CN107988195A - A kind of method for significantly improving linoleate isomerase amount of soluble expression in recombination bacillus coli - Google Patents

A kind of method for significantly improving linoleate isomerase amount of soluble expression in recombination bacillus coli Download PDF

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CN107988195A
CN107988195A CN201711170917.6A CN201711170917A CN107988195A CN 107988195 A CN107988195 A CN 107988195A CN 201711170917 A CN201711170917 A CN 201711170917A CN 107988195 A CN107988195 A CN 107988195A
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pai
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recombination bacillus
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诸葛斌
黄孟楠
陆信曜
宗红
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Jiangnan University
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    • C12Y502/01005—Linoleate isomerase (5.2.1.5)

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Abstract

The invention discloses a kind of method for significantly improving linoleate isomerase amount of soluble expression in recombination bacillus coli, belong to field of genetic engineering.The present invention is to be used as starting strain using recombination bacillus coli E.coli BL21 (DE3) (pET 22b (+)/pai), by co-expressing molecular chaperone protein, aid in the folding of linoleate isomerase, increase the Soluble expression levels of linoleate isomerase, so as to increase the method for linoleate isomerase enzyme activity.Finally preferably go out chaperone systems GroEL GroES, the enzyme activity of linoleate isomerase is improved 56%.

Description

One kind significantly improves linoleate isomerase amount of soluble expression in recombination bacillus coli Method
Technical field
The present invention relates to a kind of method for significantly improving linoleate isomerase amount of soluble expression in recombination bacillus coli, Belong to field of genetic engineering.
Technical background
Conjugated linoleic acid is as a kind of newfound nutrient, at present in American-European healthy food circle, almost into Prevent the cure-all medicine of modern civilization diseases, from anticancer to prevention of cardiovascular disease, diabetes, onto body weight control, almost live In the indispensable healthy food of 21st century modern.At present, conjugated linoleic acid is mainly by being chemically synthesized, no Only cause serious chemical pollution, and synthesis obtain be various structures conjugated linoleic acid mixture, downstream separation into This is very high.Recent years, the focus of research be gradually transferred to synthesized using bioanalysis in single-minded conjugated linoleic acid come.
At present, it is by from the Asia oil of propionibacterium acnes using the method for bioanalysis synthesis specificity conjugated linoleic acid Acid isomer enzyme heterogenous expression in other hosts, then with the direct catalytic production conjugated linoleic acid of recombinant bacterium or extract intracellular expression Enzyme, with the production of enzymatic conjugated linoleic acid.Wherein engineering bacteria is built as host produce linoleate isomerase using Escherichia coli When, restructuring linoleate isomerase mainly exists in the form of inclusion body, and the soluble recombinant protein with enzymatic activity is seldom, seriously The process using enzyme law catalysis production conjugated linoleic acid is limited, improves restructuring linoleate isomerase amount of soluble expression and enzyme activity Power becomes the key using biotransformation method production conjugated linoleic acid.
Molecular chaperones be it is a kind of can assist in other oroteins correctly fold, assemble, transhipment, degraded it is important because Son, they help the structure of other oroteins to complete the formation of correct conformation in the cell, and divide therewith after functioning From not forming the components of these protein perform functions.At present, there are three kinds of main molecular chaperones systems in Escherichia coli： DnaK-DnaJ-GrpE, GroEL-GroES and TF, they play separate and complementary work during protein folding With optimal folding effect can be obtained in experimentation by the combination of different molecular companion.5 are compared in this patent Influence of kind different chaperone combined systems to linoleate isomerase amount of soluble expression, when being introduced separately into partner system The enzyme activity of linoleate isomerase significantly improves during GroES-GroEL.
