CN101338280A - Aspergillus usamii for producing keratinase complex enzyme and enzyme-producing method thereof by fermentation - Google Patents
Aspergillus usamii for producing keratinase complex enzyme and enzyme-producing method thereof by fermentation Download PDFInfo
- Publication number
- CN101338280A CN101338280A CNA2008100627385A CN200810062738A CN101338280A CN 101338280 A CN101338280 A CN 101338280A CN A2008100627385 A CNA2008100627385 A CN A2008100627385A CN 200810062738 A CN200810062738 A CN 200810062738A CN 101338280 A CN101338280 A CN 101338280A
- Authority
- CN
- China
- Prior art keywords
- grams
- milliliters
- aspergillus usamii
- keratinase
- 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
Abstract
The invention discloses an aspergillus usami L88 which can produce keratinase compound enzyme and a method of producing enzyme with fermentation. The Aspergillus usami L88 is separated from the laboratory and preserved in general microscobial center of China microscobial preservation and management committee with the preservation number of CGMCC No. 2296. The bacterium is a starting strain. The fermentation production of the keratinase compound enzyme is realized by the strain processing slant culture, shake flask seed culture and fermentation tank culture. The Aspergillus usami L88 discloses the morphological characteristics of the strain and the DNA total order of the 5.8S ribosomal. The disclosed fermentation culture medium comprises corn flour, soybean flour, fish flour, ammonium salts, silkworm hydrolysate, keratin, calcium salts and phosphates. The produced keratinase compound enzyme has the reasonable proportion of acid protease and keratinase, high enzyme survival rate and low cost. The keratinase compound enzyme has significant environmental protection and high economical value in industries of keratin waste recycle, tanning, woven fabric post treatment and so on.
Description
Technical field
The present invention relates to the microbial engineering field, particularly a kind of Aspergillus usamii and enzymatic production method thereof of producing the M-Zyme prozyme.
Background technology
The major ingredient of M-Zyme prozyme is M-Zyme and common proteolytic ferment, can be than M-Zyme hydrolysis of keratin more quickly and efficiently, and have widely in industry such as fodder production, tanning, wool processing and to use.The feed saving energy, the cost that this method is produced is low, quality good, can be utilized by animal preferably.In containing keratic feed, interpolation M-Zyme prozyme also can improve animal and absorb keratic, increases economic efficiency.The M-Zyme prozyme can the selective hydrolysis wool, the Keratin sulfate of leather surface, is expected to replace traditional wool stripping squama and tanning process, realizes the environmental protection transformation of conventional industries.The M-Zyme prozyme can improve makeup, the externally applied medicine permeability in skin, also all has a wide range of applications in industry such as medicine, makeup.Traditional method is produced M-Zyme and proteolytic ferment separately, then they is combined with each other, and production process complexity, cost are higher relatively.
Aspergillus usamii Aspergillus usami L88 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.2296.
Summary of the invention
The purpose of this invention is to provide a kind of Aspergillus usamii and enzymatic production method thereof of producing the M-Zyme prozyme.
Produce the Aspergillus usamii Aspergillus usami L88 of M-Zyme prozyme, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.2296.
Described Aspergillus usamii morphological feature is: mycelia brown or brown-black, 2 layers of stigmas; Conidium brown or brown-black, coarse or level and smooth, general big and spherical in shape, undeveloped sometimes.
The 5.8S rrna of described Aspergillus usamii has the dna sequence dna of SEQ ID NO.1,
The enzymatic production method of producing the Aspergillus usamii of M-Zyme prozyme comprises the steps:
1) Aspergillus usamii L88 is inoculated on the slant medium,, obtains the inclined-plane of a large amount of spores of surface coverage in 20～37 ℃ of cultivations 72～360 hours; The slant medium composition is: contain 100～900 milliliters in 100～900 milliliters of worts, agar powder 10～20 grams and water in per 1000 milliliters;
2) with Aspergillus usamii L88 spore inoculating in liquid seed culture medium, in 20～37 ℃, cultivated under 80～220rpm condition 20～72 hours, obtain ferment-seeded; The seed culture medium composition is: contain Semen Maydis powder 10～30 grams, soyflour 20～60 grams, ammonium chloride 5～30 grams, Sodium phosphate dibasic 3～15 grams, calcium chloride 3～15 grams, fish meal 5～40 grams in per 1000 milliliters, 10～100 milliliters of silkworm chrysalis hydrolyzed solutions, Keratin sulfate 0.1～20 gram;
3) ferment-seeded is pressed 3～15% of fermention medium volume and inserted in the fermentor tank, jar temperature 20～37 ℃, tank pressure 0.2～1.0kg/cm
2Stir speed (S.S.) 80～220rpm, air flow is 1: 0.5～1: 2, fermented incubation time 72～360 hours, the fermention medium composition is: contain Semen Maydis powder 10～30 grams, soyflour 20～60 grams, ammonium chloride 5～30 grams, Sodium phosphate dibasic 3～15 grams, calcium chloride 3～15 grams, fish meal 5～40 grams in per 1000 milliliters, 10～100 milliliters of silkworm chrysalis hydrolyzed solutions, Keratin sulfate 0.1～20 gram;
4) the fermentation beginning is after 40～150 hours, the supplemented medium of the initial volume 5～20% of fermented liquid will be accounted for, continued to add in the fermention medium in 0.1～8 hour, the supplemented medium composition is to contain 10～990 milliliters of silkworm chrysalis hydrolyzed solutions, 10～990 milliliters in water in per 1000 milliliters.
