CN104878031A - Alginate lyase SHA-2 gene and expression vector thereof - Google Patents
Alginate lyase SHA-2 gene and expression vector thereof Download PDFInfo
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Abstract
The invention discloses an alginate lyase SHA-2 gene. A nucleotide sequence is as shown in SEQ ID NO:1. A prokaryotic expression vector of the alginate lyase SHA-2 gene is constructed; an expression product alginate lyase SHA-2 can be obtained by the prokaryotic expression vector within a relatively short period of time, and has wide substrate specificity; poly-mannuronate (PolyM) and poly-guluronate (PolyG) can also be utilized as substrates; the enzyme activity can reach 5.2U/mg; the alginate lyase SHA-2 gene is a bifunctional enzyme with a wide application prospect; the prokaryotic expression vector and an overall expression system are easy to operate; and industrialized production is facilitated.
Description
Technical field
The invention belongs to microbiological genetic engineering field, be specifically related to a kind of alginate lyase SHA-2 gene and prokaryotic expression carrier pGEX-4T-1-thereof
sHA-2, this carrier high-efficiency expresses alginate lyase Protein S HA-2.
Background technology
The development and utilization of marine resources in recent years becomes the focus of research gradually, and Lalgine is with a wide range of applications in fields such as food, medicine and chemical industry because of the physico-chemical property of its uniqueness.Lalgine oligosaccharides, because having multiple physiologically active, becomes the focus point of developing new drug.Lalgine is as one of the abundantest marine biomass simultaneously, and because having following advantage: (1) photosynthetic efficiency is high, growth is fast, and output is high, aboundresources; (2) growth does not occupy cultivated land; (3) hardly containing xylogen, cellulosic content is few, and pre-treatment is simple, is convenient to utilization and the fermentation of microorganism; Thus receive in field of biological energy source and pay close attention to widely.At present, the method for degraded Lalgine can be divided into three major types: the first kind is chemical degradation method, and what extensively adopt at present is acid-hydrolysis method, and this method complex operation step, reaction conditions is violent.Equations of The Second Kind is physical degradation methods, such as ultrasonic degradation Lalgine.3rd class is alginate lyase enzymolysis process, enzymic degradation Lalgine mild condition, process control, yield is high, green safety, and the mechanism of action is clear and definite, product is determined, can select single zymin according to specific purposes product requirements or use the zymin of differing substrate specificity combination.
Alginate lyase, primarily of generations such as Lalgine decomposer and some marine animal and plants, has great application prospect.But wild-type Lalgine decomposer yield of enzyme is low and cost is high, be difficult to reach actual application requiring.Therefore, carrying out heterogenous expression by genetic engineering means to alginate lyase gene is the most effective way improving alginate lyase output.1993, Boyd etc. cloned first
pseudomonas alginovorathe encoding gene algL of alginate lyase also expresses in intestinal bacteria, and its crude enzyme liquid activity reaches 146U/mg.Frederic in 1996 etc. will
pseudomonas alginovorathe gene aly of middle coding alginate lyase is at expression in escherichia coli, and its catalytic activity reaches 97U/mg.2009, the people such as Gaofei Duan existed
pseudoalteromonassp. in CY24, clone obtains alginate lyase gene alyPI, and expresses in intestinal bacteria, and obtaining a catalytic activity is 121.6U/mg, and molecular weight is the albumen of 58KD.2012, the utilizations such as Hwan Hee Park
sphingomonassp. the genome of MJ-3 builds gene library, and screening obtains the gene of one section of alginate lyase, and by it at expression in escherichia coli, obtains a kind of to all activated bifunctional enzyme of polyM and polyG.
Summary of the invention
The object of the invention is to provide a kind of alginate lyase SHA-2 gene, and its nucleotide sequence is as shown in SEQ ID NO:1, and this alginate lyase SHA-2 gene source is in anaerobism Lalgine decomposer
marinicatena alginatilyticasH-52(deposit number is CCTCC NO:M2013073), obtained by this laboratory genome sequencing, by BLAST comparison, result display with
zobellia galactanivorans strainthe alginate lyase sibship similarity of DsiJT is 74%.