The content of the invention
It is with E.coli BL21 present invention firstly provides the recombination bacillus coli of five kinds of coexpression molecular chaperone proteins (DE3) (pET-22b (+)/pai) is starting strain, be transferred to respectively the plasmid pG-KJE8 containing molecular chaperone protein gene, PGro7, pTf16, pKJE7, pG-Tf2, built-up recombination bacillus coli E.coli BL21 (DE3) (pET-22b (+)/ Pai) (pG-KJE8), E.coli BL21 (DE3) (pET-22b (+)/pai) (pGro7), E.coli BL21 (DE3) (pET- 22b (+)/pai) (pTf16), E.coli BL21 (DE3) (pET-22b (+)/pai) (pKJE7), E.coli BL21 (DE3) (pET-22b(+)/pai)(pG-Tf2)。
Wherein, the restructuring PAI expression bacterial strain E.coli BL21 (DE3) (pET-22b (+)/pai), are with E.coli BL21 (DE3) is host, with the expression of T7 promoter regulations PAI.Specific construction method：Using propionibacterium acnes genome as mould Plate, designs primer according to pai gene orders and adds upper Nde I and Hind III digestions site, PCR respectively at 5 ' ends and 3 ' ends Amplify and carry out pai genes, pai genes and expression plasmid pET-22b (+) while through Nde I and Hind III double digestions, connection Overnight, pET-22b (+)/pai recombinant plasmids are become, Transformed E .coli BL21 (DE3) competent cell, is configured to recombinate PAI expression bacterial strain E.coli BL21 (DE3) (pET-22b (+)/pai).
Plasmid pG-KJE8, pGro7, pTf16, pKJE7, pG-Tf2 of the present invention contain different molecular chaperone base Cause.Wherein, pG-KJE8 contains DnaK-DnaJ-GrpE and GroES-GroEL encoding genes, with promoterControl GroES Expressed with GroEL, with the expression of promoter ParaB controls chaperones protein DnaK, DnaJ and GrpE；PG-Tf2 carries Trigger Factor and GroES-GroEL, by promoterControl table reaches；And plasmid pGro7, pTf16, pKJE7 are then respectively provided with GroES-GroEL, Trigger factor, DnaK-DnaJ-GrpE albumen, are reached by promoter ParaB control tables.
The DnaK encoding genes such as NCBI-Gene ID:Shown in 944750, DnaJ encoding genes such as NCBI-Gene ID: Shown in 944753, GrpE genes such as NCBI-Gene ID:Shown in 947097.The GroES encoding genes such as NCBI-Gene ID:Shown in 948655, GroEL encoding genes such as NCBI-Gene ID:Shown in 948665, tig genes such as NCBI-Gene ID: Shown in 945081.
Molecular chaperone protein amino acid sequence is as follows：
GroES：
MNIRPLHDRVIVKRKEVETKSAGGIVLTGSAAAKSTRGEVLAVGNGRILENGEVKPLDVKVGDI VIFNDGYGVKSEKIDNEEVLIMSESDILAIVEA
GroEL：
MAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREIEL EDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVAAGMNPMDLKRGIDKAVTAAVE ELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEAMDKVGKEGVITVEDGTGLQDELDVVEGMQFDRG YLSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAVAKAGKPLLIIAEDVEGEALATLVVNTMRGI VKVAAVKAPGFGDRRKAMLQDIATLTGGTVISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEA AIQGRVAQIRQQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVE EGVVAGGGVALIRVASKLADLRGQNEDQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGNY GYNAATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKNDAADLGAAGGMGGMGG MGGMM
DnaK：
MGKIIGIDLGTTNSCVAIMDGTTPRVLENAEGDRTTPSIIAYTQDGETLVGQPAKRQAVTNPQNT LFAIKRLIGRRFQDEEVQRDVSIMPFKIIAADNGDAWVEVKGQKMAPPQISEVLKKMKKTAEDYLGEPVT EAVITVPAYFNDAQRQATKDAGRIAGLEVKRIINEPTAAALAYGLDKGTGNRTIAVYDLGGGTFDISIIEID EVDGEKTFEVLATNGDTHLGGEDFDSRLINYLVEEFKKDQGIDLRNDPLAMQRLKEAAEKAKIELSSAQT DVNLPYITADATGPKHMNIKVTRAKLESLVEDVNRSIEPLKVALQDAGLSVSDIDDVILVGGQTRMPMQ KKVAEFFGKEPRKDVNPDEAVAIGAAVQGGVLTGDVKDVLLLDVTPLSLGIETMGGVMTTLIAKNTTIP TKHSQVFSTAEDNQSAVTIHVLQGERKRAADNKSLGQFNLDGINPAPRMPQIEVTFDIDADGILHVSAKD KNSGKEQKITIKASSGLNEDEIQKMVRDAEANAEADRKFEELVQTRNQGDHLLHSTRKQVEEAGDKLPA DDKTAIESALTALETALKGEDKAAIEAKMQELAQVSQKLMEIAQQQHAQQQTAGADASANNAKDDDV VDAFEEVKDKK
DnaJ：
