CN107603994A - A kind of κ carrageenases and its gene and application - Google Patents
A kind of κ carrageenases and its gene and application Download PDFInfo
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- CN107603994A CN107603994A CN201710901361.7A CN201710901361A CN107603994A CN 107603994 A CN107603994 A CN 107603994A CN 201710901361 A CN201710901361 A CN 201710901361A CN 107603994 A CN107603994 A CN 107603994A
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Abstract
The invention discloses a kind of κ carrageenases and gene and application, the κ carrageenase precursor-genes car 30 of obtained food pelvetia silquosa Pseudoalteromonas will be cloned, convert in Escherichia coli, the recombinant bacterium of production restructuring κ carrageenases is obtained, the optimum temperature and pH of restructuring κ carrageenases are respectively 45 DEG C and 6.5；30 min are incubated at 40 DEG C, remnant enzyme activity is more than 80.0%, and 60 min are handled in pH 6.5 7.5 buffer solution, and enzyme activity remains to keep more than 50.0%.The present invention constructs the gene of carrageenase containing κ car 30 recombinant vector, realizes heterogenous expression, while industrialized production for the enzyme and application provide good basis.The κ carrageenases of expression have preferable degradation effect, degraded κ carragheen generations disaccharides, tetrose etc. to κ carragheens, can be applied in antiseptic, antivirotic, immunomodulator, antioxidant etc. is prepared.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of from food pelvetia silquosa Pseudoalteromonas（P. carrageenovora ASY5）Kappa-carrageenan enzyme gene car 30 clone and restructuring kappa-carrageenan enzyme structure and application.
Background technology
Carragheen is a kind of structural polysaccharide being present in marine red alga cell membrane, is D- galactolipins and 3,6- dehydration half
Galactose unit alternately connects what is formed by β -1,3 and α -1,4- glycosidic bonds.Numerous studies confirmation, the catabolite OK a karaoke club of carragheen
Glue widow's carbohydrates and their derivative has a variety of physiologically actives such as antiviral, antitumor, AntiHIV1 RT activity, immunological regulation, has higher medicinal
Development volue.
At present, the preparation method of carrageenan oligosaccharide mainly has reductive water solution, free radical depolymerization, chemical degradation method and enzyme
Solution.Undoubtedly, enzymatic isolation method has the characteristics that high specificity, product are single, reaction condition is gentle, and therefore, prepared by enzymatic isolation method
Carrageenan oligosaccharide is a kind of promising approach.
The bacterial strain direct fermentation obtained using nature seed selection produces carrageenase, yields poorly, condition of culture is complicated, producing enzyme
Unstable, enzyme purification cost height, therefore, it is difficult to for largely producing carrageenases.Using the method for genetic engineering, by nature
In isolated carrageenase gene carry out heterogenous expression, to improve the expression quantity of carrageenase and realize it is a large amount of prepare,
It is the focus direction studied at present.But it is existing it has been reported that restructuring carrageenase temperature stability it is poor, it is big at 0-30 DEG C
Most carrageenases can keep greater activity, and enzyme activity loss is larger under the conditions of temperature is higher than 40 DEG C, when pH is more than 6.0
Enzymatic activity is begun to decline, and therefore, being obtained from nature has preferable heat resistance carrageenase and the big scale in Escherichia coli
Reach, the industrialized production of this extensive preparation and carrageenan oligosaccharide for carrageenase has great importance.
The content of the invention
It is an object of the invention to provide a kind of kappa-carrageenan enzyme and its gene and application, by false alternately from food pelvetia silquosa
A kind of kappa-carrageenan enzyme gene is cloned in monad and is converted to e. coli bl21 (DE3), to obtain a kind of efficient table
Up to the genetic engineering bacterium of kappa-carrageenan enzyme.
To achieve these goals, the technical scheme adopted by the invention to solve the technical problem is that：
A kind of gene car 30 for encoding kappa-carrageenan enzyme, its nucleotide sequence is as shown in SEQ ID NO.1.The ATG of the gene
For initiation codon, TAA is terminator codon.
A kind of kappa-carrageenan enzyme, its amino acid sequence is as shown in SEQ ID NO.2.
A kind of recombinant plasmid containing coding kappa-carrageenan enzyme gene car 30.
