CN103212319B - Novel use of substance relevant to regulatory protein PhaR - Google Patents
Novel use of substance relevant to regulatory protein PhaR Download PDFInfo
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- CN103212319B CN103212319B CN201310126407.4A CN201310126407A CN103212319B CN 103212319 B CN103212319 B CN 103212319B CN 201310126407 A CN201310126407 A CN 201310126407A CN 103212319 B CN103212319 B CN 103212319B
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- phar
- inclusion body
- pha
- modulin
- phac
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- 239000000126 substance Substances 0.000 title claims abstract description 66
- 102000034356 gene-regulatory proteins Human genes 0.000 title abstract 10
- 108091006104 gene-regulatory proteins Proteins 0.000 title abstract 10
- 210000003000 inclusion body Anatomy 0.000 claims abstract description 247
- 102000004190 Enzymes Human genes 0.000 claims abstract description 90
- 108090000790 Enzymes Proteins 0.000 claims abstract description 90
- 108090000623 proteins and genes Proteins 0.000 claims description 137
- 239000007864 aqueous solution Substances 0.000 claims description 100
- 238000004945 emulsification Methods 0.000 claims description 96
- 102000004169 proteins and genes Human genes 0.000 claims description 90
- 235000018102 proteins Nutrition 0.000 claims description 88
- 230000002194 synthesizing effect Effects 0.000 claims description 83
- 102000038379 digestive enzymes Human genes 0.000 claims description 67
- 108091007734 digestive enzymes Proteins 0.000 claims description 67
- 239000003921 oil Substances 0.000 claims description 51
- 230000014509 gene expression Effects 0.000 claims description 29
- 239000012071 phase Substances 0.000 claims description 29
- 241000588724 Escherichia coli Species 0.000 claims description 23
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 22
- 108091006146 Channels Proteins 0.000 claims description 18
- 230000004927 fusion Effects 0.000 claims description 17
- 239000008346 aqueous phase Substances 0.000 claims description 16
- 235000014304 histidine Nutrition 0.000 claims description 15
- 150000002411 histidines Chemical class 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 239000008157 edible vegetable oil Substances 0.000 claims description 6
- 239000003995 emulsifying agent Substances 0.000 claims description 6
- 239000002569 water oil cream Substances 0.000 claims description 6
- 239000000839 emulsion Substances 0.000 abstract description 48
- 239000000194 fatty acid Substances 0.000 abstract description 2
- 230000000593 degrading effect Effects 0.000 abstract 2
- -1 Hydroxy Fatty Acid Chemical class 0.000 abstract 1
- 235000014113 dietary fatty acids Nutrition 0.000 abstract 1
- 229930195729 fatty acid Natural products 0.000 abstract 1
- 239000010410 layer Substances 0.000 description 54
- 235000019198 oils Nutrition 0.000 description 44
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 description 38
- 229920000903 polyhydroxyalkanoate Polymers 0.000 description 38
- 238000012360 testing method Methods 0.000 description 38
- 239000002283 diesel fuel Substances 0.000 description 36
- 239000013642 negative control Substances 0.000 description 31
- 239000000047 product Substances 0.000 description 25
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- 229910021642 ultra pure water Inorganic materials 0.000 description 19
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- 238000001556 precipitation Methods 0.000 description 17
- 238000002156 mixing Methods 0.000 description 16
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 15
- 239000004094 surface-active agent Substances 0.000 description 15
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 14
- 229940098773 bovine serum albumin Drugs 0.000 description 14
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 13
- 230000010355 oscillation Effects 0.000 description 13
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- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
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- 239000002245 particle Substances 0.000 description 9
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- 238000010438 heat treatment Methods 0.000 description 8
- 239000010687 lubricating oil Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 108010041952 Calmodulin Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 229910021529 ammonia Inorganic materials 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
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- 102000000584 Calmodulin Human genes 0.000 description 5
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- 239000012634 fragment Substances 0.000 description 5
- 230000002209 hydrophobic effect Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000003549 soybean oil Substances 0.000 description 5
- 235000012424 soybean oil Nutrition 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 102000008300 Mutant Proteins Human genes 0.000 description 3
- 108010021466 Mutant Proteins Proteins 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
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- 239000002244 precipitate Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000002525 ultrasonication Methods 0.000 description 3
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 102000004895 Lipoproteins Human genes 0.000 description 2
- 108090001030 Lipoproteins Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
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- 239000000872 buffer Substances 0.000 description 2
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- 239000000287 crude extract Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
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- 230000008014 freezing Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 108010028921 Lipopeptides Proteins 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 125000001183 hydrocarbyl group Chemical group 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 101150028013 phaP gene Proteins 0.000 description 1
- 108010033574 phasin Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
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- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
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- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses novel use of a substance relevant to regulatory protein PhaR. The novel use is an application of the substance relevant to the regulatory protein PhaR in preparing an oil aqueous emulsion. The substance relevant to the regulatory protein PhaR is any one of A1-A6, wherein A1 is a product composed of a regulatory protein PhaR inclusion body, a PHA (Hydroxy Fatty Acid) synthetic enzyme PhaC inclusion body and a PHA degrading enzyme PhaR inclusion body; A2 is a product composed of the regulatory protein PhaR inclusion body and the PHA synthetic enzyme PhaC inclusion body; A3 is a product composed of the regulatory protein PhaR inclusion body and the PHA degrading enzyme PhaR inclusion body; A4 is a product composed of the regulatory protein PhaR inclusion body; A5 is a product composed of the regulatory protein PhaR and the PHA synthetic enzyme PhaC; and A6 is the regulatory protein PhaR.
Description
Technical field
The present invention relates to the novelty teabag with modulin PhaR related substances.
Background technology
Biosurfactant is that a large class obtains amphipathic small molecules material (Mulligan etc., Environ Pollut, 133 (2005), 183-98 by microorganism; Khire etc., Adv Exp Med Biol, 672 (2010), 146-57; Morita etc., Biotechnol Appl Biochem, 53 (2009), 39-49), generally comprise low-molecular-weight glycolipid, lipopeptid, phosphatide, neutral lipid etc. and HMW containing lipopolymer, as (Syldatk etc., ZNaturforsch G such as lipopolysaccharides (glycolipids), lipoprotein (lipopeptides or lipoproteins), polysaccharide-protein-fatty acid complexes, 40 (1985), 61-7; 0gawa etc., Biosci Biotechnol Biochem, 64 (2000), 2466-8; Morita etc., J Biosci Bioeng, 104 (2007), 78-81; Wicke etc., J Nat Prod, 63 (2000), 621-6).Their molecular structure is made up of two parts, a part is the hydrophilic polar group of oleophobic, as monose, glycan, amino acid and phosphate etc., the non-polar group that another part is made up of the hydrocarbon chain of hydrophobic oleophilic oil, as saturated or unsaturated fatty alcohol and aliphatic acid etc.Just because of having this not only oleophylic but also hydrophilic amphipathic molecule structure, biosurfactant significantly could reduce interfacial tension, or be adsorbed on hydrophilic/lipophilic performance interface being formed and aligns to change interface closely, make oil/water two-phase be able to good dispersion.What wherein represent the most is glycolipid class (Mata-Sandoval etc., Microbiol Res, 155 (2001), 249-56; Guilmanov etc., Biotechnol Bioeng, 77 (2002), 489-94; ).
