CN104770624B - A kind of application of bigcatkin willow acid decarboxylase degraded ginkgoic acid - Google Patents
A kind of application of bigcatkin willow acid decarboxylase degraded ginkgoic acid Download PDFInfo
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- CN104770624B CN104770624B CN201510052113.0A CN201510052113A CN104770624B CN 104770624 B CN104770624 B CN 104770624B CN 201510052113 A CN201510052113 A CN 201510052113A CN 104770624 B CN104770624 B CN 104770624B
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- Prior art keywords
- acid
- ginkgoic
- ginkgoic acid
- sdc
- bigcatkin willow
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- YXHVCZZLWZYHSA-FPLPWBNLSA-N Ginkgoic acid Chemical compound CCCCCC\C=C/CCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-FPLPWBNLSA-N 0.000 title claims abstract description 97
- 239000002253 acid Substances 0.000 title claims abstract description 20
- 240000000203 Salix gracilistyla Species 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 241000218628 Ginkgo Species 0.000 abstract description 11
- 235000011201 Ginkgo Nutrition 0.000 abstract description 11
- 235000008100 Ginkgo biloba Nutrition 0.000 abstract description 11
- 239000007788 liquid Substances 0.000 abstract description 11
- 238000000034 method Methods 0.000 abstract description 11
- 241000588724 Escherichia coli Species 0.000 abstract description 8
- 102000004190 Enzymes Human genes 0.000 abstract description 7
- 108090000790 Enzymes Proteins 0.000 abstract description 7
- 230000015556 catabolic process Effects 0.000 abstract description 4
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 239000013612 plasmid Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 9
- 230000029087 digestion Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 4
- 108010042407 Endonucleases Proteins 0.000 description 3
- 102000004533 Endonucleases Human genes 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- YXHVCZZLWZYHSA-UHFFFAOYSA-N (Z)-6-[8-pentadecenyl]salicylic acid Natural products CCCCCCC=CCCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-UHFFFAOYSA-N 0.000 description 2
- 238000006065 biodegradation reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- -1 C13:1 Chemical class 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical class CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 108010029541 Laccase Proteins 0.000 description 1
- 244000018633 Prunus armeniaca Species 0.000 description 1
- 235000009827 Prunus armeniaca Nutrition 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000019225 fermented tea Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention relates to a kind of application of bigcatkin willow acid decarboxylase degraded ginkgoic acid, the gene order of the enzyme is as described in sequence 1, the enzyme is bigcatkin willow acid decarboxylase (Sdc), the clasmatosis liquid of pET21a (+) sdc/E.coli BL21 (DE3) containing Sdc can obviously reduce the content of ginkgoic acid, the enzyme of the present invention can play the degradation to ginkgoic acid in the range of wider temperature and pH, more quick convenient method is provided to take out ginkgoic acid in ginkgo, more possibilities are provided for extensive utilize of ginkgo.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of degraded application of bigcatkin willow acid decarboxylase (Sdc) to ginkgoic acid.
Background technology
Ginkgo is very wide and with a long history in China's distribution, and there is very high application value at the position such as its leaf, seed.But at this
A kind of toxic component of a little position generally existings --- ginkgoic acid.
Ginkgoic acid is a kind of septichen class compound, including C13:1、C15:1、C17:2、C15:0、C17:1 etc.,
There is very big harm to human body.
Physical removal methods and chemical minimizing technology be degrade ginkgoic acid common method, external document report compared with
Few, the country, which was once used the methods of microwave and supercritical extract, removes ginkgoic acid, and these methods are required to using organic solvent to silver
Apricot acid is dissolved, complex process.In terms of biodegradation, Li Yinliang et al. ginkgo acid contents in artificial fermentation's ginkgo leaf
Once report makes fermented tea and can reduce containing for wherein ginkgoic acid by the artificial infection coronoid process dissipate capsule bacterium ginkgo leaf that ferments in research
Amount, but it is time-consuming longer, and step is cumbersome.
By retrieval, a patent document related to present patent application is found：A kind of biodegradation of ginkgolic acid
Method (CN103053876), this method are to add 0.004U/ml- into the degeneration system containing finite concentration ginkgolic acid
(PH4.0-6.0 of degraded system, degradation temperature is 45 DEG C -60 DEG C, during degradation treatment while 0.006U/ml laccase LacC
Between be 10-24h), add amboceptor material, particularly add ABTS, degradation rate reaches as high as 100%.
By contrast, document disclosed above has relatively big difference, the skill of acquisition with present patent application in terms of technical scheme
Art effect is different.
The content of the invention
It is an object of the invention to provide a kind of application of enzyme degraded ginkgoic acid, there is provided the degraded way of another ginkgoic acid
Footpath.