Another object of the present invention is to provide the prokaryotic expression carrier of alginate lyase SHA-2 gene, and this carrier contains Ptac promotor, terminator and alginate lyase gene
sHA-2, bacterial ribosome binding site RBS, GST label,
sHA-2the upstream of gene is Ptac promotor, and the downstream of Ptac promotor is the operon sequence can induced by IPTG, near
sHA-2gene start codon upstream be a GST sequence label, amalgamation and expression albumen can be generated, the excision of GST label can not affected the structure-activity of target protein by zymoplasm afterwards.
Another object of the present invention is applied in by the prokaryotic expression carrier of alginate lyase SHA-2 gene to prepare in alginate lyase SHA-2.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
1,
marinicatena alginatilyticasH-52 alginate lyase gene
sHA-2acquisition and the structure of prokaryotic expression carrier
(1) basis
marinicatena alginatilyticasH-52 alginate lyase
sHA-2genes encoding frame sequence and prokaryotic expression carrier pGEX-4T-1 multiple clone site, design 1 pair of special primer as follows:
SHA-2-F:5’-
GGATCCATGAAAAAATCAAGCTTTTTAC-3’
SHA-2-R:5 '-
gCGGCCGCtCAATCCTGACCAGGCG-3 ', adds BamH I and Not I restriction enzyme site (underscore is restriction enzyme site) respectively at 5 ' end of upstream and downstream primer; Extract
marinicatena alginatilyticathe genome of SH-52, uses above-mentioned primer to increase;
(2) also purifying alginate lyase is reclaimed
sHA-2full-length gene fragment, and be connected on pMD19-T carrier, adopt SDS-alkaline lysis method of extracting plasmid DNA, detected by double digestion and obtain recombinant plasmid pMD19T-
sHA-2;
(3) prokaryotic expression carrier pGEX-4T-1-is built
sHA-2, with BamH I and Not I double digestion pMD19T-
sHA-2and pGEX-4T-1, and reclaim purifying alginate lyase
sHA-2gene fragment and pGEX-4T-1 carrier segments, then connect, transform, extracting plasmid carries out double digestion checking, obtains prokaryotic expression carrier pGEX-4T-1-
sHA-2, after checking order, sequencing result is carried out bioinformatic analysis.
2, the prokaryotic expression of alginate lyase SHA-2
Use heat shock method by pGEX-4T-1-
sHA-2proceed in e. coli bl21 (DE3), induced down by IPTG and screen optimum condition of the expression, and carry out great expression under optimum condition;
3, the protein purification of alginate lyase SHA-2
Collect thalline, carry out ultrasonication, the supernatant obtained carries out purifying by GST sepharose post, collects protein expression detection and next stage experiment that the albumen after purifying is used for SHA-2.
4, the characteristic research of restructuring alginate lyase SHA-2
Carry out following characteristic research to the alginate lyase SHA-2 after purifying: optimum temperuture, optimal pH etc., the restructuring alginate lyase SHA-2 that the present invention obtains, its optimum temperuture is 65 DEG C, and optimal pH is 7.5; The measuring method adopted in the present invention is conventional alginate lyase activity determination method, and assaying reaction substrate is at A
235nmthe change of place's light absorption value.
The present invention is to deriving from anaerobism Lalgine decomposer
marinicatena alginatilyticanovel alga lyase gene in SH-52
sHA-2increase, and carry out prokaryotic expression and protein purification further, obtain SHA-2 albumen.The wild strain of prior art alginate lyase by fermentation culture to product enzyme, at least need the time of 3 days, and engineering strain of the present invention only needs 6h can obtain the target protein of maximum, the restructuring alginate lyase SHA-2 albumen that the present invention obtains has substrate specificity widely, can utilize PolyM that PolyG also can be utilized for substrate, enzyme work can reach 5.2U/mg, it is a kind of bifunctional enzyme with broad prospect of application, prokaryotic expression carrier of the present invention and overall expression system easily operate, and are convenient to suitability for industrialized production; The invention solves the problem that existing product alginate lyase bacterial strain production of enzyme is lower, also for carrying out mechanism to alginate lyase SHA-2 further and Mechanism Study is laid a good foundation.