Polyhydroxyalkanoate (Polyhydroxyalkanoic acids, be called for short PHA) be the Storage polymer (Anderson etc. of a kind of cell internal carbon source of synthesizing under non-equilibrium growth conditions of many microorganisms and the energy, Microbial Rev., 54 (1990) 450-472; Lee etc., Biotech.Bioeng., 49 (1996) 1-14).Research finds, the PHA under natural conditions exists with the form of insoluble lipochondrion (granule).PHA molecule forms hydrophobic core, particle surface is wrapped in the special single-layer membrane structure of one deck, (Steinb ü chel etc., Can.J.Microbiol.41 (1995) 94-105 is formed by particle associated proteins (PhaP/Phasin), modulin (PhaR), pha synthesizing enzyme (PhaC), PHA digestive enzyme (PhaZ) and single phospholipid membrane etc.; York etc., J.Bacteriol.183 (2001) 2394-2397).PHA digestive enzyme, PhaZ plays a part PHA macromolecules degradation to be become Small molecular oligomer (Jendrossek etc., Appl.Microbiol.Biotechnol., 46 (1996) 451-463; Briese etc., J.Environ.Polym.Degrad., 2 (1994) 75-87).Modulin, there are two kinds of calmodulin binding domain CaMs in PhaR, DNA calmodulin binding domain CaM and particle calmodulin binding domain CaM (Maehara etc., J.Bacteriol.184 (2002) 3992-4002).DNA calmodulin binding domain CaM the specific phaR of being attached to gene and phaP upstream region of gene can be rich in the region of TGC, thus regulation and control PhaP albumen and the expression of himself.And hydrophobic particle calmodulin binding domain CaM is combined in PHA particle surface by hydrophobic effect.The same with PhaP, show that the hydrophobicity combination of PhaR is nonspecific (Yamashita etc., Biomacromolecules, 7 (2006) 2449-2454) by the external binding tests of PhaR and other hydrophobic materials.Pha synthesizing enzyme, PhaC is the key enzyme in PHA biosynthetic process.
Summary of the invention
Technical problem to be solved by this invention is to provide the novelty teabag with modulin PhaR related substances.
Provided by the present invention is 1 with the novelty teabag of modulin PhaR related substances)-4) in any one:
1) preparing the application in water-oil emulsion with modulin PhaR related substances, described is any one in A1-A6 with modulin PhaR related substances:
Described A1 is the product be made up of modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body and PHA digestive enzyme PhaZ inclusion body;
Described A2 is the product be made up of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body;
Described A3 is the product of described modulin PhaR inclusion body and described PHA digestive enzyme PhaZ inclusion body composition;
Described A4 is by described modulin PhaR inclusion body;
Described A5 is the product be made up of modulin PhaR and pha synthesizing enzyme PhaC;
Described A6 is described modulin PhaR.
2) preparing the application in emulsifying agent with modulin PhaR related substances, described is any one in A1-A6 with modulin PhaR related substances:
Described A1 is the product be made up of modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body and PHA digestive enzyme PhaZ inclusion body;
Described A2 is the product be made up of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body;
Described A3 is the product of described modulin PhaR inclusion body and described PHA digestive enzyme PhaZ inclusion body composition;
Described A4 is by described modulin PhaR inclusion body;
Described A5 is the product be made up of modulin PhaR and pha synthesizing enzyme PhaC;
Described A6 is described modulin PhaR.
3) prepare the method for water-oil emulsion, comprise and aqueous phase and oil phase equal-volume are mixed the step of carrying out emulsification and preparing water-oil emulsion, described aqueous phase is the aqueous solution with modulin PhaR related substances;
Described is any one in A1-A6 with modulin PhaR related substances:
Described A1 is the product be made up of modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body and PHA digestive enzyme PhaZ inclusion body;
Described A2 is the product be made up of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body;
Described A3 is the product of described modulin PhaR inclusion body and described PHA digestive enzyme PhaZ inclusion body composition;
Described A4 is by described modulin PhaR inclusion body;
Described A5 is the product be made up of modulin PhaR and pha synthesizing enzyme PhaC;
Described A6 is described modulin PhaR;
Described aqueous phase meet 1)-6) and condition:
1) described aqueous phase is the aqueous solution of described A1, and the total concentration of protein of the aqueous solution of described A1 is 3000 μ g/ml;
2) described aqueous phase is the aqueous solution of described A2, and the total concentration of protein of the aqueous solution of described A2 is 3000 μ g/ml;
3) described aqueous phase is the aqueous solution of described A3, and the total concentration of protein of the aqueous solution of described A3 is 3000 μ g/ml;
4) described aqueous phase is the aqueous solution of described A4, and the protein concentration of the aqueous solution of described A4 is 3000 μ g/ml;
5) described aqueous phase is the aqueous solution of described A5, and the total concentration of protein of the aqueous solution of described A5 is 500 μ g/ml;
6) described aqueous phase is the aqueous solution of described A6, and the protein concentration of the aqueous solution of described A6 is 500 μ g/ml.
4) emulsifying agent, any one product in A1-A3, A5:
Described A1 is the product be made up of modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body and PHA digestive enzyme PhaZ inclusion body;
Described A2 is the product be made up of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body;
Described A3 is the product of described modulin PhaR inclusion body and described PHA digestive enzyme PhaZ inclusion body composition;
Described A5 is the product be made up of modulin PhaR and pha synthesizing enzyme PhaC.
In above-mentioned novelty teabag, each component of described A1, described A2, described A3 and described A5 can be packed separately, also can mix.
In above-mentioned novelty teabag, described emulsifying agent is the material making oil and water form emulsion.Described modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body or PHA digestive enzyme PhaZ inclusion body refer to the expression product of described modulin PhaR gene, pha synthesizing enzyme PhaC gene or PHA digestive enzyme PhaZ gene accumulate in the bacterium (as Escherichia coli) and the formation that flocks together without the exposed structure of film.
In above-mentioned novelty teabag, described modulin PhaR can be following a1), a2) or protein a3):
A1) protein of amino acid sequence as shown in SEQ ID No.2;
A2) before the 1st of SEQ ID No.2, add the fusion that 6 histidines obtain;
A3) SEQ ID No.2 through the mutant protein with the identical activity of protein of amino acid sequence as shown in SEQ ID No.2 replacement and/or lack and/or add one or several amino acid residue and obtain;
Described pha synthesizing enzyme PhaC can be following b1), b2) or protein b3):
B1) protein of amino acid sequence as shown in SEQ ID No.4;
B2) before the 1st of SEQ ID No.4, add the fusion that 6 histidines obtain;
B3) SEQ ID No.4 through the mutant protein with the identical activity of protein of amino acid sequence as shown in SEQ ID No.4 replacement and/or lack and/or add one or several amino acid residue and obtain;
PHA digestive enzyme PhaZ in described PHA digestive enzyme PhaZ inclusion body can be following c1), c2) or protein c3):
C1) protein of amino acid sequence as shown in SEQ ID No.6;
C2) before the 1st of SEQ ID No.6, add the fusion that 6 histidines obtain;
C3) SEQ ID No.6 through the mutant protein with the identical activity of protein of amino acid sequence as shown in SEQ ID No.6 replacement and/or lack and/or add one or several amino acid residue and obtain;
In above-mentioned novelty teabag, in described A5, the mass ratio of described modulin PhaR and described pha synthesizing enzyme PhaC can be 1: 1; In described A1, the mass ratio of described modulin PhaR inclusion body, described pha synthesizing enzyme PhaC inclusion body and described PHA digestive enzyme PhaZ inclusion body can be 1: 1: 1; In described A2, the mass ratio of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body can be 1: 1; The mass ratio of modulin PhaR inclusion body described in described A3 and described PHA digestive enzyme PhaZ inclusion body can be 1: 1.
In above-mentioned novelty teabag, modulin PhaR channel genes Escherichia coli make described modulin PhaR gene expression obtain by described modulin PhaR and described modulin PhaR inclusion body, pha synthesizing enzyme PhaC channel genes Escherichia coli make described pha synthesizing enzyme PhaC gene expression obtain by described pha synthesizing enzyme PhaC and described pha synthesizing enzyme PhaC inclusion body, and PHA digestive enzyme PhaZ channel genes Escherichia coli make described PHA digestive enzyme PhaZ gene expression obtain by described PHA digestive enzyme PhaZ inclusion body.
In above-mentioned novelty teabag, the coded sequence of described modulin PhaR gene specifically can be SEQ ID No.1, the coded sequence of described pha synthesizing enzyme PhaC gene specifically can be SEQ ID No.3, and the coded sequence of described PHA digestive enzyme PhaZ gene specifically can be SEQ ID No.5.
