CN107325977A - A kind of method for white matter glutaminase of laying eggs, purifying and application - Google Patents
A kind of method for white matter glutaminase of laying eggs, purifying and application Download PDFInfo
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- CN107325977A CN107325977A CN201610272509.0A CN201610272509A CN107325977A CN 107325977 A CN107325977 A CN 107325977A CN 201610272509 A CN201610272509 A CN 201610272509A CN 107325977 A CN107325977 A CN 107325977A
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- glutaminase
- chryseobacterium
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- 102000009127 Glutaminase Human genes 0.000 title claims abstract description 99
- 108010073324 Glutaminase Proteins 0.000 title claims abstract description 97
- 235000013601 eggs Nutrition 0.000 title claims abstract description 23
- 210000004885 white matter Anatomy 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 65
- 102000004190 Enzymes Human genes 0.000 claims abstract description 64
- 230000001580 bacterial effect Effects 0.000 claims abstract description 57
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 57
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 56
- 241000611330 Chryseobacterium Species 0.000 claims abstract description 45
- 230000017854 proteolysis Effects 0.000 claims abstract description 44
- 230000000694 effects Effects 0.000 claims abstract description 37
- 241000249126 Chryseobacterium proteolyticum Species 0.000 claims abstract description 35
- 238000004519 manufacturing process Methods 0.000 claims abstract description 13
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- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 25
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- 102000002322 Egg Proteins Human genes 0.000 claims description 22
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- 238000010612 desalination reaction Methods 0.000 claims description 7
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- 238000012545 processing Methods 0.000 claims description 6
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 5
- 238000005341 cation exchange Methods 0.000 claims description 5
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 5
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- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 claims description 4
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- GUJOJGAPFQRJSV-UHFFFAOYSA-N dialuminum;dioxosilane;oxygen(2-);hydrate Chemical compound O.[O-2].[O-2].[O-2].[Al+3].[Al+3].O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O GUJOJGAPFQRJSV-UHFFFAOYSA-N 0.000 claims description 3
- 239000012154 double-distilled water Substances 0.000 claims description 3
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- 238000004255 ion exchange chromatography Methods 0.000 claims description 3
- 229910052901 montmorillonite Inorganic materials 0.000 claims description 3
- 239000004575 stone Substances 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 101710184006 Glutaminase 1 Proteins 0.000 claims 1
- 101710094902 Legumin Proteins 0.000 claims 1
- NEKNNCABDXGBEN-UHFFFAOYSA-L disodium;4-(4-chloro-2-methylphenoxy)butanoate;4-(2,4-dichlorophenoxy)butanoate Chemical compound [Na+].[Na+].CC1=CC(Cl)=CC=C1OCCCC([O-])=O.[O-]C(=O)CCCOC1=CC=C(Cl)C=C1Cl NEKNNCABDXGBEN-UHFFFAOYSA-L 0.000 claims 1
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- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
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- 239000012138 yeast extract Substances 0.000 description 2
- KKVFPVZIZAJCBZ-UHFFFAOYSA-N (2-hydroxyphenyl) hypochlorite Chemical compound OC1=CC=CC=C1OCl KKVFPVZIZAJCBZ-UHFFFAOYSA-N 0.000 description 1
- XOYSDPUJMJWCBH-VKHMYHEASA-N (3s)-3,5-diamino-5-oxopentanoic acid Chemical compound NC(=O)C[C@H](N)CC(O)=O XOYSDPUJMJWCBH-VKHMYHEASA-N 0.000 description 1
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- 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 1
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- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
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- 150000002475 indoles Chemical class 0.000 description 1
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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Classifications
-
- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
- A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
- A21D2/24—Organic nitrogen compounds
- A21D2/26—Proteins
- A21D2/267—Microbial proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
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- 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)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01044—Protein-glutamine glutaminase (3.5.1.44)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Nutrition Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
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Abstract
The present invention relates to the preparation and application of a kind of protein glutaminase (Protein glutaminase, PG) producing strains and its tunning.The protein glutaminase producing strains are proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains, and culture presevation numbering is CGMCC NO.10532.Bacterial strain enrichment isolation from soil is obtained, safety non-toxic, fermented white matter glutaminase of laying eggs, and enzyme activity is higher.By preparing, purifying, the purity of protein glutaminase is higher, for food industry manufacture field, can significantly improve solubility, foaming characteristic, emulsibility and the stability of protein, have broad application prospects.
Description
Technical field
The invention belongs to food microorganisms screening applied technical field, more particularly to a kind of sieve of protein glutaminase producing strains
Choosing and its preparation of tunning protein glutaminase, purifying and application.
Background technology
Native protein contains more glutamine and asparagine residue, and they cause egg in the form of hydrogen bond and other amino acid crosslinks
The dissolubility reduction of white matter, and then influence the operational characteristic of albumen, such as emulsibility, foaming characteristic, gelation (Fennema OR. food
Learn [Z] Beijing：China Light Industry Press, 2003316-317).Led which limit protein in food, beverage, health products, medicine etc.
Many applications in domain.Deamidation is to solve the important channel of this problem, and research shows, by desamidation, the amide groups in protein
Carboxyl is converted into, negative electrical charge increase reduces the isoelectric point of protein, while deamidation also changes the space structure of protein molecule, made
Originally the hydrophilic radical for being wrapped in inside is exposed, and lesser degree of desamidation (2%-6%) is with regard to that can significantly improve the work(of albumen
Can characteristic (Hamada J S, Marshall W E.Preparation and functional properties of enzymatically deamidated
soyproteins[J].Journal of Food Science.1989,54(3):598-601)。
The deamidation method of document report has acid system and enzyme process.Though acid deamidation can significantly improve the dissolubility of protein, in processing procedure
In, protein peptide chain hydrolysis and the degraded of other amino acid residues are inevitable, and this is larger to protein flavor effect.Glutamine turns
Move the deamidation that enzyme, protease and peptidoglutaminase may be incorporated for protein.But have certain deficiency：Transglutaminase mistake
Amount is using can condense protein；Protease can be by protein integral hydrolysis, and Substratspezifitaet is poor, easily produces bitter taste；Peptide paddy ammonia
Amidase does not play a role to the peptide and protein of HMW, and deamidation degree is not high.