Accompanying drawing explanation
Fig. 1 is the present invention
marinicatena alginatilyticathe detection of SH-52 genomic dna, in figure: M is DNA marker; 1 and 2 is genomic dnas;
Fig. 2 is alginate lyase of the present invention
sHA-2the TA Strategies For The Cloning schematic diagram of gene;
Fig. 3 is recombinant plasmid pMD19T-of the present invention
sHA-2electrophoresis detection schematic diagram, in figure: M is DNA marker; 1-4 is pMD19T-
sHA-2;
Fig. 4 is recombinant plasmid pMD19T-of the present invention
sHA-2double digestion detect schematic diagram, in figure: M is DNA marker; 1-4 is the pMD19T-of BamH I and Not I double digestion
sHA-2plasmid;
Fig. 5 is recombinant plasmid pMD19T-of the present invention
sHA-2pCR check schematic diagram, in figure: M is DNA marker; 6 is negative control, and 1-5 is with pMD19T-
sHA-2for template, be the PCR primer of primer amplification with SHA-2-F, SHA-2-R;
Fig. 6 is alginate lyase of the present invention
sHA-2the Prokaryotic expression vector construction schematic diagram of gene;
Fig. 7 is recombinant plasmid pGEX-4T-1-of the present invention
sHA-2electrophoresis detection schematic diagram, in figure: M is DNA marker; 1-2 is pGEX-4T-1-
sHA-2;
Fig. 8 is recombinant plasmid pGEX-4T-1-of the present invention
sHA-2enzyme cut detection schematic diagram, in figure: M is DNA marker; 1-2 be BamH I and Not I double digestion pGEX-4T-1-
sHA-2plasmid;
Fig. 9 is that the SDS-PAGE that alginate lyase SHA-2 of the present invention expresses detects schematic diagram, in figure: M is albumen marker; 1 be pGEX-4T-1 plasmid at 15 DEG C, without IPTG induction after, the total bacterial protein of 6h; 2 is pGEX-4T-1-
sHA-2plasmid at 15 DEG C, without IPTG induction after, the total bacterial protein of 6h; 3-9 is pGEX-4T-1-
sHA-2plasmid at 15 DEG C, after 1mM IPTG induces, 0,2,4,6,8,10, the total bacterial protein of 12h;
Figure 10 is alginate lyase SHA-2 of the present invention expression at 15 DEG C, in figure: M is albumen marker; 1 is pGEX-4T-1-
sHA-2plasmid is induced through 1mM IPTG at 15 DEG C, the total bacterial protein of 0h; 2-7 is pGEX-4T-1-
sHA-2plasmid 15 DEG C, to be respectively 0.1 through final concentration, 0.2,0.4,0.6,0.8,1.0mMIPTG induces the total bacterial protein of 6h;
Figure 11 is the expression of alginate lyase SHA-2 of the present invention under differing temps induction, in figure: M is albumen marker; 1 is pGEX-4T-1-
sHA-2plasmid is induced without IPTG at 15 DEG C, the total bacterial protein of 6h; 2-6 is pGEX-4T-1-
sHA-2plasmid is that 0.8mM IPTG induces the total bacterial protein of 6h at 15,20,25,28,30 DEG C through final concentration respectively;
Figure 12 is the purifying electrophoresis schematic diagram of alginate lyase of the present invention, in figure: M is albumen marker; 1 is the bacterial supernatant albumen before purifying; 2 is the stream river liquid after purifying; 3-5 is the elutriant one after using reductive glutathione wash-out, elutriant two, elutriant three;
Figure 13 is the optimal pH schematic diagram of alginate lyase SHA-2 of the present invention, in figure: diamond curve is the active schematic diagram of SHA-2 albumen in pH6.0-8.0 phosphate buffered saline buffer; Square curve is the active schematic diagram of SHA-2 albumen in pH8.0-9.0 Tris-HCl damping fluid;
Figure 14 is the optimum temperuture schematic diagram of alginate lyase SHA-2 of the present invention;
Figure 15 is the metal ion effect schematic diagram of alginate lyase SHA-2 of the present invention;
Figure 16 is the substrate specificity schematic diagram of alginate lyase SHA-2 of the present invention.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail; but scope is not limited to described content; method employing ordinary method if no special instructions in embodiment, the reagent use conventional commercial reagent if no special instructions of use or the reagent prepared according to a conventional method.