In one embodiment of the invention, described Escherichia coli are specially E.Coli BL21 (DE3).Described modulin PhaR channel genes Escherichia coli make described modulin PhaR gene carry out expressing obtaining at 16-18 DEG C by described modulin PhaR, described pha synthesizing enzyme PhaC channel genes Escherichia coli make described pha synthesizing enzyme PhaC gene obtain 16-18 DEG C of expression by described pha synthesizing enzyme PhaC, described modulin PhaR channel genes Escherichia coli make described modulin PhaR gene obtain 25 DEG C of expression by described modulin PhaR inclusion body, shown pha synthesizing enzyme PhaC channel genes Escherichia coli make described pha synthesizing enzyme PhaC gene obtain 25 DEG C of expression by described pha synthesizing enzyme PhaC inclusion body, shown PHA digestive enzyme PhaZ channel genes Escherichia coli make described PHA digestive enzyme PhaZ gene obtain 25 DEG C of expression by described PHA digestive enzyme PhaZ inclusion body.Above-mentioned expression is all carried out under the IPTG of 0.3mM-0.5mM induces.
In one embodiment of the invention, the coded sequence of described modulin PhaR gene is SEQ ID No.1, and the coded sequence of described pha synthesizing enzyme PhaC gene is SEQ ID No.3, and the coded sequence of described PHA digestive enzyme PhaZ gene is SEQ ID No.5.
In above-mentioned novelty teabag, described oil phase can be petrochemical industry oil or edible oil.Described petrochemical industry oil can be diesel oil or lubricating oil; Described edible oil can be soybean oil.
In above-mentioned novelty teabag, described emulsification can adopt vortex oscillation, and described vortex oscillation speed can be 1300rpm, and duration of oscillation is 120 seconds.
In above-mentioned novelty teabag, in described A1, the mass ratio of described modulin PhaR and described pha synthesizing enzyme PhaC is 1: 1; In described A2, the mass ratio of described modulin PhaR inclusion body, described pha synthesizing enzyme PhaC inclusion body and described PHA digestive enzyme PhaZ inclusion body is 1: 1; In described A3, the mass ratio of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body is 1: 1; The mass ratio of modulin PhaR inclusion body described in described A4 and described PHA digestive enzyme PhaZ inclusion body is 1: 1.
Experiment proves that PhaZ, PhaC and PhaR inclusion body protein all has the ability of diesel oil emulsification, lubricating oil and edible oil, and static long-time emulsion layer keeps stable, also has good emulsifying capacity after heat treatment.Emulsifiability and the inclusion body of soluble protein are similar, but in a heated condition, are easy to degraded, cause its heat endurance to decline, and emulsifying capacity reduces.And the heat-flash stability of inclusion body, can at very high temperatures, still holding structure is stablized, and ensure that the emulsifying capacity of albumen under high temperature.In preservation process, soluble protein is also very easily degraded, and inclusion body protein is then more stable.In transportation, soluble protein easily shakes and very easily spumes, and inclusion body is then relatively stable.
Accompanying drawing explanation
Fig. 1 is the physical map of recombinant expression plasmid pET28a-PhaR.
Fig. 2 is PhaZ, PhaR and PhaC protein expression SDS-PAGE electrophoretogram.
In figure, swimming lane 1:PhaZ expresses bacterial strain and adds IPTG induction; Swimming lane 2:PhaZ expresses bacterial strain and does not add IPTG induction; Swimming lane 3:PhaR expresses bacterial strain and adds IPTG induction; Swimming lane 4:PhaR expresses bacterial strain and does not add IPTG induction; Swimming lane 5:PhaC expresses bacterial strain and does not add IPTG induction; Swimming lane 6:PhaC expresses bacterial strain and adds IPTG induction; Swimming lane 7:Marker.
Fig. 3 is the SDS-PAGE electrophoretogram of PhaR, PhaC after purifying.
A is PhaC, b is PhaR.
Fig. 4 is the SDS-PAGE electrophoretogram of the PhaP after E.coliBL21 (DE3) holoprotein of amalgamation and expression PhaP and purifying.
Wherein, swimming lane M: albumen marker, swimming lane 1: the thalline holoprotein not adding IPTG abduction delivering, swimming lane 2-4: the thalline holoprotein adding IPTG abduction delivering PhaP, swimming lane 5: the PhaP after purifying.
Detailed description of the invention
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embody emulsifying capacity by emulsification value in following embodiment, concrete experimental technique is as follows: the test substance aqueous solution and isopyknic various oil phase substance be mixed in the vial of same specification at 20 DEG C and carry out emulsification treatment.Emulsification treatment mode is turbula shaker oscillation treatment.Turbula shaker oscillation treatment adopts turbula shaker (XH-C, Jin Yi, Medical Instruments factory of Jintan City) within 120 seconds, to process with the velocity fluctuation of 1300 revolution per seconds.
After emulsification treatment completes standing a period of time, the height of observed and recorded oil reservoir (oil layer), emulsion layer (emulsion layer) and water layer (water layer).And calculate relevant emulsification value (Emulsion Index) by formula (1).Emulsification value computing formula is as follows:
Emulsification value=emulsion layer/(oil reservoir+emulsion layer+water layer) × 100% (1)
Wherein, all layer height overalls refer to the total height of oil reservoir, emulsion layer and water layer three layers.But water layer or oil reservoir may disappear after emulsification sometimes, then height overall is the height sum of remaining two-layer (layers).Emulsification value is larger, illustrates that emulsifying capacity is stronger.
Emulsification experiment in following embodiment all establishes three repetitions, and emulsification value represents with mean+SD.
The prokaryotic expression of embodiment 1, modulin PhaR and inclusion body thereof, pha synthesizing enzyme PhaC and inclusion body thereof and PHA digestive enzyme PhaZ inclusion body, particle associated proteins PhaP and purifying
One, the structure of expression vector pET28a-PhaR, pET28a-PhaC, pET28a-PhaZ, pET28a-PhaP
Wherein, the amino acid sequence of modulin PhaR is SEQ ID No.2, the amino acid sequence of pha synthesizing enzyme PhaC is SEQ ID No.4, the amino acid sequence of the PHA digestive enzyme PhaZ in PHA digestive enzyme PhaZ inclusion body is SEQ ID No.6, and the amino acid sequence of particle associated proteins PhaP is SEQ ID No.8.
The modulin PhaR gene that coded sequence is SEQ ID No.1 is prepared by PCR; the sequence of modulin PhaR gene is before the 1st of SEQ ID No.1, add NdeI recognition site and protection base (sequence is ggaattccat), and after the 552nd of SEQ ID No.1, adding Hind III recognition site and protection base, (sequence is for (sequence is aagcttgggc).
The pha synthesizing enzyme PhaC gene that coded sequence is SEQ ID No.3 is prepared by PCR; the sequence of pha synthesizing enzyme PhaC gene is before the 3rd of SEQ ID No.1, add NdeI recognition site and protection base (sequence is ggaattccat), adds Hind III recognition site and protection base (sequence is aagcttgggc) after the 1776th of SEQ ID No.3.
The PHA digestive enzyme PhaZ gene that coded sequence is SEQ ID No.5 is prepared by PCR; the sequence of PHA digestive enzyme PhaZ gene is before the 1st of SEQ ID No.5, add NdeI recognition site and protection base (sequence is ggaattccat), adds Hind III recognition site and protection base (sequence is aagcttgggc) after the 738th of SEQ ID No.5.
The gene that coded sequence is the particle associated proteins PhaP of SEQ ID No.7 is prepared by PCR; the sequence of particle associated proteins PhaP gene is before the 1st of SEQ ID No.7, add NdeI recognition site and protection base (sequence is ggaattccat), adds Hind III recognition site and protection base (sequence is aagcttgggc) after the 348th of SEQ ID No.7.