The beneficial effect that the present invention compared with prior art has:
1) the present invention can disposable production M-Zyme and the prozyme of proteolytic ferment, has that production process is simple, cost is low; The enzyme work good, that can avoid causing owing to reaction between the prozyme of the consistency of gained prozyme reduces even inactivation.
2) fermention medium among the present invention, the nutritive ingredient reasonable ratio can guarantee growth and the high yield aspartic protease needs of Aspergillus usamii L88 can induce a large amount of M-Zymes again, thereby it is reasonable to produce ratio, the M-Zyme prozyme that catalytic capability is strong.
3) utilized in the fermenting process of the present invention the disadvantageous Keratin sulfate of environmental protection is substrate, cost is low, can solve environmental problem again when producing M-Zyme, and the Keratin sulfate after the degraded is used for industry such as feed can produce higher economic value.
4) the M-Zyme prozyme of the present invention's production when being used for wool rear finishing duplex industry, can effectively be avoided environmental pollution, and anti-felting effect is good, and product color is bright-coloured, the share of market height.
5) the M-Zyme prozyme of the present invention's production, when being used for the Keratin sulfate offal treatment, keratin degrading speed is fast, degradation rate is high, the amino acid destructiveness is low, the nutrition homogeneous, is easy to be absorbed by animal and utilize.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further detail.
Embodiment 1
Described Aspergillus usamii morphological feature is: mycelia brown or brown-black, 2 layers of stigmas; Conidium brown brown-black, coarse or level and smooth, general big and spherical in shape, undeveloped sometimes.
The 5.8S rrna of described Aspergillus usamii has the dna sequence dna of SEQ ID NO.1.
Comprehensive sequence and morphological feature, the microorganism of gained product M-Zyme prozyme is an Aspergillus usamii as can be known, called after Aspergillus usami L88, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation day is on December 18th, 2007, preserving number is CGMCC No.2296.
Embodiment 2
1) Aspergillus usamii L88 is inoculated on the slant medium,, obtains the inclined-plane of a large amount of spores of surface coverage in 20 ℃ of cultivations 360 hours; The slant medium composition is: contain 100 milliliters in 900 milliliters of worts, agar powder 10 grams and water in per 1000 milliliters;
2) with Aspergillus usamii L88 spore inoculating in liquid seed culture medium, in 20 ℃, cultivated under the 220rpm condition 72 hours, obtain ferment-seeded; The seed culture medium composition is: contain Semen Maydis powder 10 grams, soyflour 20 grams, ammonium chloride 5 grams, Sodium phosphate dibasic 3 grams, calcium chloride 3 grams, fish meal 5 grams in per 1000 milliliters, 10 milliliters of silkworm chrysalis hydrolyzed solutions, feather keratin 0.1 gram;
3) ferment-seeded is pressed 3% of fermention medium volume and inserted in the fermentor tank, jar temperature 20 ℃, tank pressure 0.2kg/cm
2Stir speed (S.S.) 220rpm, air flow is 1: 0.5, fermented incubation time 360 hours, the fermention medium composition is: contain Semen Maydis powder 10 grams, soyflour 20 grams, ammonium chloride 5 grams, Sodium phosphate dibasic 3 grams, calcium chloride 3 grams, fish meal 5 grams in per 1000 milliliters, 10 milliliters of silkworm chrysalis hydrolyzed solutions, feather keratin 0.1 gram;
4) the fermentation beginning will account for the supplemented medium of the initial volume 20% of fermented liquid after 150 hours, continued to add in the fermention medium in 8 hours, and material substratum composition is to contain 990 milliliters of silkworm chrysalis hydrolyzed solutions, 10 milliliters in water in per 1000 milliliters.