In the present embodiment, reagent is mainly divided into molecular biology experiment reagent, various restriction enzyme, pfu archaeal dna polymerase, dNTP etc. are Japanese precious biotechnology company limited (Dalian) and border biological gene Science and Technology Ltd. of the village, Beijing ally product, plasmid extraction kit is purchased from Sangon Biotech (Shanghai) Co., Ltd., and all the other reagent are domestic analytical pure; Instrument is molecular biology and genetic engineering laboratories common instrument; The all raw work synthesis in Shanghai of all primer sequences.
embodiment 1:
marinicatena alginatilyticathe preparation of SH-52 genomic dna and detection
The present invention is used
marinicatena alginatilyticasH-52 is this laboratory screening bacterial strain, and the preparation of SH-52 genomic dna adopts common bacteria Extraction Methods of Genome, and particular content is as follows: get 2mL incubated overnight bacterium liquid in 4 DEG C, the centrifugal 2min of 4000rpm, abandons most supernatant liquor, collects thalline; Add 100 μ l Solution I and hang bacterium, 30ul 10%SDS and 1 μ l 20mg/ml Proteinase K, mixing, hatches 1 hour for 37 DEG C; Add 100 μ l 15mol/L NaCl, mixing; Add 20 μ l CTAB/NaCl solution (CTAB 10%, NaCl 0.7mol/L), mixing, 65 DEG C, 10 minutes; Add equal-volume phenol/chloroform/primary isoamyl alcohol (25:24:1) mixing, centrifugal 5 minutes of 12000rpm; Get supernatant, add 2 times of volume dehydrated alcohols, 0.1 times of volume 3mol/L NaOAC, place 30 minutes for-20 DEG C; Centrifugal 10 minutes of 12000rpm; Precipitation adds 70% washing with alcohol; After precipitation drying, be dissolved in 20ul TE ,-20 DEG C of preservations.Get 2ul genomic dna with 1% sepharose carry out electrophoresis detection, result (Fig. 1) illustrates the genomic dna satisfactory quality extracted.
embodiment 2: alginate lyase
sHA-2the amplification of gene and TA clone
Alginate lyase
sHA-2the amplification of gene and clone as shown in Figure 2, first search from genome sequencing result
sHA-2full-length gene order, and design a pair Auele Specific Primer, sequence is as follows:
SHA-2-F：
GGATCCATGAAAAAATCAAGCTTTTTAC
SHA-2-R：
GCGGCCGCTCAATCCTGACCAGGCG
5 ' end primer has GGATCC characteristic sequence, and forms BamH I restriction enzyme site thus; 3 ' end adds GCGGCC characteristic sequence, forms Not I restriction enzyme site.