Protein glutaminase (Protein-glutaminase, EC 3.5.1.44) abbreviation PG, is a kind of new type water being just studied in recent years
Enzyme is solved, it can slough amino (Yamaguchi S, Jeenes D J, the Archer D B. of multiple proteins, polypeptide or small peptide
Protein-glutaminase from Chryseobacterium proteolyticum,an enzyme that deamidates glutaminyl residues
in proteins[J].European Journal of Biochemistry,2001,268(5):1410-1421), L- β-glutamine can be hydrolyzed into L-
Glutamic acid and ammonia, and the glutamine group of protein or peptide is acted only on, and asparagine residue and free glutamine are not influenceed,
Crosslinking and hydrolysis (Marcoa C, the Rosell C M.Effect of different protein isolates and of protein will not be caused simultaneously
transglutaminse on rice flour properties[J].Journal of Food Engineering,2008,84(1):132-139), so as to improve egg
Solubility and emulsibility of white matter etc., and the enzyme can also work in acid condition, are a kind of enzymes for having very much an application prospect in food service industry.
But because production bacterial strain is rare, so far, the research to protein glutaminase both at home and abroad is concentrated mainly on its structure and application aspect,
And very few are studied to other bacterial strains that can produce protein glutaminase, this limits the work of protein glutaminase to a certain extent
Industry application.
In existing report, the bacterium of the white matter that can lay eggs glutaminase have proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum),
Glutinous Chryseobacterium sp (Chryseobacterium gleum) and production indoles Chryseobacterium sp (Chryseobacterium indologenes).At present only
One is approved for the only proteolysis Chryseobacterium sp, but its enzymatic productivity is relatively low of industrialized production, and enzyme activity is about 0.2IU/mL.
Therefore, screening can produce high enzyme activity protein glutaminase other bacterial strains it is very necessary.Present invention aim to solve
The existing problem that white matter glutaminase bacterial strain is few, enzyme activity is low of laying eggs is safe and efficient applied to food there is provided one plant of energy, improves protein molten
Xie Du, foaming characteristic, emulsibility, the white matter glutaminase new strains of laying eggs of stability.
The content of the invention
It is an object of the invention to provide a kind of protein glutaminase (Protein-glutaminase, PG) producing strains, i.e., one plant can secrete table
Up to the new strains of the proteolysis Chryseobacterium sp of protein glutaminase, and the strain fermentation product albumen matter glutaminase preparation and its
Application in protein deamidation, will the bacterial strain prepare protein glutaminase be applied to cake production, to wherein egg white carry out deacylation
Amine is acted on, and improves the foaming characteristic of egg white, the consumption of egg white is reduced while increase cake soft degree；Opened applied to soybean protein drinks
Hair, desamidation is carried out to wherein soybean protein isolate, improves the degree of hydrolysis of soybean protein isolate, so as to add soybean protein function drink
The clarity of material, and bad flavor will not be caused；The protein glutaminase and soybean polyoses are compounded, as a kind of new protein
Stabilizer, can increase the solubility and stability of protein in protein drink, and cause flavor taste finer and smoother salubrious.
Present invention firstly provides one plant of proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains, in 2015
It is preserved within 6 days 2 months China Committee for Culture Collection of Microorganisms's common micro-organisms center year, preservation address is Chaoyang District, Beijing City North Star west
The institute 3 of road 1, deposit number is CGMCC NO.10532, and its 16S rDNA sequence is as shown in Seq ID No.3.
Described protein glutaminase producing strains proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains,
The bacterial strain energy secretory protein glutaminase, i.e. PG, the DNA sequence dna of the enzyme is as shown in Seq ID No.10, amino acid sequence
As shown in Seq ID No.14.
In the protein glutaminase, the DNA sequence dna of signal peptide of the enzyme is encoded as shown in Seq ID No.11；The signal peptide
Amino acid sequence as shown in Seq ID No.15.
In the protein glutaminase, the DNA sequence dna of propetide of the enzyme is encoded as shown in Seq ID No.12；The ammonia of the propetide
Base acid sequence is as shown in Seq ID No.16.
In the protein glutaminase, the DNA sequence dna of maturase of the enzyme is encoded as shown in Seq ID No.13；The maturase
Amino acid sequence as shown in Seq ID No.17.
Proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 grows in seed culture medium, growth temperature model
Enclose for 20-40 DEG C, it is preferable that the growth temperature is 25-30 DEG C；It is 6.0-9.0 to grow pH scopes, it is preferable that the most suitable growth
PH is 6.5-7.5.
It is seed culture medium to cultivate the culture medium used, and it is polyprotein peptone 10g/L, yeast extract 2g/L, anhydrous MgSO that it, which is formulated,4
1g/L, pH are 7.0.
In the present invention, performing PCR amplification is entered to the full length DNA of code for said proteins glutaminase, upstream and downstream primer is respectively such as Seq ID
Shown in No.4 and Seq ID No.5.
In the present invention, the DNA of propetide and maturase to code for said proteins glutaminase enters performing PCR amplification, upstream and downstream primer point
Not as shown in Seq ID No.6 and Seq ID No.7.
In the present invention, enter performing PCR amplification to the DNA of the maturase of code for said proteins glutaminase, upstream and downstream primer is respectively such as
Shown in Seq ID No.8 and Seq ID No.9.
Proteolysis Chryseobacterium sp bacterial strain (Chryseobacterium proteolyticum) YF810 protein glutamine enzyme gene is subjected to PCR
Expand and be sequenced, obtain its DNA sequence dna (Seq ID No.10).Its common 963bp of protein glutaminase DNA sequence dna (Seq ID
No.10), wherein 1-63bp (Seq ID No.11) is the sequence of encoded signal peptide, and 64-405bp (Seq ID No.12) is coding propetide
Sequence, 406-963bp (Seq ID No.13) is the sequence of encoding mature enzyme.Protein glutaminase totally 320 ammonia that the bacterial strain is produced
Base acid (Seq ID No.14), wherein signal peptide are 21 amino acid (Seq ID No.15), and propetide includes 114 amino acid (Seq ID
No.16), maturase is then made up of (Seq ID No.17) 185 amino acid.
