CN102382786A - Alkali protease high-yield strain and fermentation method of alkali protease and protease produced by alkali protease high-yield strain - Google Patents
Alkali protease high-yield strain and fermentation method of alkali protease and protease produced by alkali protease high-yield strain Download PDFInfo
- Publication number
- CN102382786A CN102382786A CN2011103336788A CN201110333678A CN102382786A CN 102382786 A CN102382786 A CN 102382786A CN 2011103336788 A CN2011103336788 A CN 2011103336788A CN 201110333678 A CN201110333678 A CN 201110333678A CN 102382786 A CN102382786 A CN 102382786A
- Authority
- CN
- China
- Prior art keywords
- sumizyme
- strain
- enzyme
- alkali protease
- fermentation
- 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
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to alkali protease high-yield bacterial strain and alkali protease. The category name of the bacterial strain is Bacillus alcalophilus, the preserving number is CGMCC NO. 5313, the preserving date is September 30th, 2011, the preserving unit is China General Microbiological Culture Collection Center (CGMCC), the preserving address is No 3, yard 1, Beichen west road, Chaoyang District, Beijing, and enzyme is obtained by fermenting alkali protease high-yield strain. In the invention, the alkali protease high-yield strain obtained by inducing is obtained by injecting an inducer into the bacillus alcalophilus by low-energy N+ions, so that the enzyme generating capability is improved effectively, the maximum value of the flask shaking activity is 30700U/mL, and is increased by 37.7% compared with that of the original strain.
Description
Technical field
The invention belongs to biotechnology, relate to induction mutation of bacterium, relate in particular to the fermentation process of a kind of Sumizyme MP superior strain and Sumizyme MP that is produced and proteolytic enzyme.
Background technology
Sumizyme MP (Alkaline Protease) belongs to the serine proteinase enzyme in the endopeptidase; Can be at protein hydrolysate peptide bond under the alkaline condition; Its righttest action pH is generally 9～11; Be mainly used in enzyme-containing detergent industry, also be widely used in industry such as process hides, silk, feed, medicine, food, environmental protection.
This enzyme is found in the pancreas of pig.1913, Rohm at first used trypsinase as the washing soaking agent.1945, the Dr.Jaag of Switzerland etc. found Studies on Microbial Alkaline Protease, makes proteolytic enzyme might be widely used in detergent industry.1963; Novo Nordisk Co.,Ltd (existing Novozymes Company) has found more to be applicable to the Sumizyme MP Alcalase of washing composition; Zymin is widely used in the Betengent product; Enzymatic laundry powder occurred, in subsequently 20 years, bacterium proteinoid enzyme is unique commercialization zymin that is applied to washing composition.At present, worldwide protease is a kind of enzyme with the most use in the industrial enzyme, accounts for 60% of enzyme total amount, and wherein Sumizyme MP just accounts for 25%.Its huge applications prospect in commerce and the vital role in fundamental research are attracting international and domestic many companies and research unit competitively they to be carried out many-sided research.
In recent years; Ionic fluid is developing very rapid because of its unique mutagenesis mechanism and biological effect as a kind of new mutation source aspect the selection by mutation; Compare with traditional mutation source, ion implantation except having the energy deposition effect, also have effects such as momentum transfer, quality deposition and charge neutralization and exchange; It with physical mutagenesis and chemomorphosis characteristic; Can under the situation that low dosage injects, significantly improve biological aberration rate, the acquisition damage is light, mutation rate is high, mutation spectrum is wide, the mutagenesis effect of inheritance stability.Ion implantation technique is widely used in having selected some have big application prospect in industry biological new variety, and having obtained remarkable economic efficiency and social benefit in the seed selection and improvement research of mikrobe strain excellent.
Summary of the invention
The purpose of this invention is to provide a kind of employing low energy N
+The Sumizyme MP superior strain that the ion implantation technique mutagenic and breeding obtains and the fermentation process of Sumizyme MP that is produced and proteolytic enzyme thereof, present method effectively improve the ability that Alkaliphilic bacillus produces Sumizyme MP.