In PCR reaction mixture, add 5-10ng's
marinicatena alginatilyticasH-52 genomic dna is as template, add Auele Specific Primer SHA-2-F and SHA-2-R of 30-50ng simultaneously, 2.5 μ l dNTP(10mM), the Pfu of 0.5-3.0 μ l reacts the pfu(5U/ μ l of Buffer and 0.5-1.0 μ l) polysaccharase (Beijing Quanshijin Biotechnology Co., Ltd), add distilled water and make final volume be 25 μ l.In 94 DEG C of heating 3min in PCR instrument, then according to 94 DEG C, 30s, 57 DEG C, 30s, 72 DEG C, the program of 1min carries out the reactions of 25 circulations, the program finally extending reaction 10min at 72 DEG C is carried out PCR and is reacted amplification and obtain
sHA-2gene, after having reacted, is separated pcr amplification product by agarose gel electrophoresis.Reclaim and purifying
sHA-2full-length gene DNA(1.0Kb), then the TA Cloning Kit of precious biological (TaKaRa) is used to be subcloned into the precious biotech firm in pMD19-T(Dalian) on carrier, experimental implementation is undertaken by the specification sheets of test kit, reaction uses reaction mixture transformation of E. coli competence Trans1-T1(Beijing Quanshijin Biotechnology Co., Ltd after spending the night), adopt alkaline lysis method of extracting plasmid DNA, detect its size (Fig. 3) with 1% agarose gel electrophoresis, choose recombinant plasmid that size conforms to theoretical value and carry out enzyme and cut detection.The recombinant plasmid pMD19T-of successful connection
sHA-2a size is had to be the fragment of about 3.6kb.Use the recombinant plasmid of BamH I and the successful connection of Not I double digestion further, produce two bar segment, a size is about 2.6kb, and another is about 1.0kb (Fig. 4,5).Insert in sequencing analysis proof recombinant plasmid vector
sHA-2full-length gene order is correct.Reaffirm be successful connection plasmid after, transformation of E. coli Trans1-T1 again, chooses single bacterium colony and carries out liquid culture, with kits plasmid pMD19T-
sHA-2.
embodiment 3: prokaryotic expression carrier pGEX-4T-1-
sHA-2structure
PGEX-4T-1-
sHA-2construction strategy as shown in Figure 6, use BamH I(TaKaRa) and Not I(TaKaRa) cut the prokaryotic expression carrier pGEX-4T-1(of purifying purchased from GE Healthcare company) and pMD19T-
sHA-2, be separated the carrier and Insert Fragment that have cut by agarose gel electrophoresis, from gel, reclaim the carrier segments pGEX-4T-1(4.9kb that pGEX-4T-1 is cut rear generation) and pMD19-
sHA-2cut generation
sHA-2the DNA fragmentation (about 1.0kb) of gene, then with the ligase enzyme test kit of precious biological (TaKaRa) connects pGEX-4T-1 carrier segments with
sHA-2the DNA fragmentation of gene produces prokaryotic expression carrier pGEX-4T-1-
sHA-2.High efficiency competent escherichia coli cell Trans1-T1(Beijing Quanshijin Biotechnology Co., Ltd is transformed) with ligation mixture, intestinal bacteria after conversion are applied to and are added with penbritin (Amp, on flat board 100mg/L), in 37 DEG C of incubated overnight, screening Amp resistance recon bacterium colony, plasmid (Fig. 7) is extracted from Amp resistance recon bacterium colony, use BamH I, Not I(TaKaRa) carry out double digestion detection, the plasmid of successful connection produces two bands of about 4.9kb and 1.0kb size (because BamH I on agarose gel electrophoresis figure, Not I only has single recognition site in mount, therefore double digestion plasmid should have two fragments) (Fig. 8).By the plasmid vector pGEX-4T-1-of successful connection
sHA-2again transformation of E. coli Trans1-T1, chooses single bacterium colony and carries out liquid culture, with kits plasmid pGEX-4T-1-
sHA-2.