As the preferred embodiment of embodiment, described kappa-carrageenan enzyme gene is connected on pET-28a.
A kind of genetic engineering bacterium containing recombinant plasmid.
As the preferred embodiment of embodiment, described genetic engineering bacterium is the e. coli bl21 of production kappa-carrageenan enzyme
(DE3)。
A kind of construction method for the genetic engineering bacterium for encoding kappa-carrageenan enzyme, comprises the following steps：
（1）To eat pelvetia silquosa Pseudoalteromonas（P. carrageenovora ASY5, it is preserved in Chinese industrial microbial preservation
Administrative center（CICC）, deposit number 23819）Genomic DNA is template, is usedP. carrageenovora ASY5 κ-
Carrageenase gene
Forward primer is：5ʹ-CCGGAATTCCAAGAGTTTAAAAAACTA-3；
Reverse primer is：5ʹ- CGCGGATCCAAAACTGCTGTGGGAATA-3ʹ；Expand the 16S rDNA sequences of bacterial strain；Using
Taq archaeal dna polymerases, are expanded in PCR instrument, expand temperature parameter：95 DEG C, 5 min；94 DEG C of denaturation, 60 s；50 DEG C are moved back
Fire, 45 s；72 DEG C of extensions, 60 s, 30 circulations；4 DEG C of holdings；Amplification sample is carried out with the agarose gel electrophoresis of 1% concentration
Detection；
（2）Gel extraction purpose product is used with pET-28a carriersEcoRI andBamDouble digestion, digestion products are sugared with congealed fat simultaneously by HI
Gel electrophoresis is verified；
（3）After correct to digestion products car and the checking of pET-28a carriers, connected in the case where connecting enzyme effect, linked system is as follows：
The μ L of DNA fragmentation 11 to be connected, the μ L of linear carrier plasmid 6, the μ L of buffer solution 2, ligase 1 μ L, 16 DEG C of 16 h of connection；Then
Connection product is converted to e. coli bl21 (DE3), containing 50 μ g/mL ampicillins and 50 mg/mL X-gal
Flat board carries out blue hickie screening, screens positive transformant, carries out bacterium solution PCR, then enters row agarose gel electrophoresis checking, obtains
Positive colony.
A kind of expression for the genetic engineering bacterium for encoding kappa-carrageenan enzyme, comprises the following steps：
Take 500 μ L to verify correct bacterium solution, be inoculated in the 50 mL LB culture mediums containing 50 μ g/mL kanamycins, in 37 DEG C,
200 r/min shaken cultivations are extremelyOD 600Up to 0.8, inducer isopropylthio-β-D- thiogalactosides are added（IPTG）To final concentration
For 0.05 mmol/L, in 10 DEG C of h of Fiber differentiation 24；Refrigerated centrifuge collects thalline, and thalline is resuspended with broken wall buffer solution；Ultrasound is broken
Bacterium solution refrigerated centrifuge after wall takes supernatant, and restructuring kappa-carrageenan enzyme is made.
As the preferred embodiment of embodiment, the optimum temperature of the restructuring kappa-carrageenan enzyme is 45 DEG C.
As the preferred embodiment of embodiment, the optimal pH of the restructuring kappa-carrageenan enzyme is 6.5, and 30 are incubated at 40 DEG C
Min, remnant enzyme activity are more than 80.0%, and 60 min are handled in pH 6.5-7.5 buffer solution, remain to keep more than 50.0%
Enzyme activity.
A kind of kappa-carrageenan enzyme is in the application of food pharmaceuticals industry, including following application field：Antiseptic is being prepared, it is disease-resistant
Toxic agent, immunomodulator, the application in antioxidant.
The present invention is after adopting the above technical scheme, have the advantages that：
Escherichia coli are a kind of prokaryotic expression hosts, the restructuring kappa-carrageenan enzyme can in the host induced expression, due to table
The restructuring kappa-carrageenan enzyme reached has preferable degradation effect, degraded kappa-carrageenan generation tetrose, six sugar, eight sugar etc. to kappa-carrageenan
Sulfated oligosaccharide, catabolite kappa-carrageenin oligose can be in antiseptic, antivirotic, immunomodulator, antioxidant etc. be prepared
Using.