With NdeI and Hind III respectively enzyme cut PhaR gene, PhaC gene, PhaZ gene and PhaP gene, with NdeI and Hind III digestion pET28a (the upper graceful bio tech ltd of Hypon), by PhaR gene, PhaC gene, PhaZ gene is connected with NdeI with the Hind III digestion product of pET28a respectively with NdeI with the Hind III digestion product of PhaP gene, carry out NdeI and Hind III digestion respectively to connection product to sign and order-checking qualification, by PhaR expression vector called after pET28a-PhaR (Fig. 1) that the fragment between NdeI and the Hind III with PhaR gene replacement pET28a obtains, by the PhaC expression vector called after pET28a-PhaC that the fragment between NdeI and the Hind III with PhaC gene replacement pET28a obtains, by the PhaZ expression vector called after pET28a-PhaZ that the fragment between NdeI and the Hind III with PhaZ gene replacement pET28a obtains, by the PhaP expression vector called after pET28a-PhaP that the fragment between NdeI and the Hind III with PhaP gene replacement pET28a obtains.
Genes of interest is inserted between NdeI and HindIII restriction enzyme site, be placed in the T7 promoter downstream that pET28a expression system carries, again due to the his label of MCS upstream and downstream, the gene inserted efficiently can start through T7 promoter, and have on the protein sequence obtained and all add his label with downstream, be convenient to purification process.
Two, with E. coli BL21 (DE3) Restruction albumen and inclusion body
By recombinant plasmid pET28a-PhaR, pET28a-PhaC, pET28a-PhaZ, pET28a-PhaP Transformed E .coli BL21 (DE3) competent cell (purchased from Beijing Tian Gen biochemical technology Co., Ltd) respectively, picking carries out Fiber differentiation containing the monoclonal of recombinant plasmid, run whole bacterial protein electrophoresis and carry out protein expression qualification, result display recombinant plasmid can normal expression PhaP, PhaZ, PhaR and PhaC albumen.But, after the thalline ultrasonication thalline of PhaZ abduction delivering, find not ultrasonic to limpid, there is a large amount of precipitation in centrifugal rear discovery lower floor, find that PhaZ albumen major part is positioned at lower floor through SDS-PAGE electrophoresis detection, upper strata does not almost have PhaZ albumen, determines to define inclusion bodies of protein.After the various reaction condition of optimization, PhaZ still expresses with inclusion bodies, and PhaP, PhaR and PhaC are under low temperature induction condition, tends to the albumen forming solubility, and when temperature reaches 25 DEG C, also has inclusion body to produce.Grope through condition, find that the formation impact of IPTG on inclusion body is less, especially responsive to temperature.In follow-up test, find some class surfactant properties of inclusion body, again due to some superiority of inclusion body, after great expression inclusion body protein, with its surfactant aspect performance of the mutual comparative studies of soluble protein.
1, the preparation of PhaP, PhaR or PhaC
Concrete grammar is as follows:
(1) by recombinant plasmid pET28a-PhaR, pET28a-PhaC, pET28a-PhaZ, pET28a-PhaP respectively Transformed E .ColiBL21 (DE3) competent cell (purchased from Tian Gen biochemical technology Co., Ltd), smear resistant panel (containing ammonia benzyl 100 μ g/ml) and screen positive bacterium colony.Picking positive bacterium colony, adds in 20ml LB culture medium, 220rpm, incubated overnight at 37 DEG C, using the nutrient solution of acquisition as seed liquor.
(2) seed liquor is added (containing ammonia benzyl 100 μ g/ml) in 1000ml LB fluid nutrient medium, 220rpm, 37 DEG C of shaken cultivation are 0.5-0.8 to OD600, add the IPTG that final concentration is 0.3mM-0.5mM, 220rpm, cultivate 16h, abduction delivering fusion for 18 DEG C, obtain the rear bacterium liquid of induction.Set the bacterium liquid that do not add IPTG as blank simultaneously.
(3) centrifugal thalline 1h under 5000g, and with 40ml binding buffer (0.5M NaCl, 20mM Tris-HCl, 5mM imidazoles (imidazole), pH=7.9) washing precipitation twice, thalline is resuspended in 40mlbinding buffer the most at last.By ultrasonic (400HZ) broken 15min that resuspended thalline exports with 40%.
(4) 12,000g, centrifugal ultrasonication liquid 1h at 4 DEG C.Collect supernatant, be rich protein-contg crude extract.
(5) cell crude extract is poured into the His label affinitive layer purification post (U.S. handled well, Merck), add binding buffer, wash buffer (0.5M NaCl successively, 60mM Tris-HCl, 20mM imidazole, pH=7.9) and elute buffer (0.5M NaCl, 1M Tris-HCl, 20mM imidazole, pH=7.9), collect the liquid finally obtained.
(6) ultrafiltration is utilized to remove salinity unnecessary in solution
The liquid that step (5) purifying obtains is added super filter tube (model is 3k Da for the U.S., Miliipore) to top limit, 5000rpm is after centrifugal 1 hour, to retain in super filter tube residual liquid in pipe.Add ultra-pure water to super filter tube top limit, continue centrifugal, repeatedly aforesaid operations 5 times.In super filter tube, in pipe, liquid is PhaP, PhaR or PhaC protein solution of removing excess salt.
(7) Freeze Drying Technique is utilized to obtain pure solid powdery PhaP
PhaP, PhaR or PhaC protein solution of removing excess salt step (6) obtained is freezing at-80 DEG C, puts into freeze drier and carry out freeze drying 24 hours after freezing.Obtain white solid powder shape PhaP, PhaR or PhaC albumen.
Take a small amount of solid powdery PhaP, PhaR or PhaC protein dissolution in ultra-pure water, and together carry out SDS-PAGE inspection with E.coli BL21 (DE3) holoprotein of non-abduction delivering and E.coli BL21 (DE3) holoprotein of amalgamation and expression.Protein electrophoresis the results are shown in Figure 3 and Fig. 4.As seen from the figure, PhaP adds that the His tag molecule size of amalgamation and expression is that about 14k Da, PhaR add that the His tag molecule size of amalgamation and expression is that about 25k Da, PhaC add that the His tag molecule size of amalgamation and expression is for about 64k Da.
2, the preparation of PhaZ inclusion body, PhaR inclusion body or PhaC inclusion body
(1) by recombinant plasmid pET28a-PhaR, pET28a-PhaC and pET28a-PhaZ respectively Transformed E .Coli BL21 (DE3) competent cell (purchased from Tian Gen biochemical technology Co., Ltd), smear resistant panel (containing ammonia benzyl 100 μ g/ml) and screen positive bacterium colony.Picking positive bacterium colony, adds in 20ml LB culture medium, 220rpm, incubated overnight at 37 DEG C, using the nutrient solution of acquisition as seed liquor.
(2) seed liquor is added (containing ammonia benzyl 100 μ g/ml) in 1000ml LB fluid nutrient medium, 220rpm, 37 DEG C of shaken cultivation are 0.5-0.8 to 0D600, add the IPTG that final concentration is 0.3mM-0.5mM, 220rpm, cultivate 16h, abduction delivering fusion for 25 DEG C, obtain the rear bacterium liquid of induction.
Centrifugal thalline 1h under (3) 5,000g, and precipitate twice with the pre-cold water washing of 20mL, thalline is resuspended in the pre-cold water of 20mL the most at last.By ultrasonic (400HZ) broken 15min that resuspended thalline exports with 40%.
(4) 5,000g, centrifugal ultrasonication liquid 1h at 4 DEG C.Collecting precipitation, namely obtains PhaZ inclusion body, PhaR inclusion body and PhaC inclusion body.
Meanwhile, the negative control group of inclusion bodies of protein is set, serves emulsification to prove inclusion bodies of protein playing emulsification instead of other cell fragments and impurity.
The negative control group of inclusion bodies of protein obtains as follows:
(1) by recombinant plasmid pET28a-PhaR, pET28a-PhaC and pET28a-PhaZ respectively Transformed E .ColiBL21 (DE3) competent cell (purchased from Tian Gen biochemical technology Co., Ltd), smear resistant panel (containing ammonia benzyl 100 μ g/ml) and screen positive bacterium colony.Picking positive bacterium colony, adds in 20ml LB culture medium, 220rpm, incubated overnight at 37 DEG C, using the nutrient solution of acquisition as seed liquor.
(2) seed liquor added (containing ammonia benzyl 100 μ g/ml) in 1000ml LB fluid nutrient medium, 220rpm, when 37 DEG C of shaken cultivation are 0.5-0.8 to OD600, becomes 220rpm, cultivates 16h for 25 DEG C, obtains bacterium liquid.