embodiment 4:alginate lyase
sHA-2the expression of albumen and the optimization of expression condition
Use prokaryotic expression carrier pGEX-4T-1-
sHA-2the competent cell (sky root biochemical technology) of transformation of E. coli BL21.Picking list bacterium colony adds 5mL LB(and contains Amp 100mg/L) in, 37 DEG C of incubated overnight (OD
600be about 1.5).Then be transferred to 100ml LB(and contain Amp 100mg/L) in, work as OD
600when reaching 0.6-0.8, add IPTG respectively and induce under different condition, collected by centrifugation thalline, add 5XSDS-PAGE sample-loading buffer (Sample loading buffer), boil thalline in 100 DEG C of heating 10min.In 4 DEG C of centrifugal (12000rpm) 10min after ice bath cooling, get supernatant and carry out SDS-PAGE.Predict that object fusion rotein size is about 63kDa (GST fusion tag is 26kDa) according to documents and materials and software analysis, adopt 12% separation gel.SDS-PAGE electrophoresis is referring to " Molecular Cloning: A Laboratory guide (third edition) ".It is that 0.8mM IPTG induces after 6h that SDS-PAGE electrophoresis result (Fig. 9,10,11) to represent at 15 DEG C through final concentration
sHA-2expressing quantity is the highest.
embodiment 5:alginate lyase
sHA-2the purifying of albumen, concrete steps are as follows:
(1) bacterial cell disruption: by 15 DEG C, 1L thalline under 0.8mM IPTG after a large amount of abduction delivering 6h, through ultrasonication thalline (work 5s, rest 5s) 20min;
(2) cleer and peaceful precipitation in collection: by bacterial cell disruption liquid in 4 DEG C, the centrifugal 20min of 12000rpm, retains upper cleer and peaceful precipitation respectively;
(3) albumen broken supernatant liquor 0.22um filter filters, removing impurity;
(4) GST Sefinose Resin post pre-treatment: the ethanol of preserve pillar 20% is released; PBS damping fluid (the 4.3mM Na of 10 times of column volumes
2hPO
4, 1.4mM KH
2pO
4, 137mM NaCl, 2.7mM KCl) and balance pillar, flow velocity is 0.5-1ml/min;
(5) protein sample upper prop: flow velocity is 0.5ml/min, collects effluent liquid;
(6) post is washed: use 5-10 times of volume PBS damping fluid (4.3mM Na
2hPO
4, 1.4mM KH
2pO
4, 137mM NaCl, 2.7mM KCl, PH7.4) and wash-out;
(7) wash-out: use 5 times of column volume elution buffers (10mM Glutathione(reduced form), 50mM Tris-HCl, PH8.0) wash-out, repeats 2-3 time, collects elutriant;
(8) aftertreatment of GST Sefinose Resin post: 5 times of column volume PBS damping fluid (4.3mM Na
2hPO
4, 1.4mM KH
2pO
4, 137mM NaCl, 2.7mM KCl, PH7.4); 5 times of column volume deionization washing posts; In 4 DEG C, preserve in 20% ethanol of 3 times of column volumes;
(9) SDS-PAGE detects: get each gradient effluent liquid of 20ul respectively, stream river liquid, washings, crude enzyme liquid adds 5 × SDS-PAGE sample-loading buffer (Sample loading buffer) of 5ul, within 10 minutes, boil in 100 DEG C of heating, loading, carries out SDS-PAGE analysis, purification result is shown in Figure 12, final acquisition SHA-2 purifying protein.
By above-mentioned experiment, invention achieves following result: the prokaryotic expression carrier (pGEX-4T-1-utilizing SHA-2 of the present invention
sHA-2) transformation of E. coli (BL21), the heterogenous expression of SHA-2 albumen can be realized.SHA-2 expression of recombinant proteins amount is higher, does not therefore need large scale culturing bacterium, and the operation of purifying SHA-2 albumen is quite simple, and cost is also very low, very easily reuses.
embodiment 6: the specificity analysis of alginate lyase SHA-2 albumen, particular content is as follows:
1, enzyme activity determination
The unsaturated uronic acid produced due to alginate lyase cracking sodium alginate has absorption value at 235nm place, calculates corresponding enzyme live by measuring reaction solution change at that wavelength.