Brief description of the drawings
The PCR agarose gel electrophoresis figures of Fig. 1 kappa-carrageenan enzyme genes；Wherein, M：DNA Maker；1-7：Gene
PCR primer.
Agarose gel electrophoresis figure after Fig. 2 bacterium solutions PCR；Wherein, M：DNA Maker；1-10：Recombinant plasmid pET28a-
After 30 bacterium solution PCR.
The recombinant plasmid pET28a-30 of Fig. 3 structures.
The polyacrylamide gel electrophoresis figure of the induced expression of Fig. 4 restructuring kappa-carrageenan enzymes；Wherein, M：Maker；1：pET-
28a empty plasmids turn bacterium solution after e. coli bl21 (DE3) ultrasonication；2：The ultrasounds of recombinant bacterium PLJ 30 after being induced without IPTG
Bacterium solution after broken wall；3：The bacterium solution after the ultrasonications of recombinant bacterium PLJ 30 after IPTG is induced；4：Weight through Ni posts after purification
Group bacterium PLJ 30.
The substrate specificity of the production restructuring kappa-carrageenan enzymes of Fig. 5 recombinant bacteriums PLJ 30.
Fig. 6 temperature is to recombinating kappa-carrageenan enzyme enzymatic activity（A）And stability（B）Influence and pH to recombinate kappa-carrageenan
Enzyme enzymatic activity（C）And stability（D）Influence.
Fig. 7 recombinates the ESI-TOF-MS mass spectrograms of kappa-carrageenan enzyme enzymolysis product.
Embodiment
Embodiment 1：Kappa-carrageenan enzyme nucleotides and amino acid sequence
Using genome sequencing technology to bacterial strainPseudoalteromonas carrageenovora ASY5 is sequenced,
Its whole genome sequence has been obtained, and relevant biological information credit analysis is carried out to gene.First, the assembled use of genome
Newbler version 2.8 method, from the beginning assembled, structure scafford is carried out to the sequencing data for removing joint sequence；
Then find out the distinctive gene family of this bacterium by OrthoMC softwares and classified；Using bioinformatics software to bacterial strainP. carrageenovora ASY5 genes carry out the biochemical reaction process of the GO functions enrichment analysis memory gene of gene
Analysis.Final assembling obtains complete genome group：Including 1 annular bacterial chromosome and 1 DNA.It can be obtained by splicing result
Know, bacterial strainP. carrageenovora ASY5 full-length genome is 4489108 bp, and G+C contents are 39.48%, are measured in advance
There are 4,144 genes to be classified into the genome of sequencing, be divided into 3,680 gene families, wherein there are 5 gene men
Specific to this bacterium of race.According toP. carrageenovora The genome sequencing result of ASY5 bacteriums, is therefrom screened
Carrageenase gene car 30, translate to obtain the amino acid sequence of kappa-carrageenan enzyme by bioinformatic analysis software DNAMAN.
Carrageenase gene car 30 nucleotide sequence is as shown in SEQ ID NO.1；ATG is initiation codon in sequence
Son, TAA are terminator codon.
A kind of kappa-carrageenan enzyme, its amino acid sequence is as shown in SEQ ID NO.2.
Embodiment 2：Recombinate kappa-carrageenan enzyme LJ 30 structure
To eat pelvetia silquosa Pseudoalteromonas（P. carrageenovora ASY5, it is preserved in Chinese industrial microbial preservation pipe
Reason center（CICC）, deposit number 23819）Genomic DNA is template, is usedP. carrageenovora ASY5 κ-card
Draw glue enzyme gene
Forward primer（SEQ ID NO.3）For：5ʹ-CCGGAATTCCAAGAGTTTAAAAAACTA-3（Underscore isEcoRI knows
Other sequence）,
Reverse primer（SEQ ID NO.4）For：5ʹ- CGCGGATCCAAAACTGCTGTGGGAATA-3ʹ（Underscore isBamHI
Recognition sequence）,
Expand the 16S rDNA sequences of bacterial strain.Using Taq archaeal dna polymerases, expanded in PCR instrument, expand temperature parameter：
95 DEG C, 5 min；（94 DEG C, 60 s；50 DEG C, 45 s；72 DEG C, 60 s）30 circulations；4 DEG C, hold.Coagulated with the agarose of 1% concentration
Gel electrophoresis detect to amplification sample, from figure 1 it appears that 750 bp or so band has been amplified, with expected band
Size result is consistent, preliminary to illustrate that PCR reactions have obtained target gene after expanding.