The present invention realizes that the technical scheme of purpose is following:
A kind of Sumizyme MP superior strain; It is characterized in that: name is called Y-22; Classification called after Alkaliphilic bacillus Bacillus alcalophilus, deposit number is: CGMCC No.5313, preservation date: on September 30th, 2011; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
A kind of Sumizyme MP, the fermentation of Sumizyme MP superior strain obtains.
And said basic protein enzyme molecular weight is 28kDa.
And said Sumizyme MP is when being substrate with the casein, and its righttest action pH is 10.5, and optimum temperature is 40 ℃, and is stable in the scope of pH7～12, at 40～50 ℃ thermostability preferably arranged.
A kind of fermentation process of Sumizyme MP is characterized in that: the used substratum that ferments is: fermention medium g/L: yeast soaks powder 15-20, cottonseed meal 25-35, maltodextrin 90-110, Trisodium Citrate 1-5, calcium chloride 2-5, K
2HPO
415-20, pH nature, solvent are water;
Fermentation condition is: temperature: 30-40 ℃; Rotating speed: 100～600r/min; Ventilation: 1: 0.5～1: 2.5; Dissolved oxygen maintains 20～30%, and the hydrochloric acid of auto-feeding ammoniacal liquor and 20% (v/v) in the fermenting process makes fermented liquid pH value maintain 7.0.
A kind of Sumizyme MP is as the application of detergent additives.
A kind of Sumizyme MP is in the application of leather industry.
A kind of Sumizyme MP is in the application of food service industry.
Advantage of the present invention and positively effect are:
1, mutagenic obtained product Sumizyme MP bacterial strain is through low energy N by Alkaliphilic bacillus among the present invention
+Ion implantation mutagenesis and the Sumizyme MP superior strain; Improved the enzymatic productivity of bacterial strain effectively, bottle vigor peak of shaking of its Sumizyme MP arrives 30700U/mL, has improved 37.7% than starting strain; Reduce production cost, and obtained good economic benefit.
What 2, the Sumizyme MP that obtains was fermented in the present invention shakes bottle vigor peak to 30700U/mL, has improved 37.7% than starting strain; 7L scale fermentor tank enzyme activity reaches 36000U/mL; 30L scale fermentor tank enzyme activity reaches 38600U/mL; 200L scale fermentor tank enzyme activity reaches 42000U/mL.
Description of drawings
Fig. 1 is N of the present invention
+Ion implantation influence curve to the bacterial strain survival rate;
Fig. 2 is N of the present invention
+Ion implantation influence to bacterial strain sudden change and positive mutation rate;
Fig. 3 is that the present invention is through low energy N
+The shake flask fermentation of the Sumizyme MP superior strain Y-22 that ion implantation technique obtains produces the enzyme curve;
Fig. 4 produces the enzyme curve for the 7L ferment tank of Sumizyme MP superior strain Y-22 of the present invention;
Fig. 5 produces the enzyme curve for the 30L ferment tank of Sumizyme MP superior strain Y-22 of the present invention;
Fig. 6 produces the enzyme curve for the 200L ferment tank of Sumizyme MP superior strain Y-22 of the present invention;
Fig. 7 is a Sumizyme MP electrophorogram of the present invention; (A.Marker, basic protein zymoprotein behind the B. purifying, C. fermented liquid);
Fig. 8 lives for the relative enzyme of Sumizyme MP of the present invention under pH5,6,7,8,9,10,11,12,13;
Fig. 9 lives for the relative enzyme of Sumizyme MP of the present invention behind 40 ℃ of insulations of pH5,6,7,8,9,10,11,12,13 times 1h;
Figure 10 lives for the relative enzyme of Sumizyme MP of the present invention under 30,40,50,60,70,80,90 ℃;
Figure 11 for Sumizyme MP of the present invention under pH10.5 45,50,55,60,65 ℃ be incubated 20,40,60,80,100 respectively, relative enzyme behind the 120min lives.
Embodiment
Through specific embodiment the present invention is made further detailed description below, following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.