In reaction system (20mM phosphate buffered saline buffer (pH 7.0), 0.3% sodium alginate), add the alginate lyase protein promoter reaction of 10 μ g, after 10min, measure the change of 235nm place light absorption value.
Enzyme is lived and is defined: light absorption value under wavelength 235nm, increases by 1 be defined as a Ge Meihuo unit (U) with per minute light absorption value.
By measuring, the enzyme work of the restructuring alginate lyase SHA-2 albumen after purifying can reach 5.2U/mg, lives far above the enzyme before purifying.
2, zymology Quality Research
(1) alginate lyase SHA-2 albumen optimum pH measures
Be the optimal reaction pH that substrate measures alginate lyase with sodium alginate in pH buffer system (pH6.0-8.0 sodium phosphate buffer or pH8.0-9.0 Tris-HCl damping fluid), be 10min 65 DEG C of reaction times, the enzyme activity be under optimal pH is defined as 100%, the relation curve of the activity of alginate lyase SHA-2 and stability and pH value as shown in figure 13, under strongly-acid or strong alkaline condition the vigor of enzyme and stability not high, alginate lyase SHA-2 the suitableeest enzyme reaction pH condition is 7.5.
(2) the suitableeest catalytic temperature estimation of alginate lyase SHA-2 albumen
Be the optimal reactive temperature that substrate measures enzyme with sodium alginate in the phosphate buffered saline buffer of pH7.0, reaction times is 10min, and the enzyme activity be under optimum temperuture is defined as 100%, and result as shown in figure 14, the optimal reactive temperature of enzyme 65 DEG C, has higher enzyme activity within the scope of 55 ~ 70 DEG C; When temperature drops to 30 DEG C, enzyme is lived and is declined rapidly, only has 17% of peak value.
(3) metal ion is on the impact of alginate lyase SHA-2 protein-active
Be that substrate measures different metal compound (in reaction solution, the concentration of metal ion the is 0.5mM) impact on alginate lyase SHA-2 protein-active with sodium alginate in the phosphate buffered saline buffer of pH7.0, to add the solution of same volume ponding as a control group, enzyme activity is measured at optimum temperuture 65 DEG C, the enzyme activity of control group is defined as 100%, result as shown in figure 15, Hg
2+enzyme is lived and has complete restraining effect, Zn
2+, Ba
2+with Ca
2+can live to enzyme and have strongly inhibited effect, Mg
2+, K
+with Mn
2+enzyme is lived and has small portion restraining effect, and Na
+with Co
2+enzyme is lived and has part promoter action.
(4) substrate specificity of alginate lyase SHA-2 albumen
Alginate lyase SHA-2 after purifying is added in 20mM phosphate buffered saline buffer (pH 7.0), add the different substrates (sodium alginate, PloyG, PloyM) that massfraction is 0.3% simultaneously, 10min is reacted at temperature of reaction is 65 DEG C, result as shown in figure 16, SHA-2 all has substrate specificity to PloyG and PloyM, but relatively slightly strong for the substrate specificity of PloyG.
Traditional method is generally by utilizing sodium alginate decomposer to ferment, separation, purifying obtain alginate lyase, but it is low to there is wild mushroom yield of enzyme, complex operation, the problems such as production cost is high, become the bottleneck that enzymolysis process prepares Lalgine oligose in a large number, the further genralrlization limiting alginate lyase uses.
The present invention adopts genetic engineering means, build the engineering strain of the alginate lyase gene containing anerobe, produce alginate lyase by abduction delivering, this is compared with traditional method, not only expand alginate lyase source, and the purge process of enzyme also simple easy handling.