Embodiment 3
1) Aspergillus usamii L88 is inoculated on the slant medium,, obtains the inclined-plane of a large amount of spores of surface coverage in 37 ℃ of cultivations 72 hours; The slant medium composition is: contain 900 milliliters in 100 milliliters of worts, agar powder 20 grams and water in per 1000 milliliters;
2) with Aspergillus usamii L88 spore inoculating in liquid seed culture medium, in 37 ℃, cultivated under the 80rpm condition 20 hours, obtain ferment-seeded; The seed culture medium composition is: contain Semen Maydis powder 30 grams, soyflour 60 grams, ammonium chloride 30 grams, Sodium phosphate dibasic 15 grams, calcium chloride 15 grams, fish meal 40 grams in per 1000 milliliters, 100 milliliters of silkworm chrysalis hydrolyzed solutions, feather keratin 20 grams;
3) ferment-seeded is pressed 15% of fermention medium volume and inserted in the fermentor tank, jar temperature 37 ℃, tank pressure 1.0kg/cm
2Stir speed (S.S.) 80rpm, air flow is 1: 2, fermented incubation time 72 hours, the fermention medium composition is: contain Semen Maydis powder 30 grams, soyflour 60 grams, ammonium chloride 30 grams, Sodium phosphate dibasic 15 grams, calcium chloride 15 grams, fish meal 40 grams in per 1000 milliliters, 100 milliliters of silkworm chrysalis hydrolyzed solutions, feather keratin 20 grams;
4) the fermentation beginning will account for the supplemented medium of the initial volume 5% of fermented liquid after 40 hours, continued to add in the fermention medium in 0.1 hour, and the supplemented medium composition is to contain 10 milliliters of silkworm chrysalis hydrolyzed solutions, 990 milliliters in water in per 1000 milliliters.
Embodiment 4
1) Aspergillus usamii L88 is inoculated on the slant medium,, obtains the inclined-plane of a large amount of spores of surface coverage in 25 ℃ of cultivations 168 hours; The slant medium composition is: contain 100 milliliters in 900 milliliters of worts, agar powder 20 grams and water in per 1000 milliliters;
2) with Aspergillus usamii L88 spore inoculating in liquid seed culture medium, in 25 ℃, cultivated under the 150rpm condition 42 hours, obtain ferment-seeded; The seed culture medium composition is: contain Semen Maydis powder 10 grams, soyflour 30 grams, ammonium chloride 10 grams, Sodium phosphate dibasic 5 grams, calcium chloride 5 grams, fish meal 20 grams in per 1000 milliliters, 50 milliliters of silkworm chrysalis hydrolyzed solutions, wool keratin 10 grams;
3) ferment-seeded is pressed 3～15% of fermention medium volume and inserted in the fermentor tank, jar temperature 25 ℃, tank pressure 0.3kg/cm
2Stir speed (S.S.) 150rpm, air flow is 1: 1.2, fermented incubation time 88 hours, the fermention medium composition is: contain Semen Maydis powder 10 grams, soyflour 30 grams, ammonium chloride 10 grams, Sodium phosphate dibasic 5 grams, calcium chloride 5 grams, fish meal 20 grams in per 1000 milliliters, 50 milliliters of silkworm chrysalis hydrolyzed solutions, wool keratin 10 grams;
4) the fermentation beginning will account for the supplemented medium of the initial volume 10% of fermented liquid after 40 hours, continued to add in the fermention medium in 4 hours, and material substratum composition is to contain 990 milliliters of silkworm chrysalis hydrolyzed solutions, 10 milliliters in water in per 1000 milliliters.