1, the screening of starting strain
The new Alkaliphilic bacillus of cultivating of picking one ring inserts the seed culture fluid from the inclined-plane; Cultivate 12h in 34 ℃, 200r/min, be applied to then in the solid medium, in 34 ℃ incubator, continue to cultivate 48h; Therefrom 20 single bacterium colonies of picking are transferred into the inclined-plane; Again these 20 inclined-plane bacterial strains are received in the seed culture medium single bacterium colony of every bottle (50mL seed culture medium/250mL triangular flask) inoculation, 200r/min; Cultivate 12h for 34 ℃, observe being forwarded to liquid fermentation medium by 2% (v/v) inoculum size after bacterium liquid becomes muddiness, free from extraneous odour.34 ℃, 200r/min, shaking culture 54h in the baffle flask (50mL fermention medium/250mL baffle flask), with fermented liquid centrifugal after, measure the crude enzyme liquid vigor with forint phenol method, the result is as shown in table 1.
After measuring the basic protein enzyme activity, select enzyme to live higher and more stable bacterial strain as starting strain.Original starting strain is through after the rejuvenation, and the enzyme of strain fermentating liquid MV alive is 19000U/mL～22000U/mL.YXB-13 is the highest, is 22300U/mL.
With the higher bacterial strain YXB-13 test of going down to posterity of living of enzyme in the above-mentioned test, the stability of 20 investigations of continuous passage bacterial strain inherited character.Its result is (seeing table 2): this bacterial strain high yield and good stability are suitable as the starting strain of ion implantation mutagenesis.
2, N
+Ion implantation mutagenesis
(1) pre-treatment of sample: the Alkaliphilic bacillus starting strain is cultured to the product gemma phase in producing the gemma substratum, gets 0.1mL gemma suspension and coat equably on the aseptic flat board, carry out N immediately after doing with aseptic wind
+Ion implantation;
(2) ion implanting conditions: the injection energy is 30keV, and target chamber vacuum tightness is 5-6 * 10
-3Kpa injects with the 6s pulsed, is spaced apart 60s, and the single implantation dosage is 1 * 10
14N
+Ions/cm
2Implantation dosage is respectively 3,8,10,30,50,80,100,200.Each implantation dosage is done a vacuum contrast.
Each processing and corresponding vacuum thereof are to impinging upon ion implantation preceding forvacuum 5min, to eliminate the influence that possibly bring because of vacuum-drying degree difference;
(3) aftertreatment of sample: will pass through N
+The petridish of ion implantation petridish and vacuum contrast is used the sterilized water wash-out respectively, suitably coats on the activation flat board after the dilution, puts 34 ℃ and counts behind the cultivation 48h down, to calculate survival rate.
Survival rate is through N
+The survival colony count of ion implantation processing and ratio through the survival colony count of vacuum control treatment.
(4) drafting of survival rate curve: draw the spore suspension 1mL after ion implantation, inject the test tube that the 9mL sterilized water is housed, process 10
-1Diluent; Pressure-vaccum 10 repeatedly
-1Diluent, make it mix the back and draw 1mL, inject the test tube that the 9mL sterilized water is housed, process 10
-2Diluent; Press the said procedure operation, preparation 10
-3Diluent; Select the diluent of suitable concentration, get 0.1mL respectively and coat in the plate, each extent of dilution do 3 parallel; After in 34 ℃ of constant incubators, cultivating 48h, take out counting.
With the implantation dosage is X-coordinate, and survival rate is an ordinate zou, draws the relation curve of survival rate and implantation dosage.
Survival rate (%)=N
+Inject and handle colony count/vacuum contrast colony count * 100%.Its result is a (see figure 1): at N
+Ion implantation dosage reaches 30 * 10
14N
+Ions/cm
2Before, the bacterial strain survival rate is with N
+The increase of ion implantation dosage and reducing rapidly; Implantation dosage surpasses 200 * 10
14N
+Ions/cm
2After, survival rate is zero basically.Work as N
+Ion implantation dosage is 30 * 10
14～50 * 10
14N
+Ions/cm
2In the time of in the scope, the survival rate of bacterial classification presents " saddle-shape " variation tendency of height-low-Gao.
(5) N
+Ion implantation influence to the bacterial strain mutation rate
Adopt just that sieve method detects each implantation dosage bacterium colony transparent circle diameter down and the ratio changing conditions of colony diameter, regulation transparent circle diameter is higher than the mutant strain that contrast strain 5% and is called gain mutant, less than 5% suddenly change for negative, is to suddenly change between ± 5%.Test-results is seen Fig. 2.