sequence table
<110> Kunming University of Science and Technology
<120> alginate lyase SHA-2 gene and expression vector thereof
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1050
<212> DNA
<213>
MarinicatenaalginatilyticaSH-52
<400> 1
atgaaaaaat caagcttttt acctctggta atggttggct tcgtattcgt attatccggt 60
tgtggcaata cagcaaaaaa aagtgaggct aaaaaaacga ctgaccttgt ttaccccagc 120
gatgtgattc catttatgga tgaatttaaa attctgttgg gtgacggcac ctatcaggat 180
gatttgattc agtatgagaa aaaggatttc ttctatgttt caaatgacgg ggagacaaac 240
tgggtggttt ataaaacgcc caattcaggt gttacctcca gaacttcgag taacaccaga 300
actgaattag gtcagaaaga tcactgggta cccgaaacag gaggtaagct gacgggcagg 360
tgtaaagtga tgcacgtttc cacttcaggc gacgccagag ttgctgcttc ctactcggta 420
gttgttgggc aaatacatag tgatgaaggt cacgaaaacg aaccgtgtaa aatcttttat 480
aagaagtttc cgggccatac caaaggttct gttttttgga actatgaaat aaataccgag 540
ggagataatt ccaaaagatg ggatttatct actgttgttt gggggtacga tatgtcagtt 600
gttggcgcag atccaacttc atatccgcca gaaccggaag acggcattga gttgggcgaa 660
gagtttttct acgaaattaa tgtctatgag gggatcatgt acctgacttt tgccagtgaa 720
ggtcacgaca cgattcggtt caccaaaaac ctgttggtct cgtcgtttgc aacggttgag 780
gatatacccg aacaaaccag aacgttgttt tttccgatcg ggcgggacgg agtcgaacgc 840
gaaaatgcct atgccggcga gattcaatac tttaagcaag gtgcatataa ccaaacgaat 900
ggcaaaaatc cggccgataa tatggtttgg agtactggcg ctgatgttta cggaggtgat 960
attgcgaaac agtatgaaaa cggagacttt gccgaagttt ggttcagaga agcaacggtg 1020
ggccctggag tcccgcctgg tcaggattga 1050
<210> 2
<211> 28
<212> DNA
<213> artificial sequence
<400> 2
ggatccatga aaaaatcaag ctttttac 28
<210> 3
<211> 25
<212> DNA
<213> artificial sequence
<400> 3
gcggccgctc aatcctgacc aggcg 25
Claims (2)
1. an alginate lyase SHA-2 gene, is characterized in that: its nucleotide sequence is as shown in SEQ ID NO:1.
2. the prokaryotic expression carrier of alginate lyase SHA-2 gene according to claim 1, is characterized in that: this carrier contains Ptac promotor, terminator and alginate lyase gene
sHA-2, bacterial ribosome binding site RBS, GST label, near
sHA-2gene start codon upstream be a GST sequence label,
sHA-2the upstream of gene is Ptac promotor, and the downstream of Ptac promotor is the operon sequence can induced by IPTG.
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| CN105255923A (en) * | 2015-10-28 | 2016-01-20 | 昆明理工大学 | Alginic acid lyase SHA-4 gene and prokaryotic expression vector thereof |
| CN105255922A (en) * | 2015-10-28 | 2016-01-20 | 昆明理工大学 | Alginic acid lyase SHA-5 gene and prokaryotic expression vector thereof |
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| CN103525717A (en) * | 2013-06-04 | 2014-01-22 | 昆明理工大学 | Anaerobic alginate decomposing bacterium and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255923A (en) * | 2015-10-28 | 2016-01-20 | 昆明理工大学 | Alginic acid lyase SHA-4 gene and prokaryotic expression vector thereof |
| CN105255922A (en) * | 2015-10-28 | 2016-01-20 | 昆明理工大学 | Alginic acid lyase SHA-5 gene and prokaryotic expression vector thereof |
| CN105255922B (en) * | 2015-10-28 | 2018-08-10 | 昆明理工大学 | A kind of alginate lyase SHA-5 genes and its prokaryotic expression carrier |
| CN105255923B (en) * | 2015-10-28 | 2018-08-31 | 昆明理工大学 | A kind of alginate lyase SHA-4 genes and its prokaryotic expression carrier |
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