Embodiment 5
1) Aspergillus usamii L88 is inoculated on the slant medium,, obtains the inclined-plane of a large amount of spores of surface coverage in 30 ℃ of cultivations 144 hours; The slant medium composition is: contain 100 milliliters in 900 milliliters of worts, agar powder 20 grams and water in per 1000 milliliters;
2) with Aspergillus usamii L88 spore inoculating in liquid seed culture medium, in 30 ℃, cultivated under the 150rpm condition 30 hours, obtain ferment-seeded; The seed culture medium composition is: contain Semen Maydis powder 10 grams, soyflour 30 grams, ammonium chloride 10 grams, Sodium phosphate dibasic 5 grams, calcium chloride 4 grams, fish meal 20 grams in per 1000 milliliters, 80 milliliters of silkworm chrysalis hydrolyzed solutions, wool keratin 5 grams;
3) ferment-seeded is pressed 10% of fermention medium volume and inserted in the fermentor tank, jar temperature 30 ℃, tank pressure 0.40kg/cm
2Stir speed (S.S.) 150rpm, air flow is 1: 1.2, fermented incubation time 86 hours, the fermention medium composition is: contain Semen Maydis powder 10 grams, soyflour 30 grams, ammonium chloride 10 grams, Sodium phosphate dibasic 5 grams, calcium chloride 4 grams, fish meal 20 grams in per 1000 milliliters, 80 milliliters of silkworm chrysalis hydrolyzed solutions, wool keratin 5 grams;
4) the fermentation beginning will account for the supplemented medium of the initial volume 15% of fermented liquid after 38 hours, continued to add in the fermention medium in 6 hours, and material substratum composition is to contain 990 milliliters of silkworm chrysalis hydrolyzed solutions, 10 milliliters in water in per 1000 milliliters.
At last, it is also to be noted that what more than enumerate only is part specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.
The present invention can summarize with other the specific form without prejudice to spirit of the present invention and principal character.Therefore, no matter from which point, above-mentioned embodiment of the present invention all can only be thought can not limit the present invention to explanation of the present invention, claims have been pointed out scope of the present invention, and scope of the present invention is not pointed out in above-mentioned explanation, therefore, in implication suitable and any change in the scope, all should think to be included in the scope of claims with claims of the present invention.
Sequence table
＜110〉Zhejiang University
＜120〉a kind of Aspergillus usamii and enzymatic production method thereof of producing the M-Zyme prozyme
<160>1
<170>PatentIn?version 3.1
<210>1
<211>534
<212>DNA
<213>Aspergillus?usami?L88
<400>1
taccgagtgc?gggtcctttg?ggcccaacct?cccatccgtg?tctattgtac?cctgttgctt 60
cggcgggccc?gccgcttgtc?ggccgccggg?ggggcgcctc?tgccccccgg?gcccgtgccc 120
gccggagacc?ccaacacgaa?cactgtctga?aagcgtgcag?tctgagttga?ttgaatgcaa 180
tcagttaaaa?ctttcaacaa?tggatctctt?ggttccggca?tcgatgaaga?acgcagcgaa 240
atgcgataac?taatgtgaat?tgcagaattc?agtgaatcat?cgagtctttg?aacgcacatt 300
gcgccccctg?gtattccggg?gggcatgcct?gtccgagcgt?cattgctgcc?ctcaagcccg 360
gcttgtgtgt?tgggtcgccg?tccccctctc?cggggggacg?ggcccgaaag?gcagcggcgg 420
caccgcgtcc?gatcctcgag?cgtatggggc?tttgtcacat?gctctgtagg?attggccggc 480
gcctgccgac?gttttccaac?cattctttcc?aggttgacct?cggatcaggt?aggg 534
Claims (4)
1. an Aspergillus usamii that produces the M-Zyme prozyme is characterized in that Aspergillus usamii Aspergillususami L88, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.2296.
2. a kind of Aspergillus usamii that produces the M-Zyme prozyme according to claim 1 is characterized in that described Aspergillus usamii morphological feature is: mycelia brown or brown-black, 2 layers of stigmas; Conidium brown or brown-black, coarse or level and smooth, general big and spherical in shape, undeveloped sometimes.