MAKQDYYEILGVSKTAEEREIRKAYKRLAMKYHPDRNQGDKEAEAKFKEIKEAYEVLTDSQKR AAYDQYGHAAFEQGGMGGGGFGGGADFSDIFGDVFGDIFGGGRGRQRAARGADLRYNMELTLEEAVR GVTKEIRIPTLEECDVCHGSGAKPGTQPQTCPTCHGSGQVQMRQGFFAVQQTCPHCQGRGTLIKDPCNK CHGHGRVERSKTLSVKIPAGVDTGDRIRLAGEGEAGEHGAPAGDLYVQVQVKQHPIFEREGNNLYCEVP INFAMAALGGEIEVPTLDGRVKLKVPGETQTGKLFRMRGKGVKSVRGGAQGDLLCRVVVETPVGLNER QKQLLQELQESFGGPTGEHNSPRSKSFFDGVKKFFDDLTR
GrpE：
MSSKEQKTPEGQAPEEIIMDQHEEIEAVEPEASAEQVDPRDEKVANLEAQLAEAQTRERDGILRV KAEMENLRRRTELDIEKAHKFALEKFINELLPVIDSDRALEVADKANPDMSAMVEGIELTLKSMLDVVR KFGVEVIAETNVPLDPNVHQAIAMVESDDVAPGNVLGIMQKGYTLNGRTIRAAMVTVAKAKA
Trigger factor：
MQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQ DVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDA DVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIK GHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKN MERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKR RVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVL AKAKVTEKETTFNELMNQQA
Present invention also offers the method for the application recombination bacillus coli fermenting and producing PAI, will be trained under 37 DEG C, 200rpm The recombination bacillus coli for supporting 12h is transferred to fermentation medium with 2% inoculum concentration, while adds final concentration of 1.5mg/ as needed The L-arabinose of mL, the tetracycline of final concentration of 10ng/mL, cultivates under 37 DEG C, 200rpm and arrives OD600=1.5；Addition IPTG to final concentration of 0.1mmol/L, reduces the temperature to 20 DEG C of induced expression 20h.
Recombination bacillus coli seed culture and fermentation：
Seed culture medium (g/L)：Tryptone 10, dusty yeast 5, NaCl 10.
Fermentation medium (g/L)：Tryptone 12, dusty yeast 24, glycerine 5, KH2PO42.31 K2HPO4 16.8。
Condition of culture：The seed that 12h is cultivated under 37 DEG C, 200rpm is transferred to fermentation medium with 2% inoculum concentration, in 20 DEG C, cultivate 20h under the conditions of 200rpm.
The present invention is using recombination bacillus coli E.coli BL21 (DE3) (pET-22b (+)/pai) as starting strain, is led to The folding of coexpression molecular chaperone protein auxiliary restructuring PAI is crossed, so that promote more PAI to become the enzyme with correct conformation, Inclusion body content is reduced, improves the enzymatic activity of restructuring PAI.During shake flask fermentation, E.coli BL21 (DE3) (pET-22b (+)/pai) (pGro7) brought up to compared with E.coli BL21 (DE3) (pET-22b (+)/pai) enzymatic activity by 0.251U/mg 0.337U/mg improves 34% (Fig. 1).
The present invention further illustrates shadow of the expression to PAI amount of soluble expression of chaperone GroES-GroEL Ring.By adding L-arabinose to final concentration of 0mg/mL, 1.5mg/mL, 3.0mg/mL, 4.0mg/mL control chaperone The expression quantity of GroEL-GroES, the amount of soluble expression of PAI further improve, in the final concentration of 4.0mg/mL of L-arabinose When, exist almost without insoluble restructuring PAI, E.coli BL21 (DE3) (pET-22b (+)/pai) (pGro7) are compared with E.coli BL21 (DE3) (pET-22b (+)/pai) enzymatic activitys bring up to 0.391 U/mg by 0.251U/mg and improve 56% (Fig. 2).This Invent and lay a good foundation to convert production conjugated linoleic acid using bioanalysis.Recombination bacillus coli construction method provided by the invention Simply, it is easy to use, there is good application prospect.
Brief description of the drawings
Fig. 1 is the expression that PAI is recombinated in 5 kinds of molecular chaperones coexpression systems of SDS-PAGE electrophoretic analysis.Wherein, A, Intracellular soluble part；B, the insoluble part of intracellular.Swimming lane M, standard protein molecular weight；Swimming lane 1, pET-22b (+)/pai； Swimming lane 2, pET-22b (+)/pai (pG-KJE8)；Swimming lane 3, pET-22b (+)/pai (pGro7)；Swimming lane 4, pET-22b (+)/ pai(pTf16)；Swimming lane 5, pET-22b (+)/pai (pKJE7)；Swimming lane 6, pET-22b (+)/pai (pG-Tf2).Remarks：Include It is specific enzyme activity in number；Chaperone GroES (10kDa) molecular weight is too small to fail to detect in figure.