The present invention realizes that the technical scheme of purpose is：
A kind of application of bigcatkin willow acid decarboxylase degraded ginkgoic acid.The gene order of the bigcatkin willow acid decarboxylase such as institute of sequence 1
State.
The temperature of the bigcatkin willow acid decarboxylase explanation ginkgoic acid is 5~80 DEG C, and pH is 3.0~11.0.
The ginkgoic acid is to include ginkgoic acid R=C13:0th, ginkgoic acid R=C15:0th, ginkgoic acid R=C15:1st, ginkgoic acid R
=C17:1st, ginkgoic acid R=C17:2.
A kind of genetic engineering bacterium for ginkgoic acid of degrading, the bigcatkin willow acid decarboxylase containing sequence as described in sequence 1.
The plasmid vector is pET21a (+).
The Host Strains are E.coli BL21 (DE3).
The advantages of the present invention are：
1st, it is bigcatkin willow acid decarboxylase Sdc the invention provides a kind of enzyme for the ginkgoic acid that can degrade, the enzyme, contains Sdc's
PET21a (+)-sdc/E.coli BL21 (DE3) clasmatosis liquid can obviously reduce the content of ginkgoic acid.Its cell of 1mL is broken
Broken liquid makes ginkgoic acid C15 in 40 DEG C, pH=5.5,5h:1 content reduces 39.017 ± 0.131%.
2nd, bigcatkin willow acid decarboxylase provided by the invention can play the drop to ginkgoic acid in the range of wider temperature and pH
Solution is acted on, and more quick convenient method is provided for taking-up ginkgoic acid in ginkgo, for the extensive more possible using providing of ginkgo
Property.
Brief description of the drawings
Fig. 1 is recombinant plasmid pET21a (+)-sdc structure；
Fig. 2 is the identification of pET21a (+)-sdc recombinant plasmids；M：Maker (10kb), 1：PET21a (+)-sdc single enzyme
Cut result, 2：PET21a (+)-sdc double digestion result；
Fig. 3 is the identification of control group pET21a (+) recombinant plasmid.M：Maker (10kb), 1：PET21a (+) single endonuclease digestion
As a result；
Embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit
Qualitatively, it is impossible to which protection scope of the present invention is limited with this.
The present invention is according to the gene orders of the reports such as Kirimura, artificial synthesized sdc genes, and builds the weight of the gene
Group plasmid, then be transferred in host cell, recombinant bacterium is inoculated into culture medium and cultivates certain time, is therefrom taken a certain amount of
Zymotic fluid carries out clasmatosis.A certain amount of ginkgoic acid standard items are weighed, are configured to solution.Clasmatosis liquid and standard items is molten
After liquid mixing, reaction a period of time, ginkgo acid content therein is determined with LC-MS.
Wherein, ginkgoic acid refers to a kind of 2- hydroxyls -6- alkylbenzoic acid class compounds, including ginkgoic acid (R=C13:0)、
Ginkgoic acid (R=C15:0), ginkgoic acid (R=C15:1) ginkgoic acid (R=C17:1) ginkgoic acid (R=C17:2) etc..
Concrete operations mode and step are as follows：
(1) according to the gene order of the reports such as Kirimura, artificial synthesized sdc genes, and build the restructuring matter of the gene
Grain pET21a (+)-sdc, then be transferred in E.coli BL21 (DE3).
Double digestion is carried out to sdc genes and plasmid pET21a (+) with Hind III/Nde I respectively, takes endonuclease bamhi to carry out
Coupled reaction, then E.coli BL21 (DE3) are transferred to complete the structure of plasmid (such as Fig. 1).Put down with nutrient solution coating Amp resistances
Plate, recombinant plasmid pET21a (+)-sdc contain Amp resistant gene, hence in so that be transferred to the BL21 of the recombinant plasmid with
Amp resistances, herein according to whether Amp resistances are carried to screen recon, what can be grown in Amp resistant panels is exactly to build
Successful recon.From picking single bacterium colony on LB solid plates, it is inoculated into after being incubated overnight in LB liquid medium, extracts matter
Grain, is identified by agarose gel electrophoresis.As a result such as Fig. 2.
There is two bands, respectively 5443bp and 1053bp in double digestion recombinant plasmid, with plasmid pET21a (+) and sdc bases
Cause it is in the same size, and single endonuclease digestion recombinant plasmid occur a band, be 6496bp.Correct recombinant plasmid by checking is entered
Row sequencing, obtained sequence is identical with former sequence, illustrates that carrier pET21a (+)-sdc determines to successfully construct.
