CN109182154A - The Ba Buwei rhodotorula of one plant of high proteinase yield - Google Patents
The Ba Buwei rhodotorula of one plant of high proteinase yield Download PDFInfo
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- CN109182154A CN109182154A CN201811170296.6A CN201811170296A CN109182154A CN 109182154 A CN109182154 A CN 109182154A CN 201811170296 A CN201811170296 A CN 201811170296A CN 109182154 A CN109182154 A CN 109182154A
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- 241000223252 Rhodotorula Species 0.000 title claims abstract description 40
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 23
- 102000035195 Peptidases Human genes 0.000 title claims abstract description 10
- 235000019833 protease Nutrition 0.000 title claims abstract description 10
- 241000007101 Rhodotorula babjevae Species 0.000 claims abstract description 20
- 244000005700 microbiome Species 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 230000000694 effects Effects 0.000 claims abstract description 3
- 239000004365 Protease Substances 0.000 claims description 13
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 13
- 235000019419 proteases Nutrition 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 abstract description 13
- 230000000813 microbial effect Effects 0.000 abstract description 8
- 241001465754 Metazoa Species 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 239000010802 sludge Substances 0.000 abstract description 3
- 239000006041 probiotic Substances 0.000 abstract description 2
- 235000018291 probiotics Nutrition 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 10
- 238000012216 screening Methods 0.000 description 7
- 239000007787 solid Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 235000021466 carotenoid Nutrition 0.000 description 3
- 150000001747 carotenoids Chemical class 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 235000004237 Crocus Nutrition 0.000 description 2
- 241000596148 Crocus Species 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 229940057059 monascus purpureus Drugs 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 108091005508 Acid proteases Proteins 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 108091023242 Internal transcribed spacer Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 244000170916 Paeonia officinalis Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 description 1
- 229940022405 astaxanthin Drugs 0.000 description 1
- 235000013793 astaxanthin Nutrition 0.000 description 1
- 239000001168 astaxanthin Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
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- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses the Ba Buwei rhodotorulas of one plant of high proteinase yield, the classification naming of the bacterial strain is Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801, depositary institution's preservation that the bacterial strain has been specified in State Intellectual Property Office, the deposit date is on June 21st, 2018, depositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number are CGMCC NO.15980.The present invention separates the Ba Buwei rhodotorula RY1801 of high proteinase yield from Chinese southern Yellow Sea coastal sludge, 20 DEG C, after 200r/min fermented and cultured 48h, strain bio amount reaches 70.36g/L, enzyme activity is 9.653U/ml, this provides a kind of novel microbial bacterial strain for the exploitation of animal probiotics.
Description
Technical field
The invention discloses the Ba Buwei rhodotorulas of one plant of high proteinase yield, belong to field of biotechnology.
Background technique
Rhodotorula is the unicellular eukaryote of the stronger saprophytic bacteria of a kind of resistance, opposite in the nutrition such as seawater and fresh water
In the aquatic saccharomycete of deficient water body environment, rhodotorula accounts for about 50%.Carotenoid and grease are rich in red yeast cell
Equal bioactive ingredients, growth rate with higher and stronger environmental suitability.Since its thallus can generate astaxanthin etc.
Carotenoid substance, so thallus is usually in yellow to peony color.Rhodotorula natural carotenoid biosynthesis,
Microbial oil production, Whole Cell Biocatalysis, postharvest fruit and vegetable biological control and functional feed exploitation etc. and are answered at the research in fields
With.
Protease is widely present in the internal organ of animal, the cauline leaf fruit of plant and microorganism, molecular biology with
Today that microbial fermentation technology is rapidly developed, the application of microbial protease industrially are very universal.Microorganism egg
White enzyme is the enzyme obtained using some microbial fermentations such as bacterium, yeast, mould or actinomyces.Microbial protease again may be used
To be divided into the protease, alkali protease, neutral proteinase, the thermophilic salt that adapt to extreme condition according to its otherness for adapting to environment
In addition to this protease and acid protease further include some protease with other function characteristic.With genetic engineering,
The development of fermentation technique and the research and development and extensive use of novel fermentation equipment, microbial fermentation are increasingly becoming industrial enzyme preparation
Core source.Therefore, novel high yield protease microbial strains are found to be of great significance.In red yeast cell recklessly rich in class
Radish element, cell wall contain glucan, can enhance body's immunity, and produce protease rhodotorula makes in growth of animal
With can promote feed conversion rate, enhance the disease resistance of animal.