The invention also provides a kind of sequence of encoding proteins matter glutaminase signal peptide, as shown in Seq ID No.11；The protein paddy
Transglutaminase is produced by proteolysis Chryseobacterium sp bacterial strain (Chryseobacterium proteolyticum) YF810.
Proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains of the present invention can produce egg by fermentation
White matter glutaminase, by this inoculation in fermentation medium, 30 DEG C, 200r/min, concussion and cultivate, this strain fermentation 12h, producing enzyme
Highest is measured, 0.7-2IU/mL is can reach, is 3.5-10 times of the proteolysis Chryseobacterium sp enzyme activity reported at present.Wherein, fermentative medium formula
For lactose 5g/L, soy peptone 15g/L, Na2HPO4·12H2O 3.8g/L, KH2PO40.25g/L, MgSO4·7H2O 0.25g/L,
FeSO4·7H2O 0.05g/L, porous pH stabilizers, such as zeolite 10-50g/L, pH is 7.2.
Wherein, the porous pH stabilizers of 10g/L-50g/L are added in preliminary fermentation culture medium, wherein, the porous pH stabilizers include
Zeolite, montmorillonite, activated carbon, medical stone, pelelith etc.；Preferably, the porous pH of 30g/L are added in preliminary fermentation culture medium stable
Agent zeolite.
Wherein, the activity of protein glutaminase is 0.7-2.0IU/mL in the zymotic fluid.
The invention also provides a kind of fermentation medium, the formula of the fermentation medium is (on the basis of the cumulative volume of zymotic fluid)：Lactose
5g/L, soy peptone 15g/L, Na2HPO4·12H2O 3.8g/L, KH2PO40.25g/L, MgSO4·7H2O 0.25g/L,
FeSO4·7H2O 0.05g/L, porous pH stabilizers 10-50g/L, pH 7.2.
Wherein, the porous pH stabilizers include zeolite, montmorillonite, activated carbon, medical stone, pelelith etc.；Preferably, it is described porous
PH stabilizers are zeolite.Preferably, the addition of the porous pH stabilizers is 30g/L.
In the present invention, bacterial strain white matter glutaminase of laying eggs is purified, its technique is：
The first step：It is concentrated by ultrafiltration
Zymotic fluid removes thalline by centrifugation is preliminary, and supernatant carries out press filtration in filter press with 0.22 μm of filter membrane and removes insoluble impurities,
And concentrated through ultrafiltration system；
Second step：Ethanol precipitation
Concentrate is precipitated through absolute ethyl alcohol, is centrifuged after standing, obtained albumen precipitation ddH2It is insoluble miscellaneous that O centrifuges removal again after redissolving
Matter, obtains protein glutamine enzyme extract；
3rd step：Desalination
Crude extract carries out desalting processing after 0.45 μm of membrane filtration with desalting column；Protein component is collected in the 280nm positions for having absworption peak；
Ion-exchange chromatography is carried out with cation-exchange chromatography post；
4th step：Cation exchange
The protein component collected through desalination, cation-exchange chromatography is carried out with cation-exchange chromatography post；There is the position of absworption peak in 280nm
Collect component；
5th step：Concentration
The component collected through cation exchange, is dialysed with PEG 20000 and concentrated, obtain the concentrate containing enzyme activity；
6th step：Gel permeation chromatography
The concentrated obtained concentrate containing enzyme activity, after 0.22 μm of membrane filtration, gel mistake is carried out with gel permeation chromatography post
Filtering layer is analysed, and component is collected in the 280nm positions for having absworption peak.
In a specific embodiment, the technique that white matter glutaminase of being laid eggs to the bacterial strain is purified is：Seed liquor is with 2%
(V/V) inoculum concentration access 25mL fermentation mediums, 30 DEG C of 200r/min shaking table culture 12h obtain zymotic fluid, by 10000r/min,
4 DEG C of centrifugation 20min go thalline to obtain supernatant, and press filtration removing insoluble impurities is carried out in filter press for 0.22 μm of filter membrane with size.Make
Use QuixstandTMUltrafiltration system concentrates 2-6 times, and molecular cut off is 3000-5000D, feed velocity 0.5L/min, maximum pressure differential
0.2MPa.After concentrate is precipitated with the absolute ethyl alcohol of 4 DEG C of precoolings of 1-4 times of volume, 10000r/min, 4 DEG C of centrifugation 20min, obtained egg
White precipitation uses ddH2O is centrifuged and is removed insoluble impurities again after redissolving, and obtains protein glutamine enzyme extract.Crude extract is through 0.45 μm
After membrane filtration, desalting processing is carried out with the Desalting desalting columns of HiPrep 26/10.Protein groups are collected in the 280nm positions for having absworption peak
Point.Ion-exchange chromatography is carried out with cation-exchange chromatography post.The protein component collected through desalination, with the SP Sepharose of HiPrep 16/10
Cation-exchange chromatography post carries out cation-exchange chromatography.Loading is eluted after finishing, the NaCl that the albumen combined with pillar passes through 0-1M
Linear gradient elution gets off, and is in charge of the component collected and have the position of absworption peak in 280nm.Obtaining containing enzyme activity after ion exchange
Component, with PEG 20000 dialyse concentrate, obtain the concentrate containing enzyme activity.The concentrated obtained concentrate containing enzyme activity, passes through
After 0.45 μm of membrane filtration, gel permeation chromatography is carried out with the Sephacryl S-100 HR gel permeation chromatographies posts of HiPrep 26/60,
Component is collected in the position that 280nm has absworption peak, obtains protein glutaminase.Pass through this purification step, protein glutaminase
Specific enzyme activity power is not less than 130U/mg, and the total enzyme activity rate of recovery can reach 32%.
The invention also provides the protein glutaminase is used for the application of deamidation.
The invention also provides the protein glutaminase to be used for the application of the deamidation of egg white in cake production, so as to improve its dissolving
Degree, foaming characteristic, emulsibility, the consumption of egg white in cake production can be reduced while increase cake soft degree.
In the manufacturing process of cake, the protein glutaminase obtained after purification of different proportion, the deamidation of egg white are added into egg white
Degree, emulsibility and foaming characteristic are continuously increased with the increase of addition.