Centrifugal thalline 1h under (3) 5,000g, and precipitate twice with the pre-cold water washing of 20mL, thalline is resuspended in the pre-cold water of 20mL the most at last.By ultrasonic (400HZ) broken 15min that resuspended thalline exports with 40%.
Centrifugal thalline 1h under (4) 14,000g.Collect respectively centrifugal after upper cleer and peaceful precipitation, precipitate through pre-cold sterilization milli-Q water 2-3 time, obtain cleer and peaceful PhaZ inclusion body negative control precipitation on PhaZ inclusion body negative control, cleer and peaceful PhaR inclusion body negative control precipitation on PhaR inclusion body negative control, cleer and peaceful PhaC inclusion body negative control precipitation on PhaC inclusion body negative control.
The ability of embodiment 2, PhaP, PhaC and PhaR protein solution diesel oil emulsification and emulsion layer stability
Water-soluble traditional chemical surfactant, commercialization washing agent and the bovine serum albumin with same concentrations of PhaP, PhaC and PhaR albumen is used for comparing of diesel oil emulsification ability.PhaP, PhaC and PhaR albumen is product prepared by above-described embodiment 1; The dodecyl sodium sulfate that tradition chemical surfactant is respectively high-purity (is called for short SDS, the U.S., Biosharp, purity >=95.0%), enuatrol (China, Hua Da chemical industry, purity >=95.0%) and polysorbas20 (China, exhibition is biological for morning, purity >=98.0%); Oil phase is diesel oil (China, China Petrochemical Industry).
Concrete operations are as follows:
1, the preparation of the test substance aqueous solution: PhaP, PhaC and PhaR albumen after purifying is all mixed with the 500 μ g/ml aqueous solution with ultra-pure water respectively, equally SDS, enuatrol, polysorbas20 and bovine serum albumin are all mixed with the 500 μ g/ml aqueous solution with ultra-pure water respectively, obtain 7 kinds of test substance aqueous solution.
2, take diesel oil as oil phase, the test substance aqueous solution of equal-volume (the test substance aqueous solution and oil phase are 0.5ml) and diesel oil are carried out emulsification treatment 20 DEG C of mixing, emulsification treatment adopts above-mentioned turbula shaker oscillation treatment, emulsification treatment completes and leaves standstill under lucifuge 25 DEG C of constant temperature, and leaving standstill takes pictures for 2 days and 30 days observes and records the height of oil reservoir, emulsion layer and water layer.And calculate relevant emulsification value by formula (1).In contrast with ultra-pure water simultaneously.
Result shows standing 2 days, and the sample of PhaP, PhaC and PhaR emulsification has obvious emulsion layer; SDS also has obvious emulsion layer, and enuatrol, polysorbas20 and bovine serum albumin sample sets do not have obvious emulsion layer.Leave standstill 30 days, the emulsification value of the sample of PhaP, PhaC and PhaR emulsification does not still have significant change, and the emulsion layer that SDS and diesel oil are formed obviously reduces, the sample further illustrating PhaP, PhaC and PhaR emulsification not only has the ability of very strong diesel oil emulsification, also has very strong emulsion layer stability.
The emulsification value that the contrast leaving standstill the different experiments group emulsification value of 2 days and 30 days shows PhaP, PhaC and PhaR diesel oil emulsification is higher, reaches more than 70%, has the emulsifying activity of similar clause type surfactant.Wherein the sample of PhaR emulsification leaves standstill 1.1 times that the emulsification value of 2 days is the sample of PhaP emulsification, slightly higher than the sample of PhaC emulsification; The emulsification value that the sample of PhaR emulsification leaves standstill 30 days is 1.1 times of the sample of PhaP emulsification, than slightly high (table 1) of the sample of PhaC emulsification.
The emulsification value of table 1, different test substance
Embodiment 3, PhaP, PhaC and PhaR protein solution emulsion type lubricant capacity experimental
The water-soluble traditional chemical surfactant with same concentrations of PhaP, PhaC and PhaR albumen and bovine serum albumin are used for comparing of emulsion type lubricant ability.PhaP, PhaC and PhaR albumen is product prepared by above-described embodiment 1; The dodecyl sodium sulfate that tradition chemical surfactant is respectively high-purity (is called for short SDS, the U.S., Biosharp, purity >=95.0%), enuatrol (China, Hua Da chemical industry, purity >=95.0%) and polysorbas20 (China, exhibition is biological for morning, purity >=98.0%); Oil phase is lubricating oil (U.S., Mobil).
Concrete operations are as follows:
1, the preparation of the test substance aqueous solution: PhaP, PhaC and PhaR albumen after purifying is all mixed with the 500 μ g/ml aqueous solution with ultra-pure water respectively, obtains PhaP sample sets, PhaC sample sets, PhaR sample sets respectively; Equally SDS, enuatrol, polysorbas20 and bovine serum albumin are all mixed with the 500 μ g/ml aqueous solution with ultra-pure water respectively, obtain SDS sample sets, enuatrol sample sets, polysorbas20 sample sets and bovine serum albumin sample sets respectively, using above-mentioned each sample group as the test substance aqueous solution.
2, take lubricating oil as oil phase, the test substance aqueous solution of equal-volume (the test substance aqueous solution and oil phase are 0.5ml) and lubricating oil are carried out emulsification treatment 20 DEG C of mixing, emulsification treatment adopts above-mentioned turbula shaker oscillation treatment, emulsification treatment completes and leaves standstill under lucifuge 25 DEG C of constant temperature, and leaving standstill takes pictures for 2 days and 30 days observes and records the height of oil reservoir, emulsion layer and water layer.And calculate relevant emulsification value by formula (1).In contrast with ultra-pure water simultaneously.
Result shows standing 2 days, and the sample sets of PhaP, PhaC and PhaR has obvious emulsion layer; Dodecyl sodium sulfate has obvious water layer, oil reservoir and emulsion layer, and emulsification value is lower.The sample sets of enuatrol, polysorbas20 and bovine serum albumin does not have obvious emulsion layer.Be all the bovine serum albumin of protein, fail emulsion type lubricant.Leave standstill 30 days, the emulsion layer that the sample sets of PhaP, PhaC and PhaR produces does not change.Illustrate that PhaP, PhaC and PhaR not only have the ability of very strong emulsion type lubricant, also there is very strong emulsion layer stability.
The emulsification value of table 2, different surfaces activating agent emulsion type lubricant
As can be seen from Table 2, the emulsification value of PhaP, PhaC and PhaR emulsion type lubricant is apparently higher than the emulsification value of enuatrol, polysorbas20 and bovine serum albumin emulsion type lubricant.
Embodiment 4, PhaP, PhaC and PhaR protein solution Deltalipid capacity experimental
The water-soluble traditional chemical surfactant with same concentrations of PhaP, PhaC and PhaR albumen and bovine serum albumin are used for comparing of emulsification edible soybean oil ability.PhaP, PhaC and PhaR albumen is product prepared by above-described embodiment 1; The dodecyl sodium sulfate that tradition chemical surfactant is respectively high-purity (is called for short SDS, the U.S., Biosharp, purity >=95.0%), enuatrol (China, Hua Da chemical industry, purity >=95.0%) and polysorbas20 (China, exhibition is biological for morning, purity >=98.0%); Oil phase selects edible soybean oil (China, Jin Longyu).
Concrete operations are as follows:
1, the preparation of the test substance aqueous solution: PhaP, PhaC and PhaR albumen after purifying is all mixed with the 500 μ g/ml aqueous solution with ultra-pure water respectively, obtains PhaP sample sets, PhaC sample sets, PhaR sample sets respectively; Equally SDS, enuatrol, polysorbas20 and bovine serum albumin are all mixed with the 500 μ g/ml aqueous solution with ultra-pure water respectively, obtain SDS sample sets, enuatrol sample sets, polysorbas20 sample sets and bovine serum albumin sample sets respectively, using above-mentioned each sample group as the test substance aqueous solution.