Gel extraction purpose product is used with pET-28a carriersEcoRI andBamHI while double digestion, digestion products congealed fat
Sugared gel electrophoresis checking.
After correct to digestion products car and the checking of pET-28a carriers, connected in the case where connecting enzyme effect, linked system is as follows：
The μ L of DNA fragmentation 11 to be connected, the μ L of linear carrier plasmid 6, the μ L of buffer solution 2, ligase 1 μ L, 16 °C of 16 h of connection.Then
Connection product is converted to e. coli bl21 (DE3), and with containing 50 μ g/mL ampicillins and 50 mg/mL
X-gal flat board carries out screening positive transformant in blue hickie screening flat board, carries out bacterium solution PCR, then carries out Ago-Gel
Electrophoresis is verified.After bacterium colony PCR checkings, positive colony is obtained as shown in Figure 2.As shown in figure 3, utilize DNAMAN softwares, structure
Build recombinant plasmid and be named as pET28a-30.
Embodiment 3：Recombinate expression of the kappa-carrageenan enzyme in Escherichia coli
Take 500 μ L to verify correct bacterium solution, be inoculated in the 50 mL LB culture mediums containing 50 μ g/mL kanamycins, in 37 DEG C,
200 r/min shaken cultivations are extremelyOD 600Up to 0.8, inducer isopropylthio-β-D- thiogalactosides are added（IPTG）To final concentration
For 0.05 mmol/L, in 10 DEG C of h of Fiber differentiation 24.Refrigerated centrifuge collects thalline, and thalline is resuspended with broken wall buffer solution.Ultrasound is broken
Bacterium solution refrigerated centrifuge after wall takes supernatant.
Fig. 4 shows, after IPTG is induced, it is observed that restructuring kappa-carrageenan expression of enzymes, by κ-OK a karaoke club in enzyme liquid
The measure of glue enzyme enzyme activity demonstrates target gene and has obtained effectively expressing.Because we purify using affinity chromatography
Destination protein, due to purified product, band is single on PAGE gel, and has the vigor of hydrolysis substrate kappa-carrageenan,
So it can confirm that band 4 is exactly purpose band.
Embodiment 4：Recombinate kappa-carrageenan enzyme zymologic property
The detection method used in embodiment：
Utilize DNS（3,5- dinitrosalicylic acids）Method measure restructuring kappa-carrageenan enzyme enzymatic activity.Reaction system include 0.2% κ-
Carragheen (with the phosphate buffered salines of pH 7.0), 500 μ L enzyme liquids, after 45 DEG C are incubated 90 min, the min of boiling water bath 5 is whole
Only react, add the min of 1 mL DNS water-baths 10, absorption value is determined under 520 nm, substituting enzyme liquid with distilled water does blank
Control experiment.Kappa-carrageenan enzyme activity is defined as：Under these conditions, the enzyme needed for 1 μm of oL reduced sugar of catalysis generation per minute
Measure as an enzyme activity unit（U）.
As shown in Figure 5, byP. carrageenovoraThe restructuring kappa-carrageenan enzyme of clonal expression has very for kappa-carrageenan
Good hydrolysis, does not have hydrolysis, explanation to ι-carragheen, agar, agarose, sodium alginate, sodium carboxymethylcellulose
The enzyme has good substrate specificity to kappa-carrageenan.
（1）Recombinate the optimal reactive temperature and temperature stability of kappa-carrageenan enzyme：In order to detect restructuring kappa-carrageenan enzyme
Optimal reactive temperature, will isolate and purify obtained enzyme in reaction temperature is 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 65 DEG C
Under the conditions of measure restructuring kappa-carrageenan enzyme enzyme activity, using highest enzyme activity as 100%.Simultaneously measurement temperature stability when, enzyme liquid 0 DEG C,
30 min and 120 min are handled at 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, samples and determines residual enzyme under optimal reactive temperature
It is living, using it is untreated when enzyme activity as 100%.It is as shown in Figure 6 that restructuring kappa-carrageenan enzyme zymologic property result is determined with DNS methods：Most thermophilic
Degree is 45 °C, and pH is 6.5, and the enzyme, which is incubated 30 min remnant enzyme activities and compared below 40 DEG C, still retains 80.0%.
