CN105255788A - Streptomyces mutabilis trehalose synthase gene and application thereof - Google Patents
Streptomyces mutabilis trehalose synthase gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical fields of genetic engineering and modern enzyme engineering and in particular relates to a trehalose synthase gene from streptomyces mutabilis. Trehalose synthase expressed by the streptomyces mutabilis can obviously improve salt tolerance of a host. Preservation number of the streptomyces mutabilis TRM45540 is CCTCC NO: M2015657. A nucleotide sequence of a trehalose synthase of the streptomyces mutabilis TRM45540 is shown in SEQ ID NO.1. An amino sequence of the trehalose synthase coded by the trehalose synthase gene of the streptomyces mutabilis TRM45540 is shown in SEQ ID NO.2. The invention also relates to an application of the streptomyces mutabilis TRM45540 in the industry.
Description
Technical field
The invention belongs to genetically engineered and modern technical field of enzyme engineering, be specifically related to a kind of trehalose synthesize enzyme gene coming from variable streptomycete, the TreP of this genetic expression can significantly improve the salt resistance ability of host.
Background technology
The research of trehalose in genetically engineered has obtained some achievements: one is the resistance ability utilizing the gene constructed special crop of trehalose to improve crop, two is the structure of utilizing works microorganism and the reasonable utilization of enzyme engineering, improve the mode of production of original trehalose, raising efficiency, reduces costs.Current Many researchers successfully by trehalose channel genes cash crop, makes plant as the bio-reactor of trehalose, and accumulates trehalose in a large number.But the research of the existing genetic engineering bacterium for producing trehalose synthase is little.The direction of many scientists research has been become in recent years by genetic engineering technique cloning and expressing trehalose synthesis involved enzyme, and produce trehalose synthesis involved enzyme by genetic engineering bacterium, and in this, as the catalytic materials of Production by Enzymes trehalose, be the method for the relative efficiency producing trehalose.
Summary of the invention
Technical problem to be solved by this invention has be to provide a kind of new actinomycetes strain that carry trehalose synthesize enzyme gene at three: one, specifically variable streptomycete StreptomycesmutabilisTRM45540; Two nucleotide sequence and the aminoacid sequences being to provide trehalose synthesize enzyme gene in this bacterial strain; Three is heterogenous expression trehalose synthesize enzyme genes, comprises this albumen of purifying, and detects the zymetology feature of TreP; Four is by building genetic engineering bacterium, and checking trehalose synthesize enzyme gene can significantly improve salt resistance ability and the industrial application of Host Strains.
Technical scheme provided by the invention: 1. variable streptomycete (Streptomycesmutabilis) TRM45540, the deposit number of this bacterial strain is CCTCCNO:M2015657.2. variable streptomycete TRM45540 trehalose synthesize enzyme gene, its nucleotide sequence is as shown in SEQIDNO.1.3. the TreP coded by variable streptomycete TRM45540 trehalose synthesize enzyme gene as described in 2, its aminoacid sequence is as shown in SEQIDNO.2.4. the variable streptomycete TRM45540 application in the industry as described in 1.
Beneficial effect: 1, clone obtains the trehalose synthesize enzyme gene in variable streptomycete
(1) growth medium
ISP4 substratum: Zulkovsky starch 10.0gL
-1, (NH
4)
2sO
42.0gL
-1, MgSO
41.0gL
-1, K
2hPO
41.0gL
-1, CaCO
32.0gL
-1, NaCl5gL
-1, FeSO
47H
2o0.001gL
-1, MnCl
27H
2o0.001gL
-1.Cultivate 7d for 28 DEG C.
(2) activate
YEME substratum: Semen Maydis powder 16.0gL
-1, peptone 5.0gL
-1, glucose 20gL
-1, NaCl20gL
-1, CaCO
31.0gL
-1, FeSO
47H
2o0.001gL
-1.Optimal conditions of fermentation is: rotating speed 180rpm, initial pH7.2-7.4, inoculum size 8%, liquid amount 80mL/250mL, leavening temperature 28 DEG C.
(3) operating process
Wait to grow abundant fresh mycelia by cultivating 7d in 28 DEG C of incubators in actinomycetes (variable streptomycete StreptomycesmutabilisTRM45540) inoculation to growth medium.Collect mycelia and make bacteria suspension in 20% glycerine mixing, put-20 DEG C of preservations; Inoculum size according to 1% by bacterial suspension inoculation in the triangular flask that YEME (final concentration 0.5% glycerine) substratum is housed, 28 DEG C of shaking tables are cultivated 36-48h and are obtained fresh bacterium liquid, the STb gene of extracting bacterial strain, the fragment of trehalose synthesize enzyme gene is obtained through pcr amplification, obtain by TA clone the clone p18T-TreA carrying this gene, and send the sequence that clone checks order clear and definite this gene.
