CN106749565A

CN106749565A - A kind of emulsified protein and its application

Info

Publication number: CN106749565A
Application number: CN201710008406.8A
Authority: CN
Inventors: 黄志勇; 高小龙; 王兴彪; 董大媛
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2017-01-05
Filing date: 2017-01-05
Publication date: 2017-05-31
Anticipated expiration: 2037-01-05
Also published as: CN106749565B

Abstract

The present invention provides a kind of emulsified protein and its application, and the amino acid sequence of the emulsified protein is SEQ ID NO:1, emulsified protein of the invention is used to dispose high viscosity, the greasy filth of heavy hydrocarbon pollution.The emulsifying agent albumen that the present invention is obtained disposes high viscosity, the greasy filth of heavy hydrocarbon pollution has good de-oiling effect; the oil content in greasy filth can be made to be reduced to present 0.59% by original 29.1%; oil content reduction by more than 97.5%; cost-saved more than 35% compared with physics, chemical method, the potentiality of great economic benefit and scale application are shown while cost-effective, environmental protection.

Description

A kind of emulsified protein and its application

Technical field

The invention belongs to bioactive molecule triage techniques field, and in particular to a kind of emulsified protein and its application.

Background technology

Biological emulsifier is a kind of large biological molecule chemical combination that important physiology and metabolic activity are shown in life process Thing；Meanwhile, there are some researches show protein, ratio changes very greatly in emulsifying agent, but its content is no notable with emulsifying activity Relation, hydrophobic amino acid sequence is used as the avtive spot for playing emulsification, the position of hydrophobic amino acid, number in protein Mesh etc. plays very big effect to emulsifying activity, it is clear that, emulsifying activity has connection closely with the molecular structure of emulsifying agent System's particularly protein structure switch all has significant effect to emulsifying activity.Protein passes through ester bond, amido link or glycosidic bond The sugared structure of one end connection, other end connection lipid, so as to form the macromolecular compound of stabilization, and can reduce interface Power, liquid is dispersed in another not miscible liquid with drops and forms uniform and stable dispersion, wherein albumen Matter played an important role during this, in addition in the research of biological emulsifier Emulsan it has also been found that, it with The esterase that a molecular weight in Acinetobactercatcoaceticus RAG-1 is about 34.5KDa interacts, can pole The earth improves emulsifying activity.The apoemulsan formed after Emulsan deproteinizeds is hydrolyzed with Proteinase K and does not have emulsifying activity； Esterase is added into apoemulsan, its emulsifying activity can be recovered again.Therefore, screening and optimizing emulsifying agent albumen particularly emulsifying agent egg The purifying and its structural research of white matter component, the chemistry/space structure to studying emulsifying agent albumen, illustrate intermolecular phase interaction With the active function mechanism for disclosing biomolecule plays vital effect；To in the neck such as water-oil separating, oily sludge de-oiling The application in domain also has huge directive significance.

Greasy filth is also oily sludge, is the environomental pollution source of the most serious produced in Petroleum Production, transport, process One of, including various poisonous and harmfuls, difficult for biological degradation material；In addition, petroleum industry produces 15000000 tons of sludge per year, this is to ecology Environment causes serious harm.So, 1998 Nian Yuan State Environmental Protection Administration list oily sludge in《National Hazard waste name Record》, national environmental protection portion is made that corresponding regulation to hazardous waste respectively again within 2011,2015；Meanwhile, new revision in 2015 《Law on Environmental Protection》Most severe requirement is made that to it, this to not perfect enough related Treatment process and measure oil exploitation and The development of processing enterprise brings unprecedented pressure.

In existing recovery technique and petroleum recovery technique, physics, chemical remediation have certain limitation, it is difficult to Required up to being fully achieved to repair, and recovery process has the problems such as efficiency is slow, and secondary pollution is serious.With chemistry, peripheral doses Compare, biological method environmental protection, input-output ratio is high, the influence very little caused to human and environment, and with low cost, no Secondary pollution and the advantage such as easy to operate can be caused.

Therefore, in the urgent need to screening and optimizing emulsifying agent albumen is processed with to greasy filth.

The content of the invention

It is an object of the invention to provide a kind of emulsified protein and its application, the emulsified protein can be for disposal high viscosity, weight The greasy filth of matter hydrocarbon contamination has good de-oiling effect, so as to make up the deficiencies in the prior art.