The invention also provides the protein glutaminase to be used for the exploitation of soybean protein drinks, for improving its solubility, carry
The clarity of high soybean protein drinks, meanwhile, bad flavor will not be brought.
In the exploitation of soybean protein drinks, the protein glutaminase obtained after purification is added into soybean protein, soybean protein
Deamidation degree and degree of hydrolysis are continuously increased with the increase of the protein glutaminase addition.
The invention also provides the protein glutaminase is used in a kind of new protein stabilizing agent.
In a kind of formula of protein stabilizing agent, protein glutaminase and soybean polyoses are compounded, egg in protein drink can be increased
The solubility and stability of white matter, and cause flavor taste finer and smoother salubrious.
Beneficial effect of the present invention is：First, involved proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810
Bacterial strain safety non-toxic, has the advantages that nutritional requirement is simple, growth is rapid, white matter of laying eggs glutaminase active is high；Second, using the bacterium
Strain fermentation is laid eggs white matter glutaminase, be can act on the protein containing glutamine residue such as soybean protein isolate, is sloughed acyl therein
Amine groups, make it preferably be applied to food production processing industry, it is advantageous that selectivity is strong and bad wind will not be brought in mechanism
The deleterious effects such as taste；3rd, laid eggs white matter glutaminase using the strain fermentation, purified technique is purified, specific enzyme activity power with it is total
The enzyme activity rate of recovery is high；4th, protein glutaminase can be fitted as solubilizer, stabilizer, nutrition enhancer, flavor improving agent etc.
In terms of making, the exploitation of soybean protein drinks and the exploitation of protein stabilizing agent for cake.Therefore, the bacterial strain is applied to food
Product field will have wide economic prospect.
Brief description of the drawings
Fig. 1 represents proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains of the present invention in embodiment 2
Colonial morphology.
Fig. 2 represents proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains of the present invention in embodiment 2
Gram's staining result.
Fig. 3 represents proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains of the present invention in embodiment 2
Scanning electron microscopic observation result.
Fig. 4 represents proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains of the present invention in embodiment 3
Growth curve.
Fig. 5 represents proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains of the present invention in embodiment 4
Producing enzyme curve.
Fig. 6 represents porous pH stabilizers of the present invention in embodiment 4, and such as zeolite addition is produced to proteolysis Chryseobacterium sp YF810 bacterial strains
The influence of enzyme.
Embodiment
With reference to specific examples below and accompanying drawing, the present invention is described in further detail.Implement the process, condition, experiment of the present invention
Method etc., is the universal knowledege and common knowledge of this area in addition to the following content specially referred to, the present invention is not particularly limited interior
Hold.
A kind of protein glutaminase producing strains YF810, Classification And Nomenclature：Proteolysis Chryseobacterium sp, Latin name：Chryseobacterium
Proteolyticum, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution is referred to as：CGMCC,
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date：On 2 6th, 2015, deposit number was CGMCC
NO.10532。
For its tunning egg of proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains involved in the present invention
The preparation and application of white matter glutaminase, are embodied using following manner.
The screening separation of the proteolysis Chryseobacterium sp of embodiment 1 (Chryseobacterium proteolyticum) YF810 bacterial strains
YF810 bacterial strains by enriched medium screening obtain, enriched medium by 54mL A liquid, 6mL 1%CBZ, 60 μ L mother liquors I,
60 μ L mother liquors II, 60 μ L mother liquors III and 120 μ L mother liquors IV are mixed.
Wherein, A formula of liquid is：5g/L glucose, 0.2g/L KH2PO4, 0.2g/L MgSO4·7H2O, 0.01%NaCl
Its formula of mother liquor I is：20g/L CaCl2
Its formula of mother liquor II is：2g/L FeSO4·7H2O, 5g/L MnSO4·4H2O
Its formula of mother liquor III is：5g/L NaWO4·4H2O, 5g/L NaMO4·2H2O
Its formula of mother liquor IV is：50g/L CuSO4·5H2O
It screens step：
The first step, weighs soil sample 10g, adds 90mL sterilized waters, and a small amount of bead is put into conical flask, shaking table vibration 10min, system
Into soil suspension.
Second step, soil supension is stood after 10min, is taken in conical flask of the 0.6mL accesses equipped with enriched medium, 30 DEG C, 200r/min
Shaking table culture 6 days.
3rd step, the bacterium solution of previous step culture is taken in conical flask of the 0.6mL accesses equipped with fresh enriched medium, each soil sample sets 2
It is individual parallel, 30 DEG C, 200r/min shaking table cultures 3 days.
4th step, takes 0.1mL into 0.9mL sterilized waters the bacterium solution that previous step is turned out, is diluted to 10-5、10-6Two gradients are carried out
Coated plate, coated plate bacterium solution amount is 100 μ L.
5th step, is observed after flat board is placed in into 30 DEG C of culture carton upside down culture 2-3d.
6th step, with 3%KOH spray solutions on yellow or crocus bacterium colony surface, is observed immediately, if bacterium colony becomes red, then is sprayed
Spill 3%HCl solution.If bacterium colony becomes yellow or crocus again again, as doubtful bacterial strain is further screened.
The identification of the proteolysis Chryseobacterium sp of embodiment 2 (Chryseobacterium proteolyticum) YF810 bacterial strains
1. Morphological Identification
(1) form of bacterium colony is observed
Doubtful bacterial strain is observed after LB flat board cultures, and bacterium colony is rounded, a diameter of 2mm-4mm, neat in edge, golden yellow or crocus,
Opaque, bacterium colony surface wettability is smooth, glossy, as shown in Figure 1.
(2) micro- sem observation
Carry out after Gram's staining and spore staining, observe, as a result show under ordinary optical microscope, bacterial strain is not moved, nothing in shaft-like
Gemma, Gram-negative, as shown in Figure 2.
Observed under ESEM, the smooth atrichia of thalline is not moved, wide 0.2 μm -0.4 μm, it is long 0.8 μm -2.2 μm, such as Fig. 3 institutes
Show.
2. Physiology and biochemistry is identified
With bacterium micro biochemical assessor (being purchased from Qingdao Hai Bo Bioisystech Co., Ltd) to proteolysis Chryseobacterium sp (Chryseobacterium
Proteolyticum) YF810 bacterial strains carry out Physiology and biochemistry identification, and its result is as shown in table 1.
Proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) the YF810 bacterial strains of table 1 Physiology and biochemistry is identified
Wherein, "+" represents that proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains are positive, "-" table
Show that proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains are negative.
According to Physiology and biochemistry qualification result, can tentatively assert that screened YF810 bacterial strains are belonged to as Chryseobacterium, qualification result with
The proteolysis Chryseobacterium sp 9670T bacterial strains that Shotaro Yamaguchi are filtered out have part variation, are shown to be one plant of new proteolysis Chryseobacterium sp bacterium
Strain.
3.16S rDNA sequencings
The total genomic dna of YF810 bacterial strains is extracted, and as masterplate, expands the 16S rDNA sequences of the bacterial strain, its primer sequence
For：
Seq ID No.1, F:5'-AGAGTTTGATCCTGGCTCAG-3'
Seq ID No.2, R:5'-CTACGGCTACCTTGTTACGA-3'
After PCR primer is detected through 1% agarose gel electrophoresis, rubber tapping is reclaimed, and delivers to commercial company's sequencing.Examining order gives birth to work by Shanghai
Biotechnology Services Co., Ltd completes.The 16S rDNA sequences measured are subjected to similitude with germy 16S rDNA sequences
Compare, as a result show YF810 bacterial strains and proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) 9670T(AB039830)
Homology highest, up to 98%, therefore the bacterial strain is proteolysis Chryseobacterium sp.
With reference to the result of colonial morphology, physiological and biochemical property and 16S rDNA gene sequencings, it may be determined that YF810 bacterial strains are one plant new
Proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum), be named as proteolysis Chryseobacterium sp YF810 (Chryseobacterium
proteolyticum YF810)。
Determined dna sequence and the amino acid sequence prediction of the encoding proteins matter glutaminase of embodiment 3
1. encoding proteins matter glutaminase full length DNA sequencing and its amino acid sequence prediction
The total genomic dna of YF810 bacterial strains is extracted, and as masterplate, expand the bacterial strain and lay eggs the total length of white matter glutaminase
DNA sequence dna, its primer sequence is：
Seq ID No.4, F:5'-CCAACCAACTTAACAAAAACTCACCATTAAAC-3'
Seq ID No.5, R:5'-GGAACCCGAACTACCGGAGCAGGATG-3'
After PCR primer is detected through 1% agarose gel electrophoresis, rubber tapping is reclaimed, and delivers to commercial company's sequencing.Examining order gives birth to work by Shanghai
Biotechnology Services Co., Ltd completes.
By the above-mentioned DNA sequence dna measured, i.e. Seq ID No.10；Translated with software Clone Manager, obtain corresponding amino acid
Sequence, i.e. Seq ID No.14.
2. encoding proteins matter glutaminase propetide and the prediction of maturase determined dna sequence and its amino acid sequence
The total genomic dna of YF810 bacterial strains is extracted, and as masterplate, expand the bacterial strain and lay eggs the total length of white matter glutaminase
DNA sequence dna, its primer sequence is：
Seq ID No.6, F:5'-CTGTGCCGATTCCAACGGGAATCAG-3'
Seq ID No.7, R:5'-GAACCCGAACTACCGGAGCAGGATG-3'
After PCR primer is detected through 1% agarose gel electrophoresis, rubber tapping is reclaimed, and delivers to commercial company's sequencing.Examining order gives birth to work by Shanghai
Biotechnology Services Co., Ltd completes.
By the above-mentioned DNA sequence dna measured, i.e. Seq ID No.12 and Seq ID No.13；Translated with software Clone Manager,
Obtain corresponding amino acid sequence, i.e. Seq ID No.16 and Seq ID No.17.
3. encoding proteins matter glutaminase maturase determined dna sequence and its amino acid sequence prediction
The total genomic dna of YF810 bacterial strains is extracted, and as masterplate, expand the bacterial strain and lay eggs the total length of white matter glutaminase
DNA sequence dna, its primer sequence is：
Seq ID No.8, F:5'-AGTCTGCAAGTCCGGAAGACGTAAG-3'
Seq ID No.9, R:5'-CAGCTGGATACATCCGGTGCAGGTG-3'
After PCR primer is detected through 1% agarose gel electrophoresis, rubber tapping is reclaimed, and delivers to commercial company's sequencing.Examining order gives birth to work by Shanghai
Biotechnology Services Co., Ltd completes.
By the above-mentioned DNA sequence dna measured, i.e. Seq ID No.13；Translated with software Clone Manager, obtain corresponding amino acid
Sequence, i.e. Seq ID No.17.
Therefore, described protein glutaminase producing strains proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810
The protein glutaminase of strain secretes, its DNA sequence dna is as shown in Seq ID No.10, and amino acid sequence is as shown in Seq ID No.14.
Wherein, the DNA sequence dna of signal peptide of the enzyme is encoded as shown in Seq ID No.11；The amino acid sequence of the signal peptide such as Seq ID
Shown in No.15；The DNA sequence dna of propetide of the enzyme is encoded as shown in Seq ID No.12；The amino acid sequence of the propetide such as Seq ID
Shown in No.16；The DNA sequence dna of maturase of the enzyme is encoded as shown in Seq ID No.13；The amino acid sequence of the maturase such as Seq
Shown in ID No.17.
The culture of the proteolysis Chryseobacterium sp of embodiment 4 (Chryseobacterium proteolyticum) YF810 bacterial strains
By proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 inoculations in 25mL seed culture mediums, its
It is formulated and is：Polyprotein peptone 10g/L, yeast extract 2g/L, anhydrous MgSO41g/L, pH 7.0.30 DEG C, 200r/min, shake
Culture 12h is swung, is transferred with 2% inoculum concentration in fresh seeds culture medium, 30 DEG C, 200r/min, shaking table culture 12h.Obtain YF810
Bacterium solution.
Its growth curve is as shown in figure 1,0-6h is the lag phase, and 6-14h is to enter stationary phase after exponential phase, 14h.Proteolysis is golden yellow
Bacillus (Chryseobacterium proteolyticum) YF810 bacterial strains nutritional requirement is simple, growth is rapid, is suitable for industrial applications.