Can know that by Fig. 2 along with the increase of ion implantation dosage, the bacterial strain mutation rate increases thereupon; Implantation dosage is 30 * 10
14～50 * 10
14N
+Ions/cm
2The positive mutation rate higher (22%～26%) of bacterial strain in the scope, implantation dosage surpasses 50 * 10
14N
+Ions/cm
2After, the mutation rate of bacterial strain continues to increase, but its positive mutation rate is the trend of reduction.Analyzing reason, possibly be because reverse mutation takes place the bacterial strain after the low dosage injection easily, and high dosage makes the damage of biomacromolecules such as DNA and microbial film seriously cause higher mutation rate and low positive variation rate.In " saddle " zone, ID is suitable, and mutation rate is higher relatively, and positive mutation rate is also higher.
3, the first screening of superior strain
Bacterium liquid is carried out N under best implantation dosage
+Ion implantation mutagenesis is handled, and suitably coats on the primary dcreening operation flat board after the dilution.
Primary dcreening operation is selected double-layer plate for use, and lower floor is the fermentation solid substratum, and the upper strata is a milk medium, and with 100 strain bacterium number consecutively Y-1-Y-100 of random choose, one-to-one point is connected to dull and stereotyped lower floor substratum; Every plate point connects 5 strain mutagenesis bacterium, every strain bacterium do one parallel, and point connects original bacterium and does contrast; Behind 34 ℃ of cultivation 54h, quantitative milk medium evenly is poured on the flat board, places 12h for 10 ℃; Calculate the ratio of transparent circle diameter and colony diameter, every plate is selected the mutant strain of 2 ratios greater than original bacterium.According to said method screen 3 and take turns, multiple sieve is carried out in tentatively selected Y-13, Y-22, Y-44, Y-66, Y-77, Y-95 six plant mutant strains.
4, the screening once more of superior strain
With primary dcreening operation sieve 6 strain bacterium carry out shake flask fermentation respectively and cultivate, with the 54h enzyme activity as sieving standard, triplicate again.Each mutant strain enzyme is lived as shown in table 3, and wherein mutant strain Y-22 product enzyme activity is the highest, reaches 30700U/mL, has improved 37.7% than initial bacterial strain.
5, the Sumizyme MP superior strain confirms
The high productive mutant that obtains of screening in the above mutagenesis testing is carried out the continuous passage test, survey its enzyme activity, investigate the stability of its inherited character, screen and finally to be applied to the bacterial strain produced.
To mutant strain Y-22 continuous passage 5 times; With 54h fermentation crude enzyme liquid vigor is standard, and the result is as shown in table 4, goes down to posterity after five times; It is stable that enzymatic productivity keeps; This with the report ion implantation mutagenesis after, but the good character genetic stability of bacterial strain is consistent, mutant strain Y-22 is the high yield alkali protein bacterial strain.
6, the Sumizyme MP superior strain shakes a bottle level fermentation and produces proteolytic enzyme
The new Alkaliphilic bacillus Y-22 that cultivates of picking one ring inserts the seed culture fluid (50mL seed culture medium/250mL triangular flask) from the inclined-plane, and 200r/min cultivates 12h for 34 ℃, and 2% (v/v) inoculum size is forwarded to liquid fermentation medium.34 ℃, shaking culture among the 200r/min, baffle flask (50mL fermention medium/250mL baffle flask), behind the certain hour of every interval that fermented liquid is centrifugal, measure the crude enzyme liquid vigor with forint phenol method, as a result shown in the accompanying drawing 3.
7, Sumizyme MP superior strain 7L ferment tank technology
The new Alkaliphilic bacillus Y-22 that cultivates of picking one ring inserts the seed culture fluid (50mL seed culture medium/250mL baffle flask) from the inclined-plane; 200r/min; Cultivate 12h, transfer into the 7L fermentor tank fermentor tank liquid amount 0.65 according to the inoculum size of 5% (v/v) for 34 ℃.34 ℃ of temperature, rotating speed: 200～500r/min, ventilation: 1: 0.5～1: 2.5, dissolved oxygen maintained 20～30%.Stream adds the hydrochloric acid of ammoniacal liquor and 20% (v/v) in the fermenting process, makes fermented liquid pH value maintain 7.0.Adopt this technology to continuously ferment three batches, shown in accompanying drawing 4, find under this stream process CONTROL PROCESS; Bacterial strain begins to produce enzyme from 20h, and 25h gets into and produces the enzyme peak period, and 55h reaches peak; Enzyme activity is on average stable at 36000U/mL, and after this, enzyme activity begins rapid decline.