Summary of the invention
The purpose of the present invention is disclosing one plant of Ba Buwei rhodotorula, can high proteinase yield, for promote cultivated animals feed turn
Change and improve the exploitation of body resistance against diseases probiotics and novel optional bacterial strain is provided.
Bacterial strain Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 of the present invention be by inventor therefrom
Screening isolates and purifies acquisition in state's southern Yellow Sea coastal sludge, and it is general to be deposited in China Committee for Culture Collection of Microorganisms
Logical microorganism center (CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is 2018 6
The moon 21, deposit number are CGMCC NO.15980.Ba Buwei rhodotorula RY1801 bacterium colony of the invention is circle, is moistened, easily
It provokes, neat in edge, surface is smooth, and it is bigger and thick than the bacterium colony of bacterium, in apparent red or crocus.
Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 of the invention is compared by ITS regional sequence to be divided
Analysis identification, closest with Ba Buwei rhodotorula affiliation, ITS sequence homology is 99%, utilizes 7 software building of MEGA
Phylogenetic tree, RY1801 and Rhodotorula babjevae culture CBS:322, Rhodotorula babjevae
Strain YS3 is same branch, according to RY1801 strain morphology feature and ITS gene order comparison result, is determined
RY1801 is Ba Buwei rhodotorula (Rhodotorula babjevae), is named as Ba Buwei rhodotorula RY1801.
Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 of the invention can high proteinase yield, in YPD
20 DEG C in fluid nutrient medium, after 200r/min fermented and cultured 48h, strain bio amount reaches 70.36g/L, enzyme activity 9.653U/
ml。
Bacterial strain Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 of the present invention belongs to Rhodotorula
(Rhodotorula), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation
Location is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is on June 21st, 2018, deposit number CGMCC
NO.15980。
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 colony morphology characteristic photo.
Fig. 2 is the aspect graph that Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 is observed under the microscope.
Fig. 3 is that the ITS regional sequence progress for the close bacterial strain included in Ba Buwei rhodotorula RY1801 and GenBank is homologous
Property compare building phylogenetic tree.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Embodiment 1: the rhodotorula of present embodiment high proteinase yield is Ba Buwei rhodotorula (Rhodotorula
Babjevae) RY1801 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation
Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is on June 21st, 2018, deposit number CGMCC
NO.15980。
Present embodiment Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 belongs to Rhodotorula, in YPD
28 DEG C of culture 48h on solid plate, bacterium colony is rounded, moistens, easily provokes, neat in edge, surface is smooth, than the bacterium colony of bacterium
It is big and thick, in apparent red or crocus.
Embodiment 2: Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 of present embodiment is by being collected in
Jiangsu Province, China South Yellow Sea coastal sludge sample isolates and purifies to obtain, and screening follows the steps below: weighing soil sample
10.0g is fitted into added in the triangular flask of bead and sterile water, after impregnating 30min, vibrates on the oscillator of 180r/min
30mim takes 1ml suspension to be added in 100mlYPD culture medium with 2.5ml injector for medical purpose, 200r/min, 28 DEG C of culture 2-3
It, takes the above-mentioned shake culture object of 1ml to obtain 10 with sterile water gradient dilution-1、10-2、10-3、10-4Bacteria suspension.Draw each ladder of 0.1ml
Spend bacteria suspension coating uniformly, 28 DEG C of inversions are cultivated 2~3 days.Suspension is inoculated in YPD solid according to streaking inoculation simultaneously
On plate, do three times in parallel, 28 DEG C of inversions are cultivated 2~3 days.Above-mentioned plate is observed, the doubtful bacterium colony on picking plate is divided
Ride culture.The different single colonie of colonial morphology is chosen again to carry out scribing line again or even cross three times, purifies purpose bacterial strain.It will
Screening to three ride of bacterial strain on Screening Protease solid medium, 28 DEG C inversion culture 2~3 days, molten egg will be generated
The bacterial strain of white circle is inoculated in YPD culture medium by 2% inoculum concentration, and 28 DEG C, after 220r/min shake culture 48h, 8000r/min
It is centrifuged 5min, collects supernatant.The punch for being 9mm with diameter punches on Screening Protease solid plate, cuts out agar
Block takes 0.1ml in 0.22 μm of disposable filtered supernatant inlet hole of syringe filter, stands 48h at 37 DEG C, do three
It is a parallel, and molten albumen circle size is measured using ImageJ software, the molten maximum bacterial strain of albumen loop diameter is chosen, Ba Bu is obtained
Tie up rhodotorula (Rhodotorula babjevae) RY1801.
