spore suspension 300 μ L, place rotary shaking table, 30 ℃, 200r/min fermentation culture 2～3 days are to the logarithm middle and later periods.
5L jar fermentation: in containing the 5L fermentor tank of 3L fermention medium, insert above-mentioned seed 150ml, 500r/min, air flow 0.8～1vvm, 30 ℃ of fermentation culture 3～4 days, to enzyme live no longer increase till.
The fermentation broth enzyme biopsy is surveyed: every 4h gets fermented liquid behind the fermentation 48h, surveys its crude enzyme liquid enzyme according to the method for embodiment 3 and lives, and reaches the highest enzyme 5600U/ml alive during 88h.
Embodiment 6 crude enzyme liquids are to the Preliminary detection of different proteins hydrolysis ability
Zein with 10%, glutenin, cytokeratin are as substrate; At pH3.0; Under 40 ℃ of conditions, detect the hydrolytic action of above-mentioned crude enzyme liquid, with trichloroacetic acid precipitation protein hydrolysate not to different proteins; Lyophilize is surveyed its content as investigating index; Obtaining the aspartic protease that Aspergillus usamii (Aspergillus sp.) M1223 produces all has certain hydrolytic action to above three kinds of protein, and the percent hydrolysis of 30min is 10～30% not wait, and hydrolysis effect is: glutenin＞zein＞cytokeratin.
Claims (2)
1. strain Aspergillus usamii mutant strain (Aspergillus sp.) M1223 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.5335, and preservation date is on October 12nd, 2011.
2. utilize Aspergillus usamii mutant strain (Aspergillus sp.) M1223 to prepare the method for acidic protein; It is characterized in that: through slant activation, liquid seeds cultivation, the liquid state fermentation of 5L fermentor tank deep layer, fermented liquid obtains crude enzyme liquid through the centrifugal thalline that goes with bacterial strain; With the casein is substrate, and the optimum temperature of above-mentioned crude enzyme liquid is 40 ℃, and the righttest action pH is 3.0; Above-mentioned crude enzyme liquid effectively degrading maize prolamine, glutenin, cytokeratin under the righttest action condition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100204974A CN102676395A (en) | 2012-01-20 | 2012-01-20 | Aspergillus usamii mutant strain and application thereof in preparation of acid protease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100204974A CN102676395A (en) | 2012-01-20 | 2012-01-20 | Aspergillus usamii mutant strain and application thereof in preparation of acid protease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102676395A true CN102676395A (en) | 2012-09-19 |
Family
ID=46808951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100204974A Pending CN102676395A (en) | 2012-01-20 | 2012-01-20 | Aspergillus usamii mutant strain and application thereof in preparation of acid protease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102676395A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146587A (en) * | 2013-03-22 | 2013-06-12 | 江南大学 | Method for producing keratinase by induced fermentation of aspergillus usaanii for originally producing acidic proteinase |
| CN105316308A (en) * | 2014-08-04 | 2016-02-10 | 湖南新鸿鹰生物工程有限公司 | High-activity acid protease |
| CN108441428A (en) * | 2018-03-22 | 2018-08-24 | 江南大学 | One plant degradation alcohol soluble protein rhizopus chinensis and its application |
| CN109097308A (en) * | 2018-09-04 | 2018-12-28 | 湖南肯基因科技有限公司 | The mutagenic strain and application thereof of high yield acid protease |
-
2012
- 2012-01-20 CN CN2012100204974A patent/CN102676395A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| SOICHI ARAI等: "Applying Protedytic Enzymes on Soybean PartÒProperties of Soy Protein Treated with Microbial Acid Protease (Molsin)", 《AGRICULTURAL AND BIOLOGICAL CHEMISTRY》 * |