8, Sumizyme MP superior strain 30L ferment tank technology
The new Alkaliphilic bacillus Y-22 that cultivates of picking one ring inserts the seed culture fluid (100mL seed culture medium/500mL baffle flask) from the inclined-plane; 200r/min; Cultivate 12h, transfer into the 30L fermentor tank fermentor tank liquid amount 0.7 according to the inoculum size of 5% (v/v) for 34 ℃.Temperature: 34 ℃; Rotating speed: 100～600r/min; Ventilation: 1: 0.5～1: 2.5; Dissolved oxygen maintains 20～30%.The hydrochloric acid of auto-feeding ammoniacal liquor and 20% (v/v) in the fermenting process makes fermented liquid pH value maintain 7.0.Adopt this technology to continuously ferment three batches, shown in accompanying drawing 5, find that after the 30h, Sumizyme MP is synthetic in a large number under this stream process CONTROL PROCESS, when arriving 50h, the highest 38600U/mL of enzyme activity.After the 54h, product enzyme speed is slack-off, and vigor descends, and very fast thalline gets into the extinction phase, and the pH value is rapid simultaneously rises.
9, Sumizyme MP superior strain 200L ferment tank technology
The new Alkaliphilic bacillus Y-22 that cultivates of picking one ring inserts the seed culture fluid (100mL seed culture medium/500mL baffle flask) from the inclined-plane; 34 ℃ of shaking culture 12h, by the inoculum size of 2% (v/v) in the 30L seed culture medium, 34 ℃; Dissolved oxygen maintains 20～30%, 10h.Transfer into the 200L fermentor tank fermentor tank liquid amount 0.7 according to the inoculum size of 5% (v/v).Temperature: 34 ℃; Rotating speed: 250～450r/min; Ventilation: 1: 0.5～1: 1.5; Dissolved oxygen: 20～30%.The hydrochloric acid of auto-feeding ammoniacal liquor and 20% (v/v) in the fermenting process makes fermented liquid pH value maintain 7.0.Shown in accompanying drawing 6, find that enzyme activity reaches peak 4.2 * 10 when this stream process CONTROL PROCESS bottom fermentation 36h
4U/mL.Cell growth lag phase shortens in the 200L fermentor tank, and growth is rapid, and the product enzyme phase is also corresponding in advance, and the whole product enzyme phase is all produced enzyme with higher speed.
Substratum
(1) preservation/activation medium (g/L): Carnis Bovis seu Bubali cream 8, yeast soak powder 2, polyprotein peptone 5, and NaCl 2, agar 17, casein 4, K
2HPO
418, the pH nature.
(2) seed culture medium (g/L): yeast soaks powder 5, Tryptones 5, glucose 10, K
2HPO
418, the pH nature.
(3) fermention medium (g/L): yeast soaks powder 17, cottonseed meal 30, maltodextrin 100, Trisodium Citrate 3, calcium chloride 2.6, K
2HPO
418, the pH nature.
(4) milk medium (g/L): skim-milk 100, agar 20, pH nature.
(5) produce gemma substratum (g/L): yeast extract paste 0.7, peptone 1, glucose 1, (NH
4)
2SO
40.2, MgSO
4.7H
2O0.2, K
2HPO
41, pH7.2, solid medium add the agar of 1.8% (w/v).
10, the mensuration of basic protein enzyme activity
Adopt " the People's Republic of China's light industry standard " general experimental technique of QB/T1805.3-93 industrial enzyme preparation, i.e. forint (Folin) method.