2, take edible soybean oil as oil phase, the test substance aqueous solution of equal-volume (the test substance aqueous solution and oil phase are 0.5ml) and edible soybean oil are carried out emulsification treatment 20 DEG C of mixing, emulsification treatment adopts above-mentioned turbula shaker oscillation treatment, emulsification treatment completes and leaves standstill under lucifuge 25 DEG C of constant temperature, and leaving standstill takes pictures for 2 days and 30 days observes and records the height of oil reservoir, emulsion layer and water layer.And calculate relevant emulsification value by formula (1).In contrast with ultra-pure water simultaneously.
Result shows standing 2 days, and PhaC, PhaR and PhaP sample sets has obvious emulsion layer; The sample sets of bovine serum albumin does not have obvious emulsion layer, is all the bovine serum albumin of protein, fails Deltalipid; The sample sets of SDS has obvious emulsion layer, water layer and oil reservoir.Leave standstill 30 days, the emulsion layer that PhaC, PhaR and PhaP sample sets produces does not change.Illustrate that PhaC, PhaR and PhaP not only have the ability of very strong Deltalipid, also there is very strong emulsion layer stability.Under 500 μ g/ml concentration, PhaC and PhaR has the ability of stronger Deltalipid, and the ability of PhaR Deltalipid is better than PhaC a little.
The emulsification value of table 3, different surfaces activating agent Deltalipid
Embodiment 5, PhaP, PhaC and PhaR albumen are tested as the heat endurance of surfactants/emulsifiers
Concrete operations are as follows:
1, the preparation of the test substance aqueous solution: PhaP, PhaC and PhaR albumen is product prepared by above-described embodiment 1, and PhaP, PhaC and PhaR albumen after purifying is all mixed with the 500 μ g/ml aqueous solution with ultra-pure water respectively.Respectively to PhaP, PhaC and PhaR protein solution 95 DEG C of heat treatment 30min and 60min of 500 μ g/ml, obtain 6 kinds of test substance aqueous solution.
2, with diesel oil (China, China Petrochemical Industry) be oil phase, the test substance aqueous solution of equal-volume (the test substance aqueous solution and oil phase are 0.5ml) and diesel oil are carried out emulsification treatment 20 DEG C of mixing, emulsification treatment adopts above-mentioned turbula shaker oscillation treatment, emulsification treatment completes and leaves standstill under lucifuge 25 DEG C of constant temperature, and leaving standstill takes pictures for 2 days observes and records the height of oil reservoir, emulsion layer and water layer.And calculate relevant emulsification value by formula (1).In contrast with ultra-pure water simultaneously.
The emulsification value of table 4, PhaP, PhaC and PhaR albumen heat treatment different time
Result shows that PhaC almost loses emulsifying capacity after heat treatment 30min, and PhaR still possesses very strong emulsifying capacity (table 4) after heat treatment 60min.Heat endurance is relevant with nature, and the heat-flash stability of PhaR itself can expand its range of application as surfactant and using value.
The effect of embodiment 6, PhaZ, PhaC and PhaR inclusion body protein diesel oil emulsification
Measure the PhaZ inclusion body of embodiment 1 preparation, PhaR inclusion body and PhaC inclusion body, PhaZ inclusion body negative control supernatant, PhaZ inclusion body negative control precipitation, PhaR inclusion body negative control supernatant, PhaR inclusion body negative control precipitation, protein content on PhaC inclusion body negative control in cleer and peaceful PhaC inclusion body negative control precipitation, be diluted with water inclusion body and inclusion body negative control supernatant all to 5000ug/ml protein concentration.And same ratio dilution inclusion body negative control precipitation.-20 DEG C save backup.
Concrete operations are as follows:
1, PhaZ, PhaC and PhaR inclusion body protein is all mixed with respectively the aqueous solution of 3000 μ g/ml, 1000 μ g/ml, 500 μ g/ml and 0 μ g/ml with ultra-pure water.PhaZ inclusion body negative control supernatant, PhaC inclusion body negative control supernatant, PhaR inclusion body negative control supernatant are all diluted to 3000 μ g/ml with ultra-pure water respectively, PhaZ inclusion body negative control precipitation, PhaC inclusion body negative control precipitation, PhaR inclusion body negative control precipitation are pressed OD with ultra-pure water respectively
600value is diluted to the cellular lysate precipitation capacity under inclusion body protein 3000 μ g/ml concentration, obtains 16 kinds of test substance aqueous solution.
2, with diesel oil (China, China Petrochemical Industry) be oil phase, the test substance aqueous solution of equal-volume (the test substance aqueous solution and oil phase are 0.5ml) and diesel oil are carried out emulsification treatment 20 DEG C of mixing, emulsification treatment adopts above-mentioned turbula shaker oscillation treatment, emulsification treatment completes and leaves standstill under lucifuge 25 DEG C of constant temperature, and leaving standstill takes pictures for 2 days and 30 days observes and records the height of oil reservoir, emulsion layer and water layer.And calculate relevant emulsification value by formula (1).
Result shows that the PhaZ inclusion body negative control supernatant water solution of 3000 μ g/ml, PhaC inclusion body negative control supernatant water solution, PhaR inclusion body negative control supernatant water solution left standstill 2 days are all identical with the emulsification value of 30 days, specifically in negative control precipitation cleer and peaceful on the negative control in table 5.When same concentrations (3000 μ g/ml), effect and the negative control of PhaZ, PhaC and PhaR inclusion body protein diesel oil emulsification contrast, and can determine that emulsifying effectiveness is produced by the interaction of inclusion body and diesel oil.The soluble protein of the identical amount of negative control supernatant has more weak diesel oil emulsification ability, gets rid of the impact of residual supernatant protein in inclusion body.Negative control precipitation has more weak diesel oil emulsification ability, gets rid of the impact of residual cellular lysate thing in inclusion body.End product shows that inclusion body protein has the ability of diesel oil emulsification really.
The emulsification value of table 5, PhaZ, PhaC and PhaR inclusion body protein and negative control diesel oil emulsification
Embodiment 7, PhaZ, PhaC and PhaR inclusion bodies of protein are as the heat endurance of surfactant
Measure the protein content in the PhaZ inclusion body of embodiment 1 preparation, PhaR inclusion body and PhaC inclusion body, add ultra-pure water dilution inclusion body to 3000ug/ml protein concentration, obtain the PhaR inclusion body aqueous solution of the PhaZ inclusion body aqueous solution of 3000 μ g/ml, the PhaC inclusion body aqueous solution of 3000 μ g/ml and 3000 μ g/ml.Respectively to the PhaR inclusion body aqueous solution 95 DEG C of heat treatment 30min and 60min of the PhaZ inclusion body aqueous solution of 3000 μ g/ml, the PhaC inclusion body aqueous solution of 3000 μ g/ml and 3000 μ g/ml, obtain 6 kinds of test substance aqueous solution.
With diesel oil (China, China Petrochemical Industry) be oil phase, the test substance aqueous solution of equal-volume (the test substance aqueous solution and oil phase are 0.5ml) and diesel oil are carried out emulsification treatment 20 DEG C of mixing, emulsification treatment adopts above-mentioned turbula shaker oscillation treatment, emulsification treatment completes and leaves standstill under lucifuge 25 DEG C of constant temperature, and leaving standstill took pictures after 2 days observes and records the height of oil reservoir, emulsion layer and water layer.And calculate relevant emulsification value by formula (1).
Result shows, and PhaZ, PhaC and PhaR inclusion bodies of protein still possesses the ability (table 6) of very strong diesel oil emulsification after heat treatment.
The emulsification value of table 6, PhaZ, PhaC and PhaR inclusion bodies of protein heat treatment different time
The heat treated stability of PhaZ, PhaC and PhaR inclusion bodies of protein is all fairly good, illustrate that inclusion bodies of protein inside is not easy to be damaged, this also just inclusion body protein compared with an advantage of soluble protein: stable existence, not by endocellular enzyme solution, is not subject to outside destroy.