（2）Recombinate optimal reaction pH and the pH stability of kappa-carrageenan enzyme：With different pH buffer system prepare 0.2% κ-
Carragheen substrate, under optimal reactive temperature, enzyme activity is determined, with highest enzyme activity 100%.Buffer solution system is respectively 50 mmol/L
Disodium hydrogen phosphate-citric acid (pH 4-8), sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 6.5-9), glycine-NaOH (pH
9.5-10.5).When the research of pH stability places one section by 4 DEG C after enzyme liquid is mixed from different pH buffer solution equal proportion
Between, survey residual enzyme activity, using it is untreated when enzyme activity as 100%.As shown in fig. 6, handle 60 in pH 6.5-7.5 buffer solution
Min, remain to keep more than 50.0% enzyme activity.
Embodiment 5：Restructuring kappa-carrageenan enzyme enzymolysis kappa-carrageenan prepares kappa-carrageenin oligose
Preparing 500 mL 0.2% (w/v) kappa-carrageenan solution, (kappa-carrageenan is dissolved in the phosphate of 0.05 mol/L pH 7.0
Buffer solution) in, 20 mL enzyme liquids are added under the conditions of 40 DEG C and react 24 h.During reaction, 10 mL enzyme liquid is added every 4 h.
Reaction places reaction solution 20 min after terminating in boiling water bath, for terminating enzyme degradation reaction.Then removed with ethanol remaining
Undegradable polysaccharide component, adds the ethanol of 1-2 times of volume, until there is precipitation to produce, 4 DEG C are placed centrifuging and taking supernatant after 2 h, so
Freezed after supernatant is concentrated afterwards, obtain the carrageenan oligosaccharide of enzyme degraded.
Shown in Fig. 7, degraded kappa-carrageenan generation disaccharides, tetrose, catabolite can prepare antiseptic, antivirotic, exempt from
Applied in epidemic disease conditioning agent, antioxidant etc., to provide foundation using recombinating kappa-carrageenan enzyme and prepare kappa-carrageenin oligose.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>It is green new（Fujian）Collects The American University of Food Co., Ltd
<120>A kind of kappa-carrageenan enzyme and its gene and application
<130> 2017
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 786
<212> DNA
<213>Eat pelvetia silquosa Pseudoalteromonas
<400> 1
atgaagggaa aatttttttt cgctttattc ttttttatat tttcacattc attatatgca 60
caagagttta aaaaactaat ttggagtgat gaatttaatt acgaaggcct tccagataaa 120
agtaaatggg gatacgaaaa aggatatgtt cgtaacggag aaaagcaatt ttataatgta 180
tctaatctag aaaattctag agtaaaaaat ggaaacctta taatagaggc tcgaaaagat 240
gctggaaatg attcaggttt atggcaagag ttatttgaag caaagaagca accttatact 300
tctgcgagcc taacgacacg caacttaaac tcatggacac atgctagaat tgaagtgcgt 360
gctaaattgc ctaaaggtgt aggtgtttgg cccgctattt ggctgctcgg tgcagataaa 420
ggcgggaaag ggtggcctgc taaaggagag attgatctca tggagtatgt tggctacaaa 480
aaaaataaag tgcatgtagc tttgcataca acaaagagaa atcatcaaaa taaaaaaggt 540
ataaataaag aatttaaatt agataattta ttaacagatt accacacata tgctgttgaa 600
aagcatccca atataattaa gttctacata gacgataagt tagtttttag ctatgaaaaa 660
gaaggcgaca gtgcagatta ttggcctttt gatgagccaa tgtatttaat tattaattta 720
gcaattggtg gttcttggga ggaaaacatg gaattgatga cgccatattc ccacagcagt 780
ttttaa 786
<210> 2
<211> 261
<212> PRT
<213>Eat pelvetia silquosa Pseudoalteromonas
<400> 2
Met Lys Gly Lys Phe Phe Phe Ala Leu Phe Phe Phe Ile Phe Ser His
1 5 10 15
Ser Leu Tyr Ala Gln Glu Phe Lys Lys Leu Ile Trp Ser Asp Glu Phe
20 25 30
Asn Tyr Glu Gly Leu Pro Asp Lys Ser Lys Trp Gly Tyr Glu Lys Gly
35 40 45
Tyr Val Arg Asn Gly Glu Lys Gln Phe Tyr Asn Val Ser Asn Leu Glu