| 周勤诚等: "宇佐美曲霉酸性蛋白酶的研究I.酸性蛋白酶537菌株的选育及其发酵条件", 《微生物学报》 * |
| 李乃强等: "霉菌酸性蛋白酶高产突变菌株9169的选育", 《无锡轻工大学学报》 * |
| 李永泉等: "宇佐美曲霉L-336酸性蛋白酶2 m3罐中试发酵研究报告", 《食品与发酵工业》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146587A (en) * | 2013-03-22 | 2013-06-12 | 江南大学 | Method for producing keratinase by induced fermentation of aspergillus usaanii for originally producing acidic proteinase |
| CN105316308A (en) * | 2014-08-04 | 2016-02-10 | 湖南新鸿鹰生物工程有限公司 | High-activity acid protease |
| CN108441428A (en) * | 2018-03-22 | 2018-08-24 | 江南大学 | One plant degradation alcohol soluble protein rhizopus chinensis and its application |
| CN109097308A (en) * | 2018-09-04 | 2018-12-28 | 湖南肯基因科技有限公司 | The mutagenic strain and application thereof of high yield acid protease |
| CN109097308B (en) * | 2018-09-04 | 2021-06-29 | 湖南肯基因科技有限公司 | Mutagenic strain for high yield of acid protease and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109439601B (en) | Bacterial strain capable of producing protease and method for preparing alkaline protease by using bacterial strain | |
| Sukkhum et al. | A novel poly (L-lactide) degrading actinomycetes isolated from Thai forest soil, phylogenic relationship and the enzyme characterization | |
| Padmapriya et al. | Purification and characterization of neutral protease enzyme from Bacillus subtilis | |
| CN110157623B (en) | Fusarium strain and method for producing D-pantolactone hydrolase by fermenting same | |
| CN104694424A (en) | Bacillus amyloliquefaciens strain separated from fermented soybeans and producing protease | |
| CN101157903B (en) | Producing Strain for beta- mannose and preparation method thereof | |
| CN100368519C (en) | Aspergillus niger lipase and its preparation method | |
| CN104388341B (en) | A kind of colloid bacillus cereus bacterial strain and its application in marine alga is degraded | |
| Nimisha et al. | Amylase activity of starch degrading bacteria isolated from soil | |
| CN102676395A (en) | Aspergillus usamii mutant strain and application thereof in preparation of acid protease | |
| Chaudhary et al. | Thermophilic actinomycetes from hot water spring capable of producing enzymes of industrial importance | |
| KR100738007B1 (en) | -68 Novel Bacillus Velezensis A-68 and Use of the Same | |
| Manivasagan et al. | Isolation, identification and characterization of multiple enzyme producing actinobacteria from sediment samples of Kodiyakarai coast, the Bay of Bengal | |
| CN104789491A (en) | Bacillus licheniformis strain and application thereof | |
| Ariole et al. | Alkaline protease production by Pseudomonas aeruginosa isolated from the gut of Pila ovata | |
| CN112195137B (en) | Bacillus belgii for antagonizing magnaporthe grisea and producing gamma-polyglutamic acid and application thereof | |
| KR101746398B1 (en) | Streptomyces atrovirens WJ2, a new microbe having great activity degrading xylan | |
| JP4618033B2 (en) | Method for producing food rich in γ-aminobutyric acid and γ-aminobutyric acid producing bacterium Lactobacillus carbatus KM14 | |
| Shumi et al. | Production of protease from Listeria monocytogenes | |
| CN106701867A (en) | Method for improving output of nikkomycin | |
| KR100882021B1 (en) | Method for production of Bacillus velezensis A-68 with rice hull | |
| RU2303066C1 (en) | Bacterium strain bacillus licheniformis as alkaline protease producer | |
| Qureshi et al. | Production of proteases by Staphylococcus epidermidis EFRL 12 using cost effective substrate (molasses) as a carbon | |
| CN103789239A (en) | Bacillus strain, and screening method and application thereof | |
| Nashima et al. | Production and optimization of lipase from wild and mutant strains of Bacillus sp. and Pseudomonas sp |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120919 |
|
| RJ01 | Rejection of invention patent application after publication |