3. a kind of Aspergillus usamii that produces the M-Zyme prozyme according to claim 1 is characterized in that the 5.8S rrna of described Aspergillus usamii has the dna sequence dna of SEQ ID NO.1:
4. the fermentation process of the Aspergillus usamii of a product M-Zyme prozyme as claimed in claim 1 is characterized in that comprising the steps:
1) Aspergillus usamii L88 is inoculated on the slant medium,, obtains the inclined-plane of a large amount of spores of surface coverage in 20～37 ℃ of cultivations 72～360 hours; The slant medium composition is: contain 100～900 milliliters in 100～900 milliliters of worts, agar powder 10～20 grams and water in per 1000 milliliters;
2) with Aspergillus usamii L88 spore inoculating in liquid seed culture medium, in 20～37 ℃, cultivated under 80～220rpm condition 20～72 hours, obtain ferment-seeded; The seed culture medium composition is: contain Semen Maydis powder 10～30 grams, soyflour 20～60 grams, ammonium chloride 5～30 grams, Sodium phosphate dibasic 3～15 grams, calcium chloride 3～15 grams, fish meal 5～40 grams in per 1000 milliliters, 10～100 milliliters of silkworm chrysalis hydrolyzed solutions, Keratin sulfate 0.1～20 gram;
3) ferment-seeded is pressed 3～15% of fermention medium volume and inserted in the fermentor tank, jar temperature 20～37 ℃, tank pressure 0.2～1.0kg/cm
2Stir speed (S.S.) 80～220rpm, air flow is 1: 0.5～1: 2, fermented incubation time 72～360 hours, the fermention medium composition is: contain Semen Maydis powder 10～30 grams, soyflour 20～60 grams, ammonium chloride 5～30 grams, Sodium phosphate dibasic 3～15 grams, calcium chloride 3～15 grams, fish meal 5～40 grams in per 1000 milliliters, 10～100 milliliters of silkworm chrysalis hydrolyzed solutions, Keratin sulfate 0.1～20 gram;
4) the fermentation beginning is after 40～150 hours, the supplemented medium of the initial volume 5～20% of fermented liquid will be accounted for, continued to add in the fermention medium in 0.1～8 hour, the supplemented medium composition is to contain 10～990 milliliters of silkworm chrysalis hydrolyzed solutions, 10～990 milliliters in water in per 1000 milliliters.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100627385A CN101338280A (en) | 2008-07-01 | 2008-07-01 | Aspergillus usamii for producing keratinase complex enzyme and enzyme-producing method thereof by fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100627385A CN101338280A (en) | 2008-07-01 | 2008-07-01 | Aspergillus usamii for producing keratinase complex enzyme and enzyme-producing method thereof by fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101338280A true CN101338280A (en) | 2009-01-07 |
Family
ID=40212450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008100627385A Pending CN101338280A (en) | 2008-07-01 | 2008-07-01 | Aspergillus usamii for producing keratinase complex enzyme and enzyme-producing method thereof by fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101338280A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935642A (en) * | 2010-06-29 | 2011-01-05 | 东华大学 | Protease composite enzyme and application thereof |
| 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 |
-
2008
- 2008-07-01 CN CNA2008100627385A patent/CN101338280A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935642A (en) * | 2010-06-29 | 2011-01-05 | 东华大学 | Protease composite enzyme and application thereof |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101433270B (en) | Preparation method of vegetable seed protein feed | |
| CN101126064B (en) | Biological modified method for erythromycin bacterium slag and reuse thereof | |
| CN110384175B (en) | Method for preparing yeast culture by using vinasse and application of yeast culture | |
| CN103636928A (en) | Method for preparing functional alga fertilizer through co-fermentation of multiple strains | |
| CN103271221B (en) | Method for producing hydrolyzed wheat protein | |
| CN104250311A (en) | Method combining biological method and chemical method for extracting chitin and proteins from shrimp crab shell | |
| CN102533553A (en) | Organic matter decomposition combined fungicide for rapidly degrading rice straws and application method thereof | |
| CN106173204A (en) | A kind of method preparing high protein feed for base material fermentation with citric acid corn starch residue and mycelia slag | |
| CN104054903A (en) | Production process of fermented cottonseed meal | |
| CN103160554A (en) | Erythromycin bacteria residue novel treatment method | |
| CN104630167A (en) | Method for producing low-temperature glucose oxidase by fermentation of marine microorganisms | |
| CN102093990B (en) | Method for producing low temperature amylases through microbial fermentation | |
| CN101338280A (en) | Aspergillus usamii for producing keratinase complex enzyme and enzyme-producing method thereof by fermentation | |
| CN102093989B (en) | Method for producing low-temperature raw diastase by fermenting microorganisms | |
| CN1329504C (en) | A strain for producing composite enzyme and process for producing compoiste emzyme using said strain fermentation | |
| CN102389024A (en) | Method for producing feeding peanut peptide by anaerobic fermentation of peanut meal | |
| CN101491289A (en) | Rape seed protein feedstuff and preparation method thereof | |
| CN107129354A (en) | Compound seed dressing nutrient solution of a kind of biogas slurry base for promoting seed to root and preparation method thereof | |
| CN110747188A (en) | Method for producing keratinase | |
| CN107541474B (en) | Solid fermentation product of bacillus amyloliquefaciens and preparation method thereof | |
| CN104839454A (en) | Production method of high-protein feed rich in valine | |
| CN104920789A (en) | Production method of high-protein feed containing arginine | |
| CN105994940B (en) | Bioactive protein feed and preparation method thereof | |
| CN100587067C (en) | Method for producing keratinase by liquid deep fermentation | |
| CN108522857A (en) | A kind of non-oven drying method of wheat alcohol grain is used as the production method and feed of feed |
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: 20090107 |