2, heterogenous expression TreP
(1) carrier of clone p18T-TreAp18T-TreA is replaced to pET28a
The plasmid DNA of extracting clone p18T-TreA and pET28a, selects BamHI and HindIII two kinds of enzymes to cut 4h to the plasmid DNA of p18T-TreA and pET28a at 37 DEG C of water-bath enzymes.By agarose gel electrophoresis, reclaim corresponding fragment.P18T-TreA reclaims the fragment of 1143bp, and pET28a reclaims the fragment of 5.3kb.Two fragments reclaimed are connected under the effect of T4DNA ligase enzyme, transforms BL21, obtain pET28a-TreA clone
(2) expression and purification TreA albumen
Inoculation pET28a-TreA clone activates in LB substratum, then 1/100 transfer be cultured to logarithmic phase in new LB substratum, add IPTG to induce, continue to cultivate 4h, collect thalline, use His label-Ni column purification TreA albumen, detected the expression of TreA albumen by SDS-PAGE.
(3) enzyme activity determination
Utilize high performance liquid chromatograph (HPLC) to analyze TreP and transform the product analysis after maltose, to be chromatographic column be analysis condition: AlltimaAmino100A5u post (4.6mm × 250mm); Detector: Alltech2000ES type light scattering detector; Moving phase: 70% acetonitrile/30%H2O; Flow velocity: 1mL/min; Column temperature: 35 DEG C.Optimum pH (4,5,6,7,8), optimum temperuture (4 DEG C, 10 DEG C, 28 DEG C, 37 DEG C, 45 DEG C, 60 DEG C), metal ion (Zn that enzyme analysis is lived
2+, Ca
2+, Mg
2+, Mn
2+and Co
2+) on enzyme live impact.
3, by building genetic engineering bacterium, trehalose synthesize enzyme gene can significantly improve the salt resistance ability of Host Strains using pUC19/DH5 α and pET28a/BL21 as negative control, using TRM45540 wild strain as positive control, recombinant bacterial strain and control strain are inoculated on the flat board of 0%, 1%, 3%, 5% respectively, detect the salt tolerance of bacterial strain.
Culture presevation illustrates: strain name: variable streptomycete TRM45540; Classification system: Streptomycesmutabilis; Deposit number: CCTCCNO:M2015657; Preservation mechanism: China typical culture collection center; Address: Wuhan, China Wuhan University, postcode: 430072, phone: (027) 68754052, fax: (027) 68754833, E-mail:cctccwhu.edu.cn; Preservation date: on November 2nd, 2015.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is further illustrated.
The clone of Fig. 1 variable streptomycete TRM45540 trehalose synthesize enzyme gene;
The heterogenous expression of the trehalose synthesize enzyme gene of the variable streptomycete TRM45540 of Fig. 2;
The best concentration of substrate of TreP of Fig. 3: variable streptomycete TRM45540;
The TreP of the variable streptomycete TRM45540 of Fig. 4 transforms the product analysis of maltose;
Fig. 5 pH is on the graphic representation of enzyme impact alive;
Fig. 6 temperature is on the graphic representation of enzyme impact alive;
Fig. 7 metal ion is on the graphic representation of enzyme impact alive;
Fig. 8 carries the salt resistance ability analysis of the recombinant bacterium of trehalose synthase gene.
Embodiment
Detailed description below by embodiment illustrates the present invention further, but is not limitation of the present invention, only does example explanation.
The clone of embodiment 1, variable streptomycete TRM45540 trehalose synthesize enzyme gene
1, the source of variable streptomycete TRM45540 and separation and Culture
From ancient city, Lou Lan, Xinjiang (height above sea level 1400m, 89 ° 22 ' 22, footpath, east ", in north latitude 40 ° 29 ' 55 ") 0-30cm pedotheque, adopt spread plate method, with containing 5%, 10%, 15%, 8 kinds of substratum of 20% and 25% sodium-chlor, 28 DEG C of constant temperature culture, isolate 7 actinomycetes 35 strains belonged to altogether, sieved again by flat board face-off method primary dcreening operation and cup-plate method, final Isolation and screening has gone out good antimicrobial effect and has had the biocontrol microorganisms TRM45540 of certain salt resistance ability, by Molecular Identification and the variable streptomycete (Streptomycesmutabilis) of morphological observation called after.