11, the purifying process of enzyme: get fermented liquid 100ml, the centrifugal 10min of 8000r/min.Get supernatant, with the solid sulphuric acid of saturation ratio 70% by saltouing the centrifugal 10min collecting precipitation of 8000r/mln.Deposition is dissolved in Tris-HCI (pH8.8) damping fluid desalination of in identical damping fluid, dialysing.Centrifugally remove a small amount of insolubles, get supernatant and cross the DEAE-SepharoseCL-6B ion exchange column and carry out purifying, carry out wash-out with 20mMTris-Hcl (pH8.8 contains 10mMNacl) damping fluid.Sumizyme MP is not adsorbed, and is eluted earlier, obtains water white liquid.Collect enzyme liquid and carry out vacuum lyophilization, obtain pure enzyme powder and further analyze.
As shown in Figure 4, it is pure that the SDS-PAGE protein electrophoresis shows that the sample behind the purifying has reached electrophoresis, and molecular weight is 28kDa.The recovery is 27.54%, and the purifying multiple is 5.23 times, can reach 18620.20U/mg than vigor.
12, the character of enzyme is studied.
When being substrate, measure the relative enzyme alive (accompanying drawing 8) of Sumizyme MP under pH5,6,7,8,9,10,11,12,13 respectively with the casein; Relative enzyme behind 40 ℃ of insulations of pH5,6,7,8,9,10,11,12,13 times 1h live (accompanying drawing 9); Relative enzyme (accompanying drawing 10) alive under 30,40,50,60,70,80,90 ℃; Under pH10.5 45,50,55,60,65 ℃ be incubated 20,40,60,80,100 respectively, the relative enzyme behind the 120min live (accompanying drawing 11).
Can obtain its righttest action pH is 10.5, and optimum temperature is 40 ℃.This enzyme is stable in the scope of pH7～12, and thermostability is preferably arranged about 45～50 ℃.
The enzyme of table 1 original strain is lived
The table 2 starting strain test-results that goes down to posterity
The multiple sieve result of table 3 Sumizyme MP superior strain
Table 4Y-22 genetic stability
| Enzyme activity (U/mL) | Relative enzyme (%) alive | |
| F1 | 30700 | 100 |
| F2 | 29588±539 | 96.4±1.8 |
| F3 | 32014±977 | 104.3±3.2 |
| F4 | 32418±910 | 105.6±3.0 |
| F5 | 31576±809 | 102.9±2.6 |
Claims (8)
1. Sumizyme MP superior strain; It is characterized in that: name is called Y-22; Classification called after Alkaliphilic bacillus Bacillus alcalophilus, deposit number is: CGMCC No.5313, preservation date: on September 30th, 2011; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
2. a Sumizyme MP is characterized in that: obtained by the described Sumizyme MP superior strain fermentation of claim 1.
3. Sumizyme MP according to claim 2 is characterized in that: said basic protein enzyme molecular weight is 28kDa.
4. Sumizyme MP according to claim 2; It is characterized in that: said Sumizyme MP is when being substrate with the casein, and its righttest action pH is 10.5, and optimum temperature is 40 ℃; Stable in the scope of pH7～12, at 40～50 ℃ thermostability is preferably arranged.
5. the fermentation process of a Sumizyme MP as claimed in claim 2, it is characterized in that: the used substratum that ferments is: fermention medium g/L: yeast soaks powder 15-20, cottonseed meal 25-35, maltodextrin 90-110, Trisodium Citrate 1-5, calcium chloride 2-5, K
2HPO
415-20, pH nature, solvent are water;
Fermentation condition is: temperature: 30-40 ℃; Rotating speed: 100～600r/min; Ventilation: 1: 0.5～1: 2.5; Dissolved oxygen maintains 20～30%, and the hydrochloric acid of auto-feeding ammoniacal liquor and 20% (v/v) in the fermenting process makes fermented liquid pH value maintain 7.0.
6. one kind like the application of the arbitrary described Sumizyme MP of claim 2-5 as detergent additives.
7. one kind like the application of the arbitrary described Sumizyme MP of claim 2-5 at leather industry.