50 55 60
Asn Ser Arg Val Lys Asn Gly Asn Leu Ile Ile Glu Ala Arg Lys Asp
65 70 75 80
Ala Gly Asn Asp Ser Gly Leu Trp Gln Glu Leu Phe Glu Ala Lys Lys
85 90 95
Gln Pro Tyr Thr Ser Ala Ser Leu Thr Thr Arg Asn Leu Asn Ser Trp
100 105 110
Thr His Ala Arg Ile Glu Val Arg Ala Lys Leu Pro Lys Gly Val Gly
115 120 125
Val Trp Pro Ala Ile Trp Leu Leu Gly Ala Asp Lys Gly Gly Lys Gly
130 135 140
Trp Pro Ala Lys Gly Glu Ile Asp Leu Met Glu Tyr Val Gly Tyr Lys
145 150 155 160
Lys Asn Lys Val His Val Ala Leu His Thr Thr Lys Arg Asn His Gln
165 170 175
Asn Lys Lys Gly Ile Asn Lys Glu Phe Lys Leu Asp Asn Leu Leu Thr
180 185 190
Asp Tyr His Thr Tyr Ala Val Glu Lys His Pro Asn Ile Ile Lys Phe
195 200 205
Tyr Ile Asp Asp Lys Leu Val Phe Ser Tyr Glu Lys Glu Gly Asp Ser
210 215 220
Ala Asp Tyr Trp Pro Phe Asp Glu Pro Met Tyr Leu Ile Ile Asn Leu
225 230 235 240
Ala Ile Gly Gly Ser Trp Glu Glu Asn Met Glu Leu Met Thr Pro Tyr
245 250 255
Ser His Ser Ser Phe
260
<210> 3
<211> 27
<212> DNA
<213>Artificial sequence
<400> 3
ccggaattcc aagagtttaa aaaacta 27
<210> 4
<211> 27
<212> DNA
<213>Artificial sequence
<400> 4
cgcggatcca aaactgctgt gggaata 27
Claims (8)
- A kind of 1. gene car 30 for encoding kappa-carrageenan enzyme, it is characterised in that：Its nucleotide sequence such as SEQ ID NO.1 institutes Show.
- A kind of 2. kappa-carrageenan enzyme of the gene code as described in claim 1, it is characterised in that：Its amino acid sequence such as SEQ ID Shown in NO.2.
- A kind of 3. recombinant plasmid of the gene car 30 containing coding kappa-carrageenan enzyme as claimed in claim 1.
- A kind of 4. recombinant plasmid as claimed in claim 3, it is characterised in that：The kappa-carrageenan enzyme gene is connected to pET- On 28a.
- A kind of 5. genetic engineering bacterium of the recombinant plasmid containing as described in claim 3 or 4, it is characterised in that：Described gene Engineering bacteria is the e. coli bl21 (DE3) of production kappa-carrageenan enzyme.
- A kind of 6. construction method of genetic engineering bacterium described in claim 5, it is characterised in that：Comprise the following steps：（1）To eat pelvetia silquosa Pseudoalteromonas genomic DNA as template；Forward primer, its nucleotide sequence such as SEQ ID Shown in NO.3；Reverse primer, its nucleotide sequence is as shown in SEQ ID NO.4；Expand the 16S rDNA sequences of bacterial strain；Using Taq archaeal dna polymerases, are expanded in PCR instrument, expand temperature parameter：95 DEG C, 5 min；94 DEG C of denaturation, 60 s；50 DEG C are moved back Fire, 45 s；72 DEG C of extensions, 60 s, 30 circulations；4 DEG C of holdings；Amplification sample is carried out with the agarose gel electrophoresis of 1% concentration Detection；（2）Gel extraction purpose product is used with pET-28a carriersEcoRI andBamDouble digestion, digestion products are sugared with congealed fat simultaneously by HI Gel electrophoresis is verified；（3）After correct to digestion products car and the checking of pET-28a carriers, connected in the case where connecting enzyme effect, linked system is as follows： The μ L of DNA fragmentation 11 to be connected, the μ L of linear carrier plasmid 6, the μ L of buffer solution 2, ligase 1 μ L, 16 DEG C of 16 h of connection；Then Connection product is converted to e. coli bl21 (DE3), and with containing 50 μ g/mL ampicillins and 50 mg/mL X- Gal flat board carries out blue hickie screening, screens positive transformant, carries out bacterium solution PCR, then enters row agarose gel electrophoresis and test Card, obtains positive colony.