Isolation medium is inorganic salt Starch Agar (ISP4) substratum: starch 10gL
-1, (NH4)
2sO
44gL
-1, MgSO
42gL
-1, K
2hPO
42gL
-1, CaCO
31gL
-1, NaCl40gL
-1, agar 18gL
-1, pH7.2-7.4.
2, the STb gene of bacterial strain TRM45540 is extracted
Inoculation TRM45540 bacterial strain cultivates 7d in 28 DEG C in ISP4 substratum (5%NaCl concentration), go out after abundant spore to be transferred by spore in YEME substratum (5%NaCl concentration) after growth 36-48h until strain growth, collection thalline extracts the STb gene of bacterial strain;
ISP4 culture medium prescription: starch: 10gL
-1, (NH4)
2sO
4: 4gL
-1, MgSO
4: 2gL
-1, K
2hPO
4: 2gL
-1, CaCO
31gL
-1, NaCl40gL
-1, agar: 18gL
-1, pH7.2-7.4;
YEME culture medium prescription: sucrose: 340gL
-1, glucose: 10gL
-1, malt extract: 3gL
-1, bacto peptone: 5gL
-1, yeast extract paste: 3gL
-1, adding distil water to 1L, 115 DEG C of sterilizing 20min;
Extract the method for streptomycete TRM45540 STb gene: collect thalline and put into aseptic EP pipe, add the 1 × TE of 480 μ L, add the N,O-Diacetylmuramidase (50mgmL of 20 μ L
-1).Put into 37 DEG C of shaking tables, 200r/min shaken overnight.Often pipe adds the SDS of 50 μ L20%, adds 5 μ L20mgmL
-1proteinase K, put into 60 DEG C of water-bath incubation 1 ~ 2h.Add the phenol of 550 μ L: chloroform: primary isoamyl alcohol (25:24:1), 12000rmin
-1centrifugal 10min, gets supernatant and moves in another centrifuge tube, repeatedly extracting 2 ~ 3 times.Get supernatant, add the dehydrated alcohol of 800 μ L, add the sodium acetate (3molL of 80 μ L
-1), put into 4 DEG C of refrigerators and precipitate DNA and be about 1h.12000rmin
-1centrifugal 10min, abandons supernatant.Add the centrifugal product of 70% ethanol purge 1 ~ 2 time of 300 μ L, 12000rmin
-1centrifugal 5min, abandons supernatant, by ethanol volatilization completely.Add the DNA of the abundant bottom of the aseptic ultrapure water of 50 μ L, electrophoresis detection DNA extraction quality, puts into-20 DEG C of refrigerators and saves backup;
Clone's trehalose synthesize enzyme gene: with the STb gene of TRM45540 bacterial strain for template, be that primer carries out pcr amplification with TreAF:CGCGGATCCGTGCGCAAGACCTTGATC, TreAR:CCCAAGCTTTCACAGCAGCGCCTTCAG.Amplification program is: 94 DEG C: 5min; 94 DEG C: 30s, 52 DEG C: 30s, 72 DEG C: 1.5min (34cycles); 72 DEG C: 10min, react rear use 1% agarose gel electrophoresis and detected;
Obtain by TA clone the clone p18T-TreA carrying this gene, and send the sequence that clone checks order clear and definite this gene; The plasmid DNA of extracting clone p18T-TreA and pET28a, selects BamHI and HindIII two kinds of enzymes to cut 4h to the plasmid DNA of p18T-TreA and pET28a at 37 DEG C of water-bath enzymes.By agarose gel electrophoresis, reclaim corresponding fragment.P18T-TreA reclaims the fragment of 1218bp, and pET28a reclaims the fragment of 5.3kb.Two fragments reclaimed are connected under the effect of T4DNA ligase enzyme, transforms BL21, obtain pET28a-TreA clone.
The TreP of embodiment 2, variable streptomycete TRM45540 transforms the product analysis of maltose.For determining that TreP can catalysis maltose trehalose synthesis further, therefore by heterogenous expression TreP, to obtain a large amount of TreP crude enzyme liquids, and the composition using maltose as substrate detection of enzymatic reactions thing.