The ability of embodiment 8, PhaR and PhaC mixed liquor emulsion type lubricant and diesel oil
1, the preparation of the test substance aqueous solution: PhaC and the PhaR albumen after embodiment 1 purifying is all mixed with the 500 μ g/ml aqueous solution with ultra-pure water respectively, obtain the PhaC aqueous solution (being called for short the PhaC aqueous solution) of 500 μ g/ml and the PhaR aqueous solution (being called for short the PhaR aqueous solution) of 500 μ g/ml, as two kinds of test substance aqueous solution.Simultaneously by 1: 1 mixing by volume of above-mentioned two kinds of solution, be mixed with the mixed surfactant aqueous solution that PhaR and PhaC is 250 μ g/ml, obtain the third test substance aqueous solution.
2, with the lubricating oil (U.S., Mobil) or diesel oil (China, China Petrochemical Industry) be oil phase, the test substance aqueous solution of equal-volume (the test substance aqueous solution and oil phase are 0.5ml) and oil phase are carried out emulsification treatment 20 DEG C of mixing, emulsification treatment adopts above-mentioned turbula shaker oscillation treatment, emulsification treatment completes and leaves standstill under lucifuge 25 DEG C of constant temperature, and leaving standstill takes pictures for 2 days observes and records the height of oil reservoir, emulsion layer and water layer.And calculate relevant emulsification value by formula (1).
Result is as shown in table 7, shows that PhaR and PhaC mixed protein is that the sample emulsion type lubricant of surfactant and diesel oil all have obvious emulsion layer; The emulsion layer of emulsion layer height in time using PhaR and PhaC emulsion type lubricant and diesel oil respectively.
The mixed liquor emulsion type lubricant of table 7, PhaR and PhaC and the emulsification value of diesel oil
The result of table 7 shows, mixed surfactant diesel oil emulsification and emulsification of lubricating oils value are used for separately the emulsification value height about 10% of oil emulsion phase time than PhaR or PhaC.The ability of diesel oil emulsification and lubricating oil and emulsifying capacity is still had to obtain effective enhancing after showing PhaR and PhaC mixing.
The ability of embodiment 9, PhaR, PhaC and PhaZ inclusion body mixed liquor diesel oil emulsification
1, the protein content in the PhaZ inclusion body of embodiment 1 preparation, PhaR inclusion body and PhaC inclusion body is measured, add ultra-pure water dilution inclusion body to 3000ug/ml protein concentration, obtain the PhaR inclusion body aqueous solution (being called for short the PhaR inclusion body aqueous solution) of the PhaZ inclusion body aqueous solution (being called for short the PhaZ inclusion body aqueous solution) of 3000 μ g/ml, the PhaC inclusion body aqueous solution (being called for short the PhaC inclusion body aqueous solution) of 3000 μ g/ml and 3000 μ g/ml respectively, as three kinds of test substance aqueous solution.Simultaneously, the PhaZ inclusion body aqueous solution and the PhaC inclusion body aqueous solution to be mixed by volume at 1: 1, be mixed with the aqueous solution that PhaZ inclusion body and PhaC inclusion body are 1500ug/ml protein concentration, obtain PhaZ and the PhaC mixing inclusion body aqueous solution, as the 4th kind of test substance aqueous solution; The PhaZ inclusion body aqueous solution and the PhaR inclusion body aqueous solution to be mixed by volume at 1: 1, be mixed with the aqueous solution that PhaZ inclusion body and PhaR inclusion body are 1500ug/ml protein concentration, obtain PhaZ and the PhaR mixing inclusion body aqueous solution, as the 5th kind of test substance aqueous solution; The PhaC inclusion body aqueous solution and the PhaR inclusion body aqueous solution to be mixed by volume at 1: 1, be mixed with the aqueous solution that PhaC inclusion body and PhaR inclusion body are 1500ug/ml protein concentration, obtain PhaC and the PhaR mixing inclusion body aqueous solution, as the 6th kind of test substance aqueous solution; The PhaR inclusion body aqueous solution, the PhaZ inclusion body aqueous solution and the PhaC inclusion body aqueous solution to be mixed by volume at 1: 1: 1, be mixed with the aqueous solution that PhaR inclusion body, PhaZ inclusion body and PhaC inclusion body are 1000ug/ml protein concentration, obtain PhaZ, PhaR and PhaC mixing inclusion body aqueous solution, as the 7th kind of test substance aqueous solution.
2, with diesel oil (China, China Petrochemical Industry) be oil phase, the test substance aqueous solution of equal-volume (the test substance aqueous solution and oil phase are 0.5m1) and diesel oil are carried out emulsification treatment 20 DEG C of mixing, emulsification treatment adopts above-mentioned turbula shaker oscillation treatment, emulsification treatment completes and leaves standstill under lucifuge 25 DEG C of constant temperature, and leaving standstill takes pictures for 2 days observes and records the height of oil reservoir, emulsion layer and water layer.And calculate relevant emulsification value by formula (1).
Result is as shown in table 8, shows that PhaZ, PhaR and PhaC mixing inclusion body still has the ability of diesel oil emulsification.After PhaZ and PhaC two kinds of inclusion body mixing, the emulsification of mixed liquor reduces.But the emulsifying capacity of the mixed liquor containing PhaR is used for separately the emulsifying effectiveness of emulsification than often kind of inclusion body significantly to be strengthened.
The emulsification value of table 8, PhaZ, PhaC and PhaR inclusion body protein and various mixed liquor diesel oil emulsification
Claims (13)
1. preparing the application in water-oil emulsion with modulin PhaR related substances, described is any one in A1-A4 with modulin PhaR related substances:
Described A1 is the product be made up of modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body and PHA digestive enzyme PhaZ inclusion body;
Described A2 is the product be made up of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body;
Described A3 is the product be made up of described modulin PhaR inclusion body and described PHA digestive enzyme PhaZ inclusion body;
Described A4 is the product be made up of modulin PhaR and pha synthesizing enzyme PhaC;
Described modulin PhaR is following a1) or protein a2):
A1) protein of amino acid sequence as shown in SEQ ID No.2;
A2) before the 1st of SEQ ID No.2, add the fusion that 6 histidines obtain;
Described pha synthesizing enzyme PhaC is following b1) or protein b2):
B1) protein of amino acid sequence as shown in SEQ ID No.4;
B2) before the 1st of SEQ ID No.4, add the fusion that 6 histidines obtain;
PHA digestive enzyme PhaZ in described PHA digestive enzyme PhaZ inclusion body is following c1) or protein c2):
C1) protein of amino acid sequence as shown in SEQ ID No.6;
C2) before the 1st of SEQ ID No.6, add the fusion that 6 histidines obtain;
In described A4, the mass ratio of described modulin PhaR and described pha synthesizing enzyme PhaC is 1:1;
In described A1, the mass ratio of described modulin PhaR inclusion body, described pha synthesizing enzyme PhaC inclusion body and described PHA digestive enzyme PhaZ inclusion body is 1:1:1;
In described A2, the mass ratio of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body is 1:1;
The mass ratio of modulin PhaR inclusion body described in described A3 and described PHA digestive enzyme PhaZ inclusion body is 1:1.
2. application according to claim 1, it is characterized in that: modulin PhaR channel genes Escherichia coli make described modulin PhaR gene expression obtain by described modulin PhaR and described modulin PhaR inclusion body, pha synthesizing enzyme PhaC channel genes Escherichia coli make described pha synthesizing enzyme PhaC gene expression obtain by described pha synthesizing enzyme PhaC and described pha synthesizing enzyme PhaC inclusion body, and PHA digestive enzyme PhaZ channel genes Escherichia coli make described PHA digestive enzyme PhaZ gene expression obtain by described PHA digestive enzyme PhaZ inclusion body.
3. application according to claim 2, it is characterized in that: the coded sequence of described modulin PhaR gene is SEQ ID No.1, the coded sequence of described pha synthesizing enzyme PhaC gene is SEQ ID No.3, and the coded sequence of described PHA digestive enzyme PhaZ gene is SEQ ID No.5.
4. according to described application arbitrary in claim 1-3, it is characterized in that: described oil phase is petrochemical industry oil or edible oil.