8. one kind like the application of the arbitrary described Sumizyme MP of claim 2-5 in food service industry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103336788A CN102382786A (en) | 2011-10-28 | 2011-10-28 | Alkali protease high-yield strain and fermentation method of alkali protease and protease produced by alkali protease high-yield strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103336788A CN102382786A (en) | 2011-10-28 | 2011-10-28 | Alkali protease high-yield strain and fermentation method of alkali protease and protease produced by alkali protease high-yield strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102382786A true CN102382786A (en) | 2012-03-21 |
Family
ID=45822620
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103336788A Pending CN102382786A (en) | 2011-10-28 | 2011-10-28 | Alkali protease high-yield strain and fermentation method of alkali protease and protease produced by alkali protease high-yield strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102382786A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925388A (en) * | 2012-10-26 | 2013-02-13 | 天津科技大学 | Alkaline proteinase high-producing strain and alkaline proteinase being produced from same |
| CN103333873A (en) * | 2013-04-24 | 2013-10-02 | 北京仁峰科技有限公司 | Method for preparing alkaline protease through fermentation method |
| CN103409347A (en) * | 2013-07-25 | 2013-11-27 | 山东隆科特酶制剂有限公司 | Bacterial strain capable of producing alkali protease and industrialized liquid fermentation method of bacterial strain |
| CN106082767A (en) * | 2016-06-06 | 2016-11-09 | 郭远臣 | A kind of string loads the self-repairing cement-base material of microorganism |
| CN106676024A (en) * | 2015-11-05 | 2017-05-17 | 中国科学院天津工业生物技术研究所 | Bacillus amyloliquefaciens for high yield of alkali protease and application of bacillus amyloliquefaciens |
| CN109182154A (en) * | 2018-09-27 | 2019-01-11 | 中国药科大学 | The Ba Buwei rhodotorula of one plant of high proteinase yield |
| CN112143676A (en) * | 2020-09-21 | 2020-12-29 | 山西大学 | Bacillus halodurans for producing alkaline protease and method and application of bacillus halodurans for producing alkaline protease |
| CN113817629A (en) * | 2021-07-30 | 2021-12-21 | 中国科学院合肥物质科学研究院 | Bacterial strain for producing alkaline protease, produced alkaline protease and method |
| CN117070394A (en) * | 2023-04-20 | 2023-11-17 | 广东药科大学 | Alkalophilic strain for producing alkaline protease, alkaline protease and application thereof |
-
2011
- 2011-10-28 CN CN2011103336788A patent/CN102382786A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 孙同毅等: "高活力碱性蛋白酶产生菌的选育与发酵放大", 《辽宁工程技术大学学报(自然科学版)》 * |
| 成堃等: "产碱性蛋白酶菌株的筛选、分子鉴定及其酶学性质的初步研究", 《中国酿造》 * |
| 殷向斌等: "N+离子注入诱变选育高产碱性蛋白酶菌株的研究", 《2007年全国发酵行业高峰论坛暨中国发酵工业协会2007年行业大会论文汇编》 * |
| 苑琳等: "嗜碱性芽孢杆菌产高碱碱性蛋白酶发酵培养基及发酵条件的研究", 《食品与发酵工业》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925388A (en) * | 2012-10-26 | 2013-02-13 | 天津科技大学 | Alkaline proteinase high-producing strain and alkaline proteinase being produced from same |
| CN102925388B (en) * | 2012-10-26 | 2015-05-20 | 天津科技大学 | Alkaline proteinase high-producing strain and alkaline proteinase being produced from same |
| CN103333873A (en) * | 2013-04-24 | 2013-10-02 | 北京仁峰科技有限公司 | Method for preparing alkaline protease through fermentation method |
| CN103409347A (en) * | 2013-07-25 | 2013-11-27 | 山东隆科特酶制剂有限公司 | Bacterial strain capable of producing alkali protease and industrialized liquid fermentation method of bacterial strain |
| CN103409347B (en) * | 2013-07-25 | 2015-10-28 | 山东隆科特酶制剂有限公司 | A kind of bacterial strain and industrialization liquid fermentation process thereof producing Sumizyme MP |
| CN106676024A (en) * | 2015-11-05 | 2017-05-17 | 中国科学院天津工业生物技术研究所 | Bacillus amyloliquefaciens for high yield of alkali protease and application of bacillus amyloliquefaciens |
| CN106082767A (en) * | 2016-06-06 | 2016-11-09 | 郭远臣 | A kind of string loads the self-repairing cement-base material of microorganism |
| CN106082767B (en) * | 2016-06-06 | 2018-01-09 | 重庆三峡学院 | A kind of string loads the self-repairing cement-base material of microorganism |