1 heterogenous expression TreP
1.1 cultivate
PET28a-TreA clone be inoculated in 5mLLB substratum, 37 DEG C, 220rpm, shaken overnight cultivates activation, 1/100 switching activation bacterium liquid is in LB substratum, and the OD value of cultivation is 0.6-0.8, and rear interpolation IPTG is 0.01mM to final concentration, 16 DEG C, 220rpm, vibration inducing culture 4h.Simultaneously not add the pET28a-TreA clone of IPTG induction and EMB28a (BL21 carries pET28 carrier) in contrast, collected by centrifugation thalline, carries out SDS-PAGE electrophoresis detection.The pET28a-TreA of induction has had more the protein band of the 40kDa of an expection than pET28a-TreA and EMB28a do not induced.The purifying of fusion rotein TreA carries out according to Ni-AgaroseResinfor6 × His-TaggedProteins purifying specification sheets, obtains TreA albumen by Ni column purification.
1.2LB substratum: peptone: 10gL
-1, yeast extract: 5gL
-1, sodium-chlor: 10gL
-1, block that microbiotic final concentration 50 μ gmL
-1.
The purification process of 1.3 fusion rotein TreA:
(1), after collecting thalline, every 100mg thalline (weight in wet base) adds 1-5mL bacterial lysate, high pressure cracker cracking thalline; (2) centrifugal, abandon supernatant, precipitation is resuspended in SolubleBindingBuffer and (if needed, can high pressure cracker break process be carried out, before process, can 1-5mMPMSF be added);
(3) repeated centrifugation, abandons supernatant, continues with high pressure cracker broken;
(4) be resuspended in InclusionBodyBindingBuffer by precipitation, ice bath 1 hour, makes solubilization of inclusion bodies;
Supernatant is the membrane filtration of 0.45 μM with aperture by (5) 10,000 × g centrifugal 20 minutes;
(6) by protein solution load upper prop, flow velocity be 10 times of column volumes/hour;
(7) use the InclusionBodyBindingBuffer of 15 times of volumes to rinse pillar, collect stream and wear peak;
(8) use the InclusionBodyElutionBuffer wash-out of 5 times of volumes, collect elution peak;
(9) after wash-out, use the deionized water wash pillar of InclusionBodyBindingBuffer and 5 times column volume of 3 times of column volumes successively, then balance (ethanol will by filler submergence), 4 DEG C of preservations with 20% ethanol of 3 times of column volumes.
2 TreP catalysis maltose trehalose synthesis
PET28a-TreA recombinant bacterium thalline is washed twice with the phosphate buffered saline buffer of pH7.0.Take the resuspended thalline of PBS damping fluid that 2g wet thallus adds the pH7.0 of 30mL precooling respectively, make in mixed solution, there is no obvious bacterium block, the fragmentation of bacterium liquid is obtained crude enzyme liquid.In crude enzyme liquid, add sterilizing 70% maltose, fully mix.Put into 40 DEG C of constant-temperature tables, 150r/min reacts 16h.After reaction terminates, add and boil 10min, stopped reaction.Then, carry out centrifugal, condition 4 DEG C, 10000r/min works 2min, gets supernatant liquor deionized water and dilutes 10 times, and by the membrane filtration of supernatant liquor with diameter 0.22 μm, and use liquid chromatography analysis result.
2.1 best concentration of substrate
Configure 10%, 20%, 30%, 40%, 50%, 60%, 70% (w/v) maltose solution is added 4 μ L enzyme liquid (32 μ g), respectively in 40 DEG C of constant-temperature tables, 150r/min reacts 16h, then uses the product of HPLC analytical reaction.
2.2 optimal reaction pH value
Getting 96 μ L pH is respectively that the maltose substrate of the phosphate buffered saline buffer configuration 30% of 4,4.5,5,5.5,6,6.5,7,7.5 and 8 adds the enzyme liquid of 4 μ L (32 μ g), respectively in 40 DEG C of constant-temperature tables, 150r/min reacts 16h, then uses the product of HPLC analytical reaction.
2.3 optimal reactive temperature
Not getting 96 μ L pH is that the maltose substrate of the phosphate buffered saline buffer configuration 30% of 7.5 adds the enzyme liquid (32 μ g) of 4 μ L, respectively in 4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 60 DEG C and 80 DEG C of constant-temperature tables, 150r/min reacts 16h, then uses the product of HPLC analytical reaction.