- A kind of 7. expression for the genetic engineering bacterium for encoding kappa-carrageenan enzyme, it is characterised in that：Comprise the following steps：Take 500 μ L build the bacterium solution of correct genetic engineering bacterium, are inoculated in the 50 mL LB culture mediums containing 50 μ g/mL kanamycins, in 37 DEG C, 200 r/min shaken cultivations extremelyOD 600Up to 0.8, inducer isopropylthio-β-D- thiogalactosides are added to final concentration of 0.05 mmol/L, in 10 DEG C of h of Fiber differentiation 24；Refrigerated centrifuge collects thalline, and thalline is resuspended with broken wall buffer solution；Ultrasonication Bacterium solution refrigerated centrifuge afterwards takes supernatant, and restructuring kappa-carrageenan enzyme is made.
- 8. the kappa-carrageenan enzyme described in a kind of claim 2 is preparing antiseptic, antivirotic, immunomodulator or antioxidant In application.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762799A (en) * | 2018-12-26 | 2019-05-17 | 青岛大学 | A kind of kappa-carrageenan enzyme and its application |
| CN110551716A (en) * | 2019-09-10 | 2019-12-10 | 集美大学 | preparation method and application of gene Glu525 |
| CN113980937A (en) * | 2021-11-30 | 2022-01-28 | 中国海洋大学 | Lambda-carrageenase OUC-G150-L7 and application thereof |
| CN114015675A (en) * | 2021-12-14 | 2022-02-08 | 中国海洋大学 | Lambda-carrageenase OUC-LuV and application thereof |
-
2017
- 2017-09-28 CN CN201710901361.7A patent/CN107603994B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762799A (en) * | 2018-12-26 | 2019-05-17 | 青岛大学 | A kind of kappa-carrageenan enzyme and its application |
| CN110551716A (en) * | 2019-09-10 | 2019-12-10 | 集美大学 | preparation method and application of gene Glu525 |
| CN110551716B (en) * | 2019-09-10 | 2021-05-07 | 集美大学 | Preparation method and application of gene Glu525 |
| CN113980937A (en) * | 2021-11-30 | 2022-01-28 | 中国海洋大学 | Lambda-carrageenase OUC-G150-L7 and application thereof |
| CN113980937B (en) * | 2021-11-30 | 2022-11-25 | 中国海洋大学 | Lambda-carrageenase OUC-G150-L7 and application thereof |
| CN114015675A (en) * | 2021-12-14 | 2022-02-08 | 中国海洋大学 | Lambda-carrageenase OUC-LuV and application thereof |
| CN114015675B (en) * | 2021-12-14 | 2023-02-21 | 中国海洋大学 | Lambda-carrageenase OUC-LuV and application thereof |
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| CN107603994B (en) | 2020-12-29 |
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Denomination of invention: one kind k- Carrageenan enzyme and its gene and Application Effective date of registration: 20210730 Granted publication date: 20201229 Pledgee: Bank of Xiamen Limited by Share Ltd. Zhangzhou branch Pledgor: GREENFRESH (FUJIAN) FOODSTUFF Co.,Ltd. Registration number: Y2021350000085 |
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Effective date of registration: 20220808 Address after: Anshan Industrial Park, Zini Town, Longhai City, Zhangzhou City, Fujian Province 363100 Patentee after: GREENFRESH (FUJIAN) FOODSTUFF CO.,LTD. Address before: Anshan Industrial Park, Zini Town, Longhai City, Zhangzhou City, Fujian Province 363100 Patentee before: GREENFRESH (FUJIAN) FOODSTUFF CO.,LTD. Patentee before: JIMEI University |