Fig. 2 is shadow of the SDS-PAGE electrophoretic analysis difference GroEL-GroES expression quantity to restructuring PAI amount of soluble expression Ring.Wherein, A, intracellular soluble part；B, the insoluble part of intracellular.Swimming lane M, standard protein molecular weight；Swimming lane 1,2,3,4, PET-22b (+)/pai (pGro7) adds the final concentration of 0, L-arabinose of 1.5mg/mL, 3.0mg/mL, 4.0mg/mL and lures Lead.Remarks：It is specific enzyme activity in bracket；Chaperone GroES (10kDa) molecular weight is too small to fail to detect in figure.
Embodiment
Example 1
From picking single bacterium colony on the fresh plate containing E.coli BL21 (DE3) (pET-22b (+)/pai), to final concentration For in the ampicillin LB culture mediums of 100 μ g/ml, 37 DEG C, 200rpm cultivate OD600During=0.4-0.6, taken out from shaking table, ice Centrifuged after bath 30min.With the CaCl of 0.1mol/L2Washing thalline 3 times.By pG-KJE8, pGro7, pTf16, pKJE7 and pG- Tf2 plasmids are added in competent cell, 42 DEG C of water-bath thermal shock 90s after ice bath 30min, add the LB culture mediums of 1ml, 37 DEG C Corresponding resistant panel is coated with after culture 1h, the correct transformant of picking after it grows, obtains the restructuring large intestine containing corresponding plasmid Bar E.coli BL21 (DE3) (pET-22b (+)/pai) (pG-KJE8), E.coli BL21 (DE3) (pET-22b (+)/pai) (pGro7), E.coli BL21 (DE3) (pET-22b (+)/pai) (pTf16), E.coli BL21 (DE3) (pET-22b (+)/ Pai) (pKJE7), E.coli BL21 (DE3) (pET-22b (+)/pai) (pG-Tf2).
Example 2
The single bacterium of picking difference recombinant bacterium falls within the LB culture mediums containing corresponding antibiotic from tablet, makees after being incubated overnight For seed liquor, it is inoculated in 2% inoculum concentration in the fermentation medium of 50mL, while it is Arabic to add final concentration of 1.5mg/ml Sugar or the expression of 10ng/ml tetracycline inducing molecules chaperone.Treat culture to OD600IPTG is added when=1.5 to final concentration of 0.1mmol, reduces the temperature to 20 DEG C, 20h is cultivated under 200rpm, collects somatic cells ultrasonication detection enzyme activity.
Example 3
The single bacterium of picking recombinant bacterium E.coli BL21 (DE3) (pET-22b (+)/pai) (pGro7), which is fallen within, from tablet contains There are the LB culture mediums of ampicillin and chloramphenicol, seed liquor is used as after being incubated overnight, be inoculated in 50mL's with 2% inoculum concentration In fermentation medium, while add the L-arabinose induction companion of final concentration of 0,1.5mg/ml, 3.0mg/ml, 4.0mg/ml Protein G roEL-GroES is expressed.Treat culture to OD600IPTG is added when=1.5 to final concentration of 0.1mmol, is reduced the temperature to 20 DEG C, 20h is cultivated under 200rpm, collect somatic cells ultrasonication detection enzyme activity.
Example 4
Escherichia coli wall-breaking method, adjustment cell concentration to 3mLOD600=30, by bacterium solution in 4 DEG C, 10000rpm is centrifuged, Collect somatic cells and washed three times with PBS buffer, recombinant Bacillus coli cells are resuspended in PBS buffer, 400W Ultrasonication 10min, smudge cells.
Example 5
Using the enzyme activity of spectrophotometry PAI.The PAI enzyme activity of 1 unit is defined as：Catalysis bottom per minute at 25 DEG C Thing linoleic acid forms the enzyme amount needed for 1 μm of ol conjugated linoleic acid (HPOD, ε=17107.3L/ (mol × cm)).Enzyme activity determination bar Part：Using linoleic acid as substrate, light absorption value under Shimadzu UV-2450 spectrophotometer on-line determinations 234nm is utilized at 25 DEG C Change, enzyme activity is calculated with the slope of light absorption value change curve initial part.
Table 1 is the enzyme activity of the recombination bacillus coli PAI containing different molecular chaperone.
Table 2 is the E.coli BL21's (DE3) (pET-22b (+)/pai) (pGro7) under the induction of different L-arabinoses PAI enzyme activity.
Although the present invention is disclosed as above with preferred embodiments, it is not limited to the present invention, any to be familiar with this technology People, without departing from the spirit and scope of the present invention, can all do various change and modification, therefore protection scope of the present invention It should be subject to what claims were defined.