The impact that 2.4 metal ions are lived on trehalose synthase enzyme
Different metal ion is selected to study: Zn
2+, Cu
2+, Fe
2+, Fe
3+, K
+, Mn
2+and Ca
2+, comprise following component in 100 μ L reaction systems: with the maltose of PBSbuffer configuration 30%, the metal ion of 20mM potassium primary phosphate-hydrogen dipotassium damping fluid (pH7.5) and above-mentioned 1mM or 10mM.Simultaneously not add the above-mentioned reaction solution of above-mentioned any metal ion in contrast.In constant-temperature table, 150r/min reacts 16h, then uses the product of HPLC analytical reaction.
The TreP of embodiment 3, variable streptomycete TRM45540 can significantly improve the salt resistance ability of Host Strains
1, structure carries the recombinant bacterium comprising trehalose synthesize enzyme gene
Clone's trehalose synthesize enzyme gene: with the STb gene of TRM45540 bacterial strain for template, with TreAF and TreAR for primer carries out pcr amplification.PCR primer is reclaimed and obtains by TA Cloning Transformation DH5 α the clone p18T-TreA carrying this gene;
The plasmid DNA of extracting clone p18T-TreA and pET28a, selects BamHI and HindIII two kinds of enzymes to cut 4h to the plasmid DNA of p18T-TreA and pET28a at 37 DEG C of water-bath enzymes.By agarose gel electrophoresis, reclaim corresponding fragment.P18T-TreA reclaims the fragment of 1143bp, and pET28a reclaims the fragment of 5.3kb.Two fragments reclaimed are connected under the effect of T4DNA ligase enzyme, transforms BL21, obtain pET28a-TreA clone
2, the Salt Tolerance Analysis of recombinant bacterium
With pUC19/DH5 α, pET28a/BL21 for negative control, TRM45540 is positive control, carries out Salt Tolerance Analysis to recombinant bacterium p18T-TreA/DH5 α and pET28a-TreA/BL21.Each bacterial strain is seeded in respectively on the flat board of 0%, 1%, 3%, 5%, detects the salt resistance ability of each bacterial strain.Found that TreA gene can improve the ability of recombinant bacterium tolerance NaCl significantly.
4 test-results
The clone of the trehalose synthesize enzyme gene of 4.1TRM45540
By pcr amplification, reclaim the fragment of 1143bp.Sequencing result DNAMAN5.2 software splicing, determines that this fragment is by 1143 based compositions, obtains the TreADNA sequence of biocontrol microorganisms TRM45540.The amino acid quantity of TreP (TreA) TreP: 386; Molecular weight: 41587.4; PI:7.25; Molecular formula: C
1834h
2944n
546o
539s
10; Transformation period: in Mammals: 100h; In yeast: > 20h; In intestinal bacteria: > 10h; Unstability index, close to 39.46, shows that albumen is very stable; Liposoluble coefficient: 89.77, shows by force fat-soluble; Hydrophobic and hydrophilicity analysis: predict the outcome as-0.151, prove that albumen has wetting ability
4.2 TrePs can change into trehalose for catalysis maltose
4.2.1 best concentration of substrate
Result shows along with the addition of maltose gets more and more, originally the transformation efficiency of enzyme remains unchanged substantially, when concentration of substrate is the highest to transformation efficiency time 30% (w/v), remain on about 10%, and decline from 40%-60% (w/v) transformation efficiency.
4.2.2 optimum pH
Result shows that the vigor of TreP can maintain higher vigor when pH is 6-8.5, and its vigor when pH is 7.5 is the highest.
4.2.3 optimum temperuture
Result shows that the vigor of TreP can maintain higher vigor when temperature is 20-40 DEG C, and its vigor when temperature is 40 DEG C is the highest.
4.2.4 metal ion is on the impact of enzyme work
Result shows Ca
2+and Zn
2+faint promoter action is had, Co to the vigor of TreP
2+, Mg
2and Mn
2+to the vigor restraining effect of TreP.
Claims (4)
1. variable streptomycete (
streptomycesmutabilis) TRM45540, the deposit number of this bacterial strain is CCTCCNO:M2015657.
2. variable streptomycete TRM45540 trehalose synthesize enzyme gene, its nucleotide sequence is as shown in SEQIDNO.1.
3. the TreP coded by variable streptomycete TRM45540 trehalose synthesize enzyme gene as claimed in claim 2, its aminoacid sequence is as shown in SEQIDNO.2.
4. variable streptomycete TRM45540 as claimed in claim 1 application in the industry.
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