Present invention firstly provides a kind of emulsified protein, it includes：

1) amino acid sequence is SEQ ID NO:1 albumen,

2) replace on amino acid sequence 1), lack, adding one or several amino acid, the albumen as derived from 1)；

Another aspect of the present invention provides a kind of gene for encoding above-mentioned emulsified protein, and one kind nucleotide sequence is SEQ ID NO:2；

The present invention also provides a kind of recombinant expression carrier；The recombinant expression carrier carries above-mentioned encoding gene；

Another aspect of the invention also provides a kind of recombinant cell, for recombinantly expressing above-mentioned emulsified protein；

Another aspect of the present invention provides the purposes of above-mentioned emulsified protein, is for disposing high viscosity, heavy hydrocarbon pollution Greasy filth；

The emulsifying agent albumen that the present invention is obtained disposes high viscosity, the greasy filth of heavy hydrocarbon pollution, and there is good de-oiling to imitate Really, the oil content in greasy filth can be made to be reduced to present 0.59% by original 29.1%, oil content reduces by more than 97.5%, Cost-saved more than 35% compared with physics, chemical method, shown while cost-effective, environmental protection great Economic benefit and the potentiality of scale application.

Brief description of the drawings

Fig. 1：The emulsifying agent of different proportion and the mixed emulsifying activity of atoleine (E24)

Fig. 2：Difference (NH₄)₂SO₄The emulsifying activity (E24) of the emulsifying agent prepared under the conditions of concentration gradient.

Fig. 3：The emulsifying activity (E24) of the emulsifying agent albumen prepared under different temperatures gradient condition.

Fig. 4：The emulsifying activity (E24) of the emulsifying agent albumen prepared using anion effect chromatography.

Fig. 5：The purity of the emulsifying agent albumen prepared using gel permeation chromatography.

Fig. 6：The purity and emulsifying activity of the emulsifying agent albumen of expression.

Specific embodiment

Emulsified protein of the invention is described in detail with reference to embodiment.

Embodiment 1：The screening and sequence analysis of emulsified protein

Applicant's screening obtains a bacillus licheniformis and belongs to XS2-450 bacterium (Geobacillus pallidus), grinds Study carefully and find that its tunning has emulsifying activity.The preserving number of the bacterial strain is CGMCC No.9430, is protected on 07 09th, 2014 Hide the Chinese microorganism strain preservation positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Administration committee's common micro-organisms center.

Purifying obtains the albumen with emulsification of the invention from the metabolite of the bacterial strain.

1) preparation of crude extract

60 DEG C of active bacterial strain Geobacillus sp.XS2-450 shaking tables, 180rpm shaken cultivations 10h are used as fermentation Seed liquor, inoculum concentration 10% (v/v), is inoculated into fermentation medium, and final concentration of 0.1% palm is added when 12h is fermented Used as the derivant for producing high-activity biological emulsifying agent, galactopoiesis agent zymotic fluid incubation time is 24h, the zymotic fluid that will be obtained to oil 30min is centrifuged at 4 DEG C, under conditions of 5000rpm and removes thalline, the supernatant for obtaining is the crude extract of emulsifying agent.Crude extract enters Row is concentrated into former supernatant to 1/5 volume, is preserved under the conditions of 4 DEG C.

Emulsifying activity to crude extract detects that emulsifying activity (E24) assay method of emulsifying agent is that emulsifying agent is thick Extract is with atoleine respectively with 3:1、3:2、1:1、3:4、3:5、1:2 ratio mixing, be vortexed concussion 3min, stands 24h, breast Change activity=emulsification layer height/liquid surface total height x100%.

Result is as shown in figure 1, determine emulsifying agent with atoleine with 3:4(v:V) it is the emulsification of emulsifying agent when ratio mixes Activity determination method, now its emulsifying activity highest, can reach 75.0%.

2) saltout treatment

Take 20ml steps 1) the emulsifying agent crude extract for preparing add ammonium sulfate it is respectively reached 10%, 20%, 30%th, 40%, 50%, 60%, 70%, 80% saturation degree, is stirred at room temperature 1h, places 4h under the conditions of 4 DEG C afterwards, will The ammonium sulfate of different saturation 30min is centrifuged at 4 DEG C, under conditions of 10000rpm and removes precipitation, and measure contains emulsifying agent The emulsifying activity of supernatant.

Result as shown in Fig. 2 with other concentration conditions under (NH₄)₂SO₄The saltout supernatant of acquisition of gradient is compared, Under conditions of 40% ammonium sulfate is saltoutd, the emulsifying activity of its supernatant reaches highest, (E24=79.8%)；So, select Saturation degree is that 40% ammonium sulfate carries out the preparation of emulsifying agent, and the sample that will be obtained is used for during subsequent experimental.

3) it is heat-treated

The optimum growth temperature of active bacterial strain Geobacillus sp.XS2-450 is 60 DEG C and its biological emulsification for producing Agent can also be maintained at 50% or so at 100 DEG C；Set accordingly different thermograde (60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 100 DEG C) treatment 30min, 30min being centrifuged at 4 DEG C, under conditions of 10000rpm and removes precipitation, the supernatant of acquisition is system of the present invention Standby emulsifying agent.Determine the emulsifying activity of contained emulsifying agent in supernatant.