The protein glutaminase fermented and cultured of embodiment 5
The fermented and cultured of first step protein glutaminase
YF810 bacterium solutions are accessed into 25mL fermentation mediums with 2% inoculum concentration, its formula is：Lactose 5g/L, soy peptone 15g/L,
Na2HPO4·12H2O 3.8g/L, KH2PO40.25g/L, MgSO4·7H2O 0.25g/L, FeSO4·7H2O 0.05g/L, pH are 7.2.
30 DEG C of 200r/min shaking table culture 12h, obtain zymotic fluid.
The enzyme activity determination of second step protein glutaminase
Zymotic fluid passes through 10000r/min, and 4 DEG C, centrifugation 20min goes thalline to obtain supernatant, and supernatant is diluted into 5 times, takes 0.1mL's
Supernatant dilution is added in test tube, 37 ± 0.5 DEG C of warm bath 1min；Then it is molten that warmed-up 0.01mol/L Cbz-Gln-Gly are added thereto
Liquid 1mL, 37 ± 0.5 DEG C of warm bath react 60min.Then 1mL solution of trichloroacetic acid is added, is well mixed, terminating reaction.Phynol method
Determine the content of ammonia in solution after reaction.A630Determine light absorption value A1.Control group is reacted after 60min again first to add after solution of trichloroacetic acid
Add substrate solution.Remaining operates same experimental group.Determine A630For A2.The amount of enzyme needed for definition 1 μm of ol ammonia of generation per minute is one
Enzyme-activity unit (IU).Enzyme activity calculation formula is as follows in zymotic fluid：
Enzyme activity (U/mL)=(A1-A2) × 2.1/0.1 × 1/17.03 × 1/60 × a × 5
A1：The light absorption value of experimental group；
A2：The light absorption value of control group；
a：The slope of the standard curve of the ammonia of phenol hypochlorite spectrophotometry.
This bacterial strain enzyme activity is measured with methods described, its producing enzyme curve as shown in Fig. 2 highest enzyme activity appears in the logarithmic growth middle and later periods,
Enzyme activity can reach 0.7IU/mL, be the proteolysis Chryseobacterium sp 9670 reported at presentT3.5 times of enzyme activity.
The protein glutaminase fermentation optimization of embodiment 6
The addition porous pH stabilizers of 10g/L-50g/L in original fermentation medium, such as zeolite, 30 DEG C of 200r/min shaking table culture 12h,
Obtain zymotic fluid.Each zymotic fluid enzyme activity is measured, its optimum results is as shown in figure 3, zeolite addition 1%, 2%, 3%, 4%, 5%
(w/v), the yield of enzyme of the bacterial strain can be improved, and when zeolite addition is 30g/L, its facilitation effect is optimal, and enzyme activity can reach 2.0
IU/mL, is 3 times before optimization, is the proteolysis Chryseobacterium sp 9670 reported at presentT10 times of enzyme activity.
30g/L zeolites are added in original fermentation medium, 30 DEG C, 200r/min shaking table culture 12h obtain zymotic fluid, and determine hair
Bacteria concentration, the ammonia density (and pH) of zymotic fluid.As a result it is as shown in table 2.
The proteolysis Chryseobacterium sp of table 2 (Chryseobacterium proteolyticum) YF810 strain fermentation medium optimizations
The advantage of the porous pH stabilizers of the type is that the porous of material can be such that thalline adsorbs in hole, so that promote thalli growth,
And then it is improved zymotic fluid unit enzyme activity；The pH stabilizations of material can be such that the pH of zymotic fluid maintains suitable for strain growth and production
The scope of enzyme.
The purifying of the protein glutaminase of embodiment 7
The first step：It is concentrated by ultrafiltration
Zymotic fluid passes through the preliminary removing thalline of centrifugation, and it is insoluble that supernatant carries out press filtration removing with the filter membrane that size is 0.22 μm in filter press
Impurity.Use QuixstandTMUltrafiltration system concentrates 2-6 times, and molecular cut off is 3000-5000Da, feed velocity 0.5L/min, most
Big pressure differential 0.2MPa.
Second step：Ethanol precipitation
After concentrate is precipitated with the absolute ethyl alcohol of 4 DEG C of precoolings of 1-4 times of volume, 10000r/min, 4 DEG C of centrifugation 20min, obtained albumen precipitation
Use ddH2O is centrifuged and is removed insoluble impurities again after redissolving, and obtains protein glutamine enzyme extract.
3rd step：Desalination
Crude extract carries out desalting processing after 0.45 μm of membrane filtration with the Desalting desalting columns of HiPrep 26/10.There is suction in 280nm
Collect protein component in the position for receiving peak.
4th step：Cation exchange
The protein component collected through desalination, cation-exchange chromatography is carried out with the SP Sepharose cation-exchange chromatography posts of HiPrep 16/10.
Component is collected in the 280nm positions for having absworption peak.
5th step：Concentration
The component containing enzyme activity is obtained after ion exchange, is dialysed and concentrated with PEG 20000, obtain the concentrate containing enzyme activity.
6th step gel permeation chromatography
The concentrated obtained concentrate containing enzyme activity, after 0.45 μm of membrane filtration, with the Sephacryl S-100 of HiPrep 26/60
HR gel permeation chromatographies post carries out gel permeation chromatography, collects component in the 280nm positions for having absworption peak, obtains protein glutamine
Enzyme.
In the present invention, by this purification step, protein glutaminase specific enzyme activity power is not less than 130U/mg, the total enzyme activity rate of recovery
It is not less than 32%.
Application of the protein glutaminase of embodiment 8 in cake production
Needed in the manufacturing process of cake by egg white foaming parcel air so as to during baking cake be made soft tasty, egg white
Through desamidation, its emulsibility and foaming characteristic can be effectively improved, increases the soft degree of cake.By protein glutamine after purification
Enzyme is applied to the desamidation of egg white, to improve its emulsibility and foaming characteristic.