Claims (7)

  1. A kind of 1. method for significantly improving linoleate isomerase amount of soluble expression in recombination bacillus coli, it is characterised in that With recombination bacillus coli E.coli BL21 (DE3) (pET-22b (+)/pai) for starting strain, importing contains molecular chaperone protein Plasmid pG-KJE8 (dnaK-dnaJ-grpE/groES-groEL), pGro7 (groES-groEL), pTf1 (tig), pKJE7 (dnaK-dnaJ-grpE) and pG-Tf2 (groES-groEL/tig).
  2. 2. recombination bacillus coli according to claim 1, it is characterised in that with recombination bacillus coli E.coli BL21 (DE3) (pET-22b (+)/pai) is for host, with promoter with E.coli BL21 (DE3) as PAI production starting strains T7 regulates and controls the expression of PAI.
  3. 3. the method for the application claim 1 or 2 recombination bacillus coli fermentation production PAI, it is characterised in that large intestine bar will be recombinated 12h is cultivated under the conditions of 37 DEG C of bacterium, 200rpm, recombinant bacterial strain is transferred to fermentation medium with 2% inoculum concentration, while as needed Add the tetracycline inducing molecule companion expression of the L-arabinose and 10ng/mL of final concentration of 1.5mg/mL, 37 DEG C, 200rpm Under the conditions of culture to OD600IPTG to final concentration of 0.1mmol/L is added when=1.5,20h inductions are cultivated under 20 DEG C, 200rpm Express PAI.
  4. 4. the method described in application claim 3, it is characterised in that the fermentation medium contains：Peptone 12g/L, yeast Powder 24g/L, glycerine 5g/L, KH2PO4 2.31g/L、K2HPO4 16.8g/L。
  5. 5. the method culture recombination bacillus coli fermentation production PAI described in application claim 3, improves restructuring PAI in Escherichia coli The most notable chaperone system of amount of soluble expression is GroEL-GroES.
  6. 6. the method culture recombination bacillus coli described in application claim 3, the expression quantity of control chaperone GroEL-GroES Further improve the amount of soluble expression of PAI in E.coli BL21 (DE3) (pET-22b (+)/pai) (pGro7), addition L- Ah Draw uncle's sugar final concentration of 0mg/mL, 1.5mg/mL, 3.0mg/mL, 4.0mg/mL.
  7. 7. application claim 6 described in method, E.coli BL21 (DE3) (pET-22b (+)/pai) (pGro7) L- I The amount of soluble expression highest of PAI is recombinated during the uncle final concentration of 4.0mg/mL of sugar.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108660102A (en) * 2018-05-22 2018-10-16 江南大学 A kind of recombination bacillus coli of solubility expression linoleate isomerase and its application
CN110656077A (en) * 2019-11-07 2020-01-07 江南大学 Method for producing sucrose phosphorylase and application thereof

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