| CN109182154A (en) * | 2018-09-27 | 2019-01-11 | 中国药科大学 | The Ba Buwei rhodotorula of one plant of high proteinase yield |
| CN109182154B (en) * | 2018-09-27 | 2021-04-30 | 中国药科大学 | Pabuvina rhodotorula strain capable of producing protease at high yield |
| CN112143676A (en) * | 2020-09-21 | 2020-12-29 | 山西大学 | Bacillus halodurans for producing alkaline protease and method and application of bacillus halodurans for producing alkaline protease |
| CN113817629A (en) * | 2021-07-30 | 2021-12-21 | 中国科学院合肥物质科学研究院 | Bacterial strain for producing alkaline protease, produced alkaline protease and method |
| CN113817629B (en) * | 2021-07-30 | 2023-05-02 | 中国科学院合肥物质科学研究院 | Alkaline protease producing strain, alkaline protease produced by strain and method |
| CN117070394A (en) * | 2023-04-20 | 2023-11-17 | 广东药科大学 | Alkalophilic strain for producing alkaline protease, alkaline protease and application thereof |
| CN117070394B (en) * | 2023-04-20 | 2024-03-29 | 广东药科大学 | Alkalophilic strain for producing alkaline protease, alkaline protease and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102382786A (en) | Alkali protease high-yield strain and fermentation method of alkali protease and protease produced by alkali protease high-yield strain | |
| CN102925388B (en) | Alkaline proteinase high-producing strain and alkaline proteinase being produced from same | |
| CN103451121B (en) | Bacillus licheniformis and application thereof | |
| CN101555461A (en) | Bacterial strain LT3 producing alkalescence cellulase and breeding method and initial optimization of cellulase production conditions thereof | |
| CN106434457B (en) | One plant of alkali protease superior strain and its produced alkali protease | |
| CN103409347A (en) | Bacterial strain capable of producing alkali protease and industrialized liquid fermentation method of bacterial strain | |
| CN110373359A (en) | A kind of streptomyces albus X-18 and the method using bacterium production epsilon-polylysine | |
| CN109439601A (en) | One plant of method for producing the bacterial strain of protease and its preparing alkali protease | |
| CN101864366B (en) | Penicillium citrinum bacterial strain and application thereof | |
| CN110042072A (en) | A kind of aflatoxin degradation B1Bacterial strain and its application | |
| CN105087399B (en) | A kind of straw-returning fiber hydrolization biocontrol fungi and microbial inoculum and application | |
| CN104328078A (en) | Brevibacillus borstelensis for producing neutral protease | |
| CN100497593C (en) | Low-temperature amylase strain, low-temperature amylase, and production therefor | |
| CN105255771A (en) | Staphylococcus succinus for generating collagenase and application thereof | |
| CN104860401B (en) | Applications of the one Rhizopus oryzae bacterial strain JHSW01 in for ferment organic liquid waste and agriculture and forestry organic waste material | |
| CN103361281A (en) | High-temperature degrading bacteria and application thereof | |
| CN103849581A (en) | Endophytic curtobacterium of zanthoxylum as well as screening and purifying method and application of endophytic curtobacterium of zanthoxylum | |
| CN103131639B (en) | Trichoderma longibrachiatum strain and application thereof | |
| CN104031860A (en) | Bacillus sphaericus for high-yield high-temperature-resistant xylanase and application thereof | |
| CN104450571A (en) | Bacillus thuringiensis strain for efficient degradation of fly larvae protein | |
| CN107557311B (en) | Lactobacillus acidophilus and application thereof in fermentation production of antibacterial polypeptide | |
| CN101173232A (en) | Microorganism preparation for degradation of hogwash garbage and technique of preparing the same | |
| CN101182513A (en) | Method for mutagenesis breeding neuter protease high yield bacterial strain by hypophrenia N+ion injection technology | |
| Turaeva et al. | Isolation and identification of bacteria with cellulolytic activity from the microflora of animal juice | |
| CN110903994B (en) | Bacillus licheniformis for producing high-temperature protease and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120321 |