(1) egg white 40g is weighed, 100mL 200mM acid buffers is added and is placed in charging cup, add the protein glutamy of different proportion
Incubated in amine enzyme, 37 DEG C of water-baths after 2h, supernatant is taken after centrifugation, determine ammonia content therein.Meanwhile, add in identical egg white solution
Sulfuric acid, through 100 DEG C of reaction 4h, then determines the ammonia content in supernatant, is total ammonia content to 1M.Ammonia content and total ammonia content in sample
Percentage is deamidation degree.
(2) egg white 40g is weighed, 100mL water is added and is placed in charging cup, with 100mL soybean salad oils, add the albumen of different proportion
Incubated in matter glutaminase, 37 DEG C of water-baths after 2h, 30s, 1500r/min are beaten with 1000r/min speed in high-speed tissue mashing machine
5min is centrifuged, the volume of emulsion layer is recorded, the volume of emulsion layer is the emulsification angle value for representing egg white.
(3) egg white 40g is weighed, 100mL water is added and is placed in charging cup, add the protein glutaminase of different proportion, act on 30min
Afterwards, 1min is beaten in high-speed tissue mashing machine, in the graduated cylinder that 500mL is poured into rapidly, foam volume is the foaming angle value for representing egg white.
The different proteins glutaminase addition of table 3 is to egg white deamidation degree, emulsifiability and foaminess, cake mellowness, retraction degree
Influence
With the increase of protein glutaminase addition, deamidation degree, emulsifiability and the foaminess of egg white are all continuously increased, the pine of cake
Softness, retraction degree also be improved significantly.
Application of the protein glutaminase of embodiment 9 in the exploitation of soybean protein drinks
, it is necessary to improve the solubility of soybean protein by certain method in soybean protein drinks development process, drinks egg is reached
The demand that Bai Hanliang is high, clarity is high, while can eliminate due to the bad flavor that soybean protein content increase is brought.By albumen after purification
Matter glutaminase is applied to the desamidation of soybean protein, to improve its solubility, eliminates bad flavor.
1g and 2g soybean protein isolates are weighed respectively loaded on different hydrolysis pipes, are settled to 100mL with 200mM acid buffers, are obtained
10g/L and 20g/L soybean protein suspension.Protein glutaminase is added into different soybean protein isolate suspensions respectively, is made
The ratio of enzyme-to-substrate reaches 20 units/gram albumen and 50 units/g albumen, and then mixed liquor is placed in 37 DEG C of water-baths and incubates 18h.
(1) take and supernatant is taken after the sample of differential responses time, centrifugation, determine ammonia content therein.Meanwhile, in the 10g/L of same volume
And add sulfuric acid to 1M in 20g/L soybean protein isolate suspensions, through 100 DEG C of reaction 4h, the ammonia content in supernatant is then determined, is total
Ammonia content.The percentage of ammonia content and total ammonia content is deamidation degree in sample.
(2) sample of differential responses time is taken, adding CCl3COOH crystal makes CCl3COOH concentration reach after 12%, protein precipitation,
Determine the protein content in supernatant.Meanwhile, sulfuric acid is added in the soybean protein isolate suspension of same volume to 1M, through 100 DEG C of reaction 4h,
Then the protein content in supernatant is determined, is total protein content.The percentage of protein content and total protein content is in supernatant after sample pellet
Degree of hydrolysis.
Influence of the different proteins glutaminase addition of table 4 to the clarity containing various concentrations soybean protein drinks and local flavor
Protein glutaminase is added in the soybean protein aqueous solution, its solubility can be significantly increased, makes the clarity of solution significantly
Improve, the soybean protein content in increase drink also disclosure satisfy that outward appearance demand of the consumer to soybean protein drinks.Meanwhile, albumen
Also high degree eliminates beany flavor to the desamidation of matter glutaminase, and meet consumer needs to the mouthfeel of soybean protein drinks
Ask.
A kind of application of the protein glutaminase of embodiment 10 in protein stabilizing agent
Soybean polyoses are a kind of conventional protein stabilizing agent, the extensive use in the exploitation of various protein drinks and sour milk beverage.By egg
White matter glutaminase is compounded with soybean polyoses, can increase the solubility and stability of protein in protein drink, and cause mouthfeel more
It is fine and smooth salubrious.
(1) drink with function that soybean protein isolate content is 0.5% and 1% is made：Respectively to the work(containing 0.5% and 1% soybean protein isolate
The protein glutaminase and soybean polyoses mixture of different compound proportions are added in energy drink, mixed liquor is then placed in 37 DEG C of water-baths
Middle incubation 2h.Sample after incubation is stood into 10d, 20d, 30d, 40d, 60d respectively.
(2) rate of deposition is determined：The sample for standing different time is taken, after shaking up, 50mL, 3000r/min is taken, centrifuges 15min, remove
Supernatant simultaneously calculates rate of deposition.Rate of deposition=(quality of centrifuged deposit and pipe-blank pipe quality)/(total matter of quality-blank pipe of sample and pipe
Amount).
(3) it is random to allow consumer to taste the drink with function containing 1% soybean protein isolate of different disposal in school, exhibition, market, and record
Consumer evaluates to the mouthfeel of drink after different disposal.
Influence of the compound action of the protein glutaminase of table 5 and soybean polyoses to protein precipitation rate
Compared with soybean protein addition 0.5% in drink with function in existing report, add after protein glutaminase is compounded with soybean polyoses
It is added in drink, can still reaches good protein stabilization effect when the addition of soybean protein increases to 1% in drink,
Therefore, the addition of protein glutaminase can cooperate with soybean polyoses stable protein, reduce the consumption of soybean polyoses, improve egg in drink
The content of white matter, strengthens its nutritive value.
Evaluation result of the consumer of table 6 to the drink with function mouthfeel of different disposal
Protein glutaminase is added in drink with soybean polyoses compounding as protein stabilizing agent, can improve the mouthfeel of drink, make drink
Product are clearly fine and smooth, more get consumer reception.
In the present invention, it is only necessary to less protein glutamine enzyme dosage and shorter enzyme action time, with regard to higher deacylation can be reached
Amine degree.And higher deamidation degree can make solubility, foaming characteristic, emulsibility and the stability of protein be greatly improved, and it can eliminate not
Good local flavor, this utilization to protein in the food industry is extremely important.
The protection content of the present invention is not limited to above example.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art's energy
The change and advantage enough expected all are included in the present invention, and using appended claims as protection domain.