Result as shown in figure 3, after Temperature Treatment emulsifying agent activity at 80 DEG C, its activity (E24=under the conditions of 30min 83.2%)；So, it is 80 DEG C from temperature, 30min is processed as the preparation method of emulsifying agent.

4) anion-exchange chromatography

3) sample that will be obtained in, adds bag filter (8K-14K) to be dialysed in 50mmol/L Tris-HCl buffer solutions At night, afterwards with 0.22 μm of membrane filtration, the filtrate regulation pH8.0 that will be obtained enters to advance from anion-exchange chromatography to it The purifying of one step, purification condition is 50mmol/L Tris-HCl sample-loading buffers, 1mol/L NaCl elution buffers, pillar type Number HiTrapQFF 7/25, loading speed 1.5ml/min, press 0.5MPa, filling pressure 0.3MPa before post, determine anion chromatography The emulsifying activity of contained emulsifying agent in each resulting component.

Three components are obtained after by anion-exchange chromatography, the activity of each component is as shown in figure 4, component 1 Activity be up to E24=83.5%, component one is dialysed desalination and carries out freeze-drying concentration, 4 DEG C of preservations, treats further Research.

5) gel permeation chromatography

By step 4) in the sample that obtains, carry out gel permeation chromatography (50mmol/L Tris-HCl) sample-loading buffer, 1mol/L NaCl elution buffers, loading speed 1.0ml/min presses 0.5MPa, filling pressure 0.15MPa before post.Determine gel In each component obtained by filtration chromatography in the emulsifying activity and emulsifying agent of contained emulsifying agent protein component purity.

Result as shown in figure 5, in component 2 only one of which main band, its E24=84.1%, stripe size is 60K Da；The protein band of correlation, its E24=0 are not detected in component 3；Can speculate that component 2 is emulsified protein accordingly.

6) the mass spectrum sequencing of emulsifying agent albumen.

The single protein bands of 60K Da with emulsifying activity are cut into glue respectively, and film dosim are carried out to protein band, All peptide fragments of albumen are extracted, the sample that will be extracted carries out tandem mass spectrum sequencing, the amino acid sequence for obtaining albumen is SEQ ID NO:1, a kind of its nucleotides sequence of encoding gene is classified as SEQ ID NO:2；

Embodiment 2

This example demonstrates that molecular size range is 60K Da recombinant expression of proteins and active verification method.

1. the structure of recombinant plasmid vector

(1) amplification of genes of interest

Design of primers is carried out using the corresponding nucleotide sequence of 60K albumen and Primer Premier 5.0 in embodiment 3

Enter performing PCR amplification by template of active bacterial strain Geobacillus sp.XS2-450 genomes.

PCR reaction systems：

Reaction condition is：

95℃15min(95℃30s,60℃30s,72℃2min)·32cycles；72℃5min.By pcr amplification product Carry out post recovery.

(2) digestion of genes of interest and pET21a+ carriers

The fragment for reclaiming is carried out respectively with pET21a+ carriers with fastdigestNdeI and fastdigestHindIII Double digestion.

Digestion system and condition：

37 DEG C of water-bath 30min.

(3) connection of genes of interest and pET21a+ carriers

Linked system and condition：

16 DEG C, 3h is reacted, obtain being connected with the recombinant plasmid vector of genes of interest fragment.

The conversion (competent cell of commercialization) of 2.DH5 α competent cells

1. the DH5 α competent cells for taking 50 μ L are placed in ice bath, after DH5 α competent cells dissolve, are experienced to DH5 α The recombinant vector containing genes of interest is added in state cell suspension, uniform, ice bath 30min is gently blown and beaten with pipettor.

2., be transferred to centrifuge tube in ice bath rapidly by 42 DEG C of thermal shock 45s, and 2~3min is stood on ice.

3. the aseptic LB culture mediums of 450 μ L are added in each centrifuge tube, 37 DEG C of shaking tables, 150rpm concussion trainings is placed in after mixing Foster 45min makes thalline recover.

4. the μ L of DH5 α competent cells 200 for having converted are taken, are added in the LB solid agar mediums of the antibiotic of benzyl containing ammonia, It is with aseptic spreading rod that cell is uniformly spreadable, flat board is placed in 37 DEG C up to liquid is absorbed, culture 12h is inverted, obtain Dan Ke It is grand.

5. the monoclonal that will be obtained carries out bacterium colony PCR checkings, plasmid extraction, sequence verification, the restructuring of conversion of succeeding Plasmid.

3. the induced expression in Escherichia coli and purifying

(1) induced expression

1. the bacterial strain E.coliBL21 (DE3) containing recombinant plasmid and pET-21a (+) empty plasmid (comparing) is taken respectively, Be inoculated in the LB culture mediums containing ammonia benzyl antibiotic of 5ml, 37 DEG C of shaking table concussion and cultivates overnight, to carry out the activation of bacterial strain.