5. preparing the application in emulsifying agent with modulin PhaR related substances, described is any one in A1-A4 with modulin PhaR related substances:
Described A1 is the product be made up of modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body and PHA digestive enzyme PhaZ inclusion body;
Described A2 is the product be made up of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body;
Described A3 is the product of described modulin PhaR inclusion body and described PHA digestive enzyme PhaZ inclusion body composition;
Described A4 is the product be made up of modulin PhaR and pha synthesizing enzyme PhaC; Described modulin PhaR is following a1) or protein a2):
A1) protein of amino acid sequence as shown in SEQ ID No.2;
A2) before the 1st of SEQ ID No.2, add the fusion that 6 histidines obtain;
Described pha synthesizing enzyme PhaC is following b1) or protein b2):
B1) protein of amino acid sequence as shown in SEQ ID No.4;
B2) before the 1st of SEQ ID No.4, add the fusion that 6 histidines obtain;
PHA digestive enzyme PhaZ in described PHA digestive enzyme PhaZ inclusion body is following c1) or protein c2):
C1) protein of amino acid sequence as shown in SEQ ID No.6;
C2) before the 1st of SEQ ID No.6, add the fusion that 6 histidines obtain;
In described A4, the mass ratio of described modulin PhaR and described pha synthesizing enzyme PhaC is 1:1;
In described A1, the mass ratio of described modulin PhaR inclusion body, described pha synthesizing enzyme PhaC inclusion body and described PHA digestive enzyme PhaZ inclusion body is 1:1:1;
In described A2, the mass ratio of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body is 1:1;
The mass ratio of modulin PhaR inclusion body described in described A3 and described PHA digestive enzyme PhaZ inclusion body is 1:1.
6. application according to claim 5, it is characterized in that: modulin PhaR channel genes Escherichia coli make described modulin PhaR gene expression obtain by described modulin PhaR and described modulin PhaR inclusion body, pha synthesizing enzyme PhaC channel genes Escherichia coli make described pha synthesizing enzyme PhaC gene expression obtain by described pha synthesizing enzyme PhaC and described pha synthesizing enzyme PhaC inclusion body, and PHA digestive enzyme PhaZ channel genes Escherichia coli make described PHA digestive enzyme PhaZ gene expression obtain by described PHA digestive enzyme PhaZ inclusion body.
7. application according to claim 6, it is characterized in that: the coded sequence of described modulin PhaR gene is SEQ ID No.1, the coded sequence of described pha synthesizing enzyme PhaC gene is SEQ ID No.3, and the coded sequence of described PHA digestive enzyme PhaZ gene is SEQ ID No.5.
8. according to described application arbitrary in claim 5-7, it is characterized in that: described oil phase is petrochemical industry oil or edible oil.
9. prepare the method for water-oil emulsion, comprise and aqueous phase and oil phase equal-volume are mixed the step of carrying out emulsification and preparing water-oil emulsion, described aqueous phase is the aqueous solution with modulin PhaR related substances;
Described is any one in A1-A4 with modulin PhaR related substances:
Described A1 is the product be made up of modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body and PHA digestive enzyme PhaZ inclusion body;
Described A2 is the product be made up of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body;
Described A3 is the product of described modulin PhaR inclusion body and described PHA digestive enzyme PhaZ inclusion body composition;
Described A4 is the product be made up of modulin PhaR and pha synthesizing enzyme PhaC;
Described aqueous phase meet 1)-4) and condition:
1) described aqueous phase is the aqueous solution of described A1, and the total concentration of protein of the aqueous solution of described A1 is 3000 μ g/ml;
2) described aqueous phase is the aqueous solution of described A2, and the total concentration of protein of the aqueous solution of described A2 is 3000 μ g/ml;
3) described aqueous phase is the aqueous solution of described A3, and the total concentration of protein of the aqueous solution of described A3 is 3000 μ g/ml;
4) described aqueous phase is the aqueous solution of described A4, and the total concentration of protein of the aqueous solution of described A4 is 500 μ g/ml;
Described modulin PhaR is following a1) or protein a2):
A1) protein of amino acid sequence as shown in SEQ ID No.2;
A2) before the 1st of SEQ ID No.2, add the fusion that 6 histidines obtain;
Described pha synthesizing enzyme PhaC is following b1) or protein b2):
B1) protein of amino acid sequence as shown in SEQ ID No.4;
B2) before the 1st of SEQ ID No.4, add the fusion that 6 histidines obtain;
PHA digestive enzyme PhaZ in described PHA digestive enzyme PhaZ inclusion body is following c1) or protein c2):
C1) protein of amino acid sequence as shown in SEQ ID No.6;
C2) before the 1st of SEQ ID No.6, add the fusion that 6 histidines obtain;
In described A5, the mass ratio of described modulin PhaR and described pha synthesizing enzyme PhaC is 1:1;
In described A1, the mass ratio of described modulin PhaR inclusion body, described pha synthesizing enzyme PhaC inclusion body and described PHA digestive enzyme PhaZ inclusion body is 1:1:1;
In described A2, the mass ratio of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body is 1:1;
The mass ratio of modulin PhaR inclusion body described in described A3 and described PHA digestive enzyme PhaZ inclusion body is 1:1.
10. method according to claim 9, it is characterized in that: modulin PhaR channel genes Escherichia coli make described modulin PhaR gene expression obtain by described modulin PhaR and described modulin PhaR inclusion body, pha synthesizing enzyme PhaC channel genes Escherichia coli make described pha synthesizing enzyme PhaC gene expression obtain by described pha synthesizing enzyme PhaC and described pha synthesizing enzyme PhaC inclusion body, and PHA digestive enzyme PhaZ channel genes Escherichia coli make described PHA digestive enzyme PhaZ gene expression obtain by described PHA digestive enzyme PhaZ inclusion body.
11. methods according to claim 10, it is characterized in that: the coded sequence of described modulin PhaR gene is SEQ ID No.1, the coded sequence of described pha synthesizing enzyme PhaC gene is SEQ ID No.3, and the coded sequence of described PHA digestive enzyme PhaZ gene is SEQ ID No.5.
12., according to described method arbitrary in claim 9-11, is characterized in that: described oil phase is petrochemical industry oil or edible oil.
13. emulsifying agents, any one product in A1-A4:
Described A1 is the product be made up of modulin PhaR inclusion body, pha synthesizing enzyme PhaC inclusion body and PHA digestive enzyme PhaZ inclusion body;
Described A2 is the product be made up of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body;
Described A3 is the product of described modulin PhaR inclusion body and described PHA digestive enzyme PhaZ inclusion body composition;
Described A4 is the product be made up of modulin PhaR and pha synthesizing enzyme PhaC;
Described modulin PhaR is following a1) or protein a2):
A1) protein of amino acid sequence as shown in SEQ ID No.2;
A2) before the 1st of SEQ ID No.2, add the fusion that 6 histidines obtain;
Described pha synthesizing enzyme PhaC is following b1) or protein b2):
B1) protein of amino acid sequence as shown in SEQ ID No.4;
B2) before the 1st of SEQ ID No.4, add the fusion that 6 histidines obtain;
PHA digestive enzyme PhaZ in described PHA digestive enzyme PhaZ inclusion body is following c1) or protein c2):
C1) protein of amino acid sequence as shown in SEQ ID No.6;
C2) before the 1st of SEQ ID No.6, add the fusion that 6 histidines obtain;
In described A4, the mass ratio of described modulin PhaR and described pha synthesizing enzyme PhaC is 1:1;
In described A1, the mass ratio of described modulin PhaR inclusion body, described pha synthesizing enzyme PhaC inclusion body and described PHA digestive enzyme PhaZ inclusion body is 1:1:1;
In described A2, the mass ratio of described modulin PhaR inclusion body and described pha synthesizing enzyme PhaC inclusion body is 1:1;
The mass ratio of modulin PhaR inclusion body described in described A3 and described PHA digestive enzyme PhaZ inclusion body is 1:1.
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| CN201310126407.4A CN103212319B (en) | 2013-04-12 | 2013-04-12 | Novel use of substance relevant to regulatory protein PhaR |
| PCT/CN2014/000377 WO2014166299A1 (en) | 2013-04-12 | 2014-04-08 | New uses for substances related to regulatory protein phar |
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