Claims (12)
- The white matter glutaminase 1. a kind of proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains are laid eggs Method, it is characterised in that methods described includes：The proteolysis Chryseobacterium sp bacterial strain is accessed into shaken cultivation 12h in fermentation medium, Obtain the zymotic fluid of the protein glutaminase；Wherein, the temperature of the culture is 30 DEG C, and the fermentation medium includes： Lactose 5g/L, soy peptone 15g/L, Na2HPO4·12H2O 3.8g/L, KH2PO40.25g/L, MgSO4·7H2O 0.25g/L, FeSO4·7H2O 0.05g/L, pH are 7.2.
- 2. the method as described in claim 1, it is characterised in that the porous pH of 10g/L-50g/L are added in preliminary fermentation culture medium Stabilizer, wherein, the porous pH stabilizers include zeolite, montmorillonite, activated carbon, medical stone, pelelith.
- 3. a kind of fermentation medium, it is characterised in that the culture medium includes：Lactose 5g/L, soy peptone 15g/L, Na2HPO4·12H2O 3.8g/L, KH2PO40.25g/L, MgSO4·7H2O 0.25g/L, FeSO4·7H2O 0.05g/L, it is porous PH stabilizers 10g/L-50g/L, pH are 7.2.
- 4. fermentation medium as claimed in claim 3, it is characterised in that the porous pH stabilizers are zeolite, the zeolite adds Dosage is 30g/L.
- 5. the protein glutaminase produced by proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains, Characterized in that, the DNA sequence dna of the protein glutaminase is as shown in Seq ID No.10, the protein glutamy The amino acid sequence of amine enzyme is as shown in Seq ID No.14；The proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its deposit number is CGMCC NO.10532, its 16Sr DNA sequence dna is as shown in Seq ID No.3.
- 6. as claimed in claim 5 produced by proteolysis Chryseobacterium sp (Chryseobacterium proteolyticum) YF810 bacterial strains Protein glutaminase, it is characterised in that the DNA sequence dna of the signal peptide of code for said proteins glutaminase such as Seq Shown in ID No.11；The amino acid sequence of the signal peptide is as shown in Seq ID No.15；Code for said proteins glutaminase Propetide DNA sequence dna as shown in Seq ID No.12；The amino acid sequence of the propetide is as shown in Seq ID No.16；Compile The DNA sequence dna of the maturase of the code protein glutaminase is as shown in Seq ID No.13；The amino acid of the maturase Sequence is as shown in Seq ID No.17.
- 7. a kind of DNA sequence dna of encoding proteins matter glutaminase signal peptide, it is characterised in that as shown in Seq ID No.11；Institute The amino acid sequence of signal peptide is stated as shown in Seq ID No.15.
- 8. a kind of purification process of protein glutaminase, it is characterised in that methods described includes：The first step：It is concentrated by ultrafiltrationZymotic fluid removes thalline by centrifugation is preliminary, and it is insoluble that supernatant carries out in filter press press filtration removing with 0.22 μm of filter membrane Impurity, and concentrated through ultrafiltration system；Second step：Ethanol precipitationConcentrate is precipitated through absolute ethyl alcohol, is centrifuged after standing, obtained albumen precipitation ddH2O is centrifuged and removed not again after redissolving Solubility impurity, obtains protein glutamine enzyme extract；3rd step：DesalinationCrude extract carries out desalting processing after 0.45 μm of membrane filtration with desalting column；Egg is collected in the 280nm positions for having absworption peak Bai Zufen；Ion-exchange chromatography is carried out with cation-exchange chromatography post；4th step：Cation exchangeThe protein component collected through desalination, cation-exchange chromatography is carried out with cation-exchange chromatography post；There is absworption peak in 280nm Position collect component；5th step：ConcentrationThe component collected through cation exchange, is dialysed with PEG 20000 and concentrated, obtain the concentrate containing enzyme activity；6th step：Gel permeation chromatographyThe concentrated obtained concentrate containing enzyme activity, after 0.22 μm of membrane filtration, is carried out with gel permeation chromatography post Gel permeation chromatography, component is collected in the 280nm positions for having absworption peak.
- 9. purification process as claimed in claim 8, it is characterised in that pass through after purification, the enzyme of the protein glutaminase Vigor is not less than 130U/mg, and the rate of recovery is not less than 32%.
- 10. as described in claim 5 or 6 cake production will be used for by protein glutaminase, strengthens the frothing capacity of egg white, reduce The consumption of egg white, while mitigating the application of the degree of caving in of cake.
- 11. as described in claim 5 or 6 soybean protein in soybean protein drinks will be used for by protein glutaminase, improve big Legumin addition and clarity is higher, while the application of bad flavor can be eliminated.
- 12. protein glutaminase will be compounded as described in claim 5 or 6 with soybean polyoses, protein in protein drink is improved Stability, while increase drink in protein content, strengthen the application of its trophic function.
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| CN113631043A (en) * | 2019-02-21 | 2021-11-09 | 天野酶制品株式会社 | Prevention of nut milk coagulation |
| CN113825403A (en) * | 2019-02-21 | 2021-12-21 | 天野酶制品株式会社 | Prevention of coagulation of vegetable milk |
| EP3928628A4 (en) * | 2019-02-21 | 2022-11-09 | Amano Enzyme Inc. | Prevention of coagulation of vegetable milk |
| EP3928627A4 (en) * | 2019-02-21 | 2022-11-09 | Amano Enzyme Inc. | Prevention of coagulation of nut milk |
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| RU2808068C2 (en) * | 2019-02-26 | 2023-11-22 | АМАНО ЭНЗАЙМ ЮЭсЭй КО., ЛТД. | Stable protein compositions |
| CN114317355A (en) * | 2021-12-30 | 2022-04-12 | 华东师范大学 | Fermentation medium for improving enzyme activity of protein glutaminase |
| CN115381083A (en) * | 2022-08-09 | 2022-11-25 | 武汉新华扬生物股份有限公司 | Complex enzyme preparation and application thereof in increasing litchi juice protein content |
| CN116138348A (en) * | 2022-11-23 | 2023-05-23 | 嘉兴未来食品研究院 | Method for masking beany flavor of legume proteins by using protein glutaminase modification |
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