2. the thalline of activation is transferred in the LB culture mediums that 50ml contains above-mentioned antibiotic with 1% inoculum concentration, 37 DEG C Shaking table culture about 1h, after OD to 0.6~0.8, adds the 1M IPTG (final concentration of 1mM) of 50 μ L, induced expression is carried out, 20 DEG C low temperature induction 12h.

(2) purifying of expression product

1. by after thalline ice bath 30min, 4 DEG C of centrifugation 10min of 5000rpm outwell supernatant；

2. with PBS (pH7.4) buffer solution re-suspended cell of 30ml, 4 DEG C of centrifugation 10min of 5000rpm outwell supernatant；

3. with PBS (pH7.4) re-suspended cell of 3ml, and re-suspension liquid is transferred in the centrifuge tube of 5ml；

4. broken wall is carried out to cell with Ultrasonic Cell Disruptor.Condition：Power 30%, ultrasonic 2ms stops 4ms, broken wall 10min；

5. by 4 DEG C of centrifugation 30min of cell pyrolysis liquid 13000rpm, supernatant is transferred in clean 5ml centrifuge tubes.

6. 0.6ml supernatants are taken to be added in His Trap columns by several times, target protein is combined with chromatography media, 4 DEG C of 100rcf 30s/ times.

7. rinsed with PBS (pH7.4) first and be not associated with the foreign protein on pillar, be repeated 10 times, then with 20,40, The imidazole buffer of 60mM is eluted to albumen respectively, is repeated 1 times, and eluent is collected respectively.

8. the eluent containing target protein is respectively placed in Millipore super filter tubes, adds PBS+5% glycerine to volume Determine volume, uniform, 4 DEG C of centrifugation 10min of 2000rpm are blown and beaten with pipettor, abandon waste liquid, repeat this step 2~3 time.

9. the protein solution after concentration is transferred in the 1.5mlEP pipes of clean and precooling respectively, -80 DEG C of preservations.

4. the emulsifying activity of target protein and purity are verified

Emulsifying activity is carried out according to emulsifying activity assay method in embodiment 1 to the target protein that embodiment 2 is obtained to test Card, purity checking is carried out using SDS-PAGE.

Result such as Fig. 6 (' 1, the 2,3 ' albumen for getting off to application 20,40,60mM buffer solution elutions respectively in figure), wherein using During the buffer solution eluted protein of 20mM, the purity of its target protein reaches ideal effect；And show good emulsifying activity, E24=84.9%.

Embodiment 3 is this example demonstrates that application of the emulsifying agent albumen in terms of greasy filth disposal.

It is of the invention main using the emulsifying agent albumen prepared in embodiment 2 treatment high viscosity, the greasy filth of heavy hydrocarbon pollution.

It is set to homogenize greasy filth stirring, allotment greasy filth, the ratio of emulsifying agent albumen reach 4:3, add 10 times The water of greasy filth, is sufficiently stirred under the conditions of 60 DEG C, makes the crude oil in its emulsification greasy filth, stands, and the dregs of fat of sedimentation carry out depth and take off Oil, the remaining glutinous composition high of removing, so as to the purpose that the oil reached in greasy filth with residue separate, the upper strata crude oil that will finally emulsify is complete Reclaimed in portion.Greasy filth oil content is monitored in processing procedure, the oil content in greasy filth is by 29.1% original reduction Present 0.59% is arrived, oil content reduction by more than 97.5% meets greasy filth disposal standard；With physics, chemical treatment method phase Than cost-saved more than 35%, it can be seen that it has very big application value.

SEQUENCE LISTING

<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences

<120>A kind of emulsified protein and its application

<130>

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<170> PatentIn version 3.5

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Claims

1. a kind of albumen, it is characterised in that described albumen includes：

1) amino acid sequence is SEQ ID NO:1 albumen,

2) replace on amino acid sequence 1), lack, adding one or several amino acid, the albumen as derived from 1).

2. a kind of gene, it is characterised in that the albumen described in described gene code claim 1.

3. gene as claimed in claim 2, it is characterised in that the nucleotides sequence of the gene is classified as SEQ ID NO:2.

4. a kind of recombinant expression carrier, it is characterised in that the described recombinant expression carrier carries the base described in claim 2 Cause.

5. a kind of recombinant host cell, it is characterised in that described recombinant host cell is converted/transfected described in claim 4 Recombinant expression carrier.

6. application of the albumen described in claim 1 in emulsifying agent is prepared.

7. a kind of emulsifying agent, it is characterised in that described emulsifying agent includes the albumen described in claim 1.

8. application of the albumen described in claim 1 in disposal high viscosity, the greasy filth of heavy hydrocarbon pollution.