YPD culture medium by 1% yeast extract, 2% peptone, 2% glucose group at.
YPD solid medium is made of 1% yeast extract, 2% peptone, 2% glucose, 2% agar.
Screening Protease solid medium is by 1% yeast extract, 2% peptone, 2% glucose, 2% agar, 1% defatted milk
Powder composition.
Molecular Identification is carried out to obtained Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 of screening, by with
Lower step carries out: the total DNA of bacterial strain is extracted, using fungi ITS universal primer: ITS1:5 '-TCCGTAGGTGAACCTGCGG-
3';ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' carries out PCR amplification by template of genomic DNA.Then it is returned using glue
Kit recovery purifying PCR product is received, Shanghai Sheng Gong Bioisystech Co., Ltd is transferred to be sequenced.
The measured ITS sequence length of Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 is 579bp,
For sequence as shown in SEQ ID NO:1, the ITS sequence in sequencing result and GenBank carries out sequence analysis, then uses software structure
Phylogenetic tree (as shown in Figure 1) is built, to determine the race relation of bacterial strain.Homology analysis the result shows that, the sequence and announce
Ba Buwei rhodotorula affiliation it is closest, similitude 99%.By combining morphological features, growth conditions
It determines that Ba Buwei rhodotorula (Rhodotorula babjevae) RY1801 is one plant of new strains of Rhodotorula, is named as bar
Cloth ties up rhodotorula (Rhodotorula babjevae) RY1801.
Claims (2)
1. the Ba Buwei rhodotorula of one plant of high proteinase yield, it is characterised in that the Ba Buwei rhodotorula of high proteinase yield is Ba Buwei
Rhodotorula (Rhodotorula babjevae) RY1801, is deposited in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center (CGMCC), preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the deposit date is on June 21st, 2018,
Deposit number is CGMCC NO.15980.
2. a kind of Ba Buwei rhodotorula (Rhodotorula babjevae) according to claim 1, which has, generates protease
Effect.
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Cited By (1)
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CN110804560A (en) * | 2019-12-11 | 2020-02-18 | 自然资源部第三海洋研究所 | Yeast 9-1 for producing cellulase and protease and application thereof, microbial preparation and preparation method and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102382786A (en) * | 2011-10-28 | 2012-03-21 | 天津科技大学 | Alkali protease high-yield strain and fermentation method of alkali protease and protease produced by alkali protease high-yield strain |
WO2017184884A1 (en) * | 2016-04-21 | 2017-10-26 | The Regents Of The University Of California | Methods of producing polyol lipids |
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2018
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102382786A (en) * | 2011-10-28 | 2012-03-21 | 天津科技大学 | Alkali protease high-yield strain and fermentation method of alkali protease and protease produced by alkali protease high-yield strain |
WO2017184884A1 (en) * | 2016-04-21 | 2017-10-26 | The Regents Of The University Of California | Methods of producing polyol lipids |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110804560A (en) * | 2019-12-11 | 2020-02-18 | 自然资源部第三海洋研究所 | Yeast 9-1 for producing cellulase and protease and application thereof, microbial preparation and preparation method and application thereof |
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