Due to resistant genes of the pET21a (+) containing Amp, so available same method carries out the screening of recon, Fig. 3
For the qualification result of control group single endonuclease digestion, there is a band in figure, be 5443bp, illustrate that pET21a (+) is successfully transferred to
E.coli BL21(DE3)。
(2) it is 100 μ g/ pET21a (+)-sdc/E.coli BL21 (DE3) of restructuring to be inoculated into Amp and IPTG concentration
In mg LB culture mediums, at a temperature of 37 DEG C, 130rpm culture 12h, nutrient solution is inoculated in IPTG by 1% inoculum concentration and Amp is dense
Degree is 100 μ gmL-1LB culture mediums in, 30 DEG C, 120rpm culture 18h.
(3) the nutrient solution 4mL of step (2) is taken, and the ultrasonication in ice bath, power 40%, work 3s, interval 5s, the time
10min, 3000rpm centrifuge 30s, abandon supernatant, add 2mL PBSs, and clasmatosis liquid is can obtain after mixing.
(4) by clasmatosis liquid and ginkgoic acid C15:The dilution 1mL of 1 standard solution is mixed in 10mL centrifuge tube A
It is even, take PBS and ginkgoic acid C15:Each 1mL of dilution of 1 standard solution mixes conduct pair in 10mL centrifuge tube B
According to.O is passed through into two pipes2About 5min, 5h is reacted in 40 DEG C, pH=5.5, by reacted each group solution vacuum freeze drying extremely
Constant weight (weighing result difference is no more than 0.002g twice in succession), then 2mL isopropanols are separately added into thereto, fully dissolving, use
0.22 μm of organic membrane filtration, and determine the ginkgoic acid C15 after each group is reacted in solution with LC-MS:1 concentration, every group of experiment weight
It is multiple 3 times.It is 0.003 ± 0.045mg/mL that can calculate with the ginkgo acid content in the reacted solution of clasmatosis liquid, and right
It is 0.006 ± 0.042mg/mL according to the ginkgo acid content in group solution.
The clasmatosis of pET21a (+)-sdc/E.coli BL21 (DE3) containing Sdc is understood according to the result of embodiment
Liquid can obviously reduce the content of ginkgoic acid.Its clasmatosis liquid of 1mL makes ginkgoic acid C15 in 40 DEG C, pH=5.5,5h:1 contains
Amount reduces 39.017 ± 0.131%.
The sequence of the sdc genes is：
1 atgcgcggaa aggtttctct cgaggaggcg ttcgagcttc ccaagttcgc tgcccagacc
61 aaggagaagg ccgagctcta catcgccccc aacaaccgcg accggtactt tgaggagatt
121 ctcaacccgt gcggcaaccg tctcgagctt tcgaacaagc acggtatcgg ctacaccatc
181 tactctatct actcgcctgg tccgcaggga tggaccgagc gcgccgagtg tgaggagtac
241 gcgcgcgagt gcaacgacta catctcgggc gagattgcca atcacaagga ccggatgggt
301 gcctttgccg ctctgtcgat gcacgacccc aagcaggcgt ccgaggagct tacccgctgc
361 gttaaagagc tcggtttcct cggcgcgctc gtcaacgacg tgcagcacgc cggacccgaa
421 ggcgagaccc acatcttcta cgaccagccc gagtgggaca tcttctggca gacttgcgtc
481 gatctcgacg ttccattcta cctccacccc gagcctccct tcggctcgta cctccgcaac
541 cagtacgagg gacgcaagta ccttattggt cctcccgtgt cttttgccaa cggcgtctcg
601 ctccacgtcc tcggcatgat cgtcaacggt gtctttgacc gcttccccaa gctcaaggtc
661 atcctcggcc accttggcga gcacattccc ggagacttct ggcgcatcga gcactggttc
721 gagcactgct cccgccctct cgccaagtcg cgcggagacg tcttcgctga gaagcccctc
781 ctccactact tccgcaacaa catctggctc accacctcgg gcaacttctc caccgagact
841 ctcaagttct gcgtcgagca cgtcggcgcc gagcgcatcc tcttctccgt cgactcgcct
901 tacgagcaca tcgacgtcgg atgcggatgg tacgacgaca acgccaaggc tatcatggag
961 gccgttggcg gtgagaaggc ctacaaggac attggccgtg acaacgccaa gaagctcttc
1021 aagctcggca agttctacga ctcggaggct tag
Claims (1)
1. a kind of application of bigcatkin willow acid decarboxylase degraded ginkgoic acid, the temperature of the bigcatkin willow acid decarboxylase degraded ginkgoic acid for 5~
80 DEG C, pH is 3.0~11.0, and the gene order of the bigcatkin willow acid decarboxylase is as described in sequence 1；
The ginkgoic acid is to include ginkgoic acid R=C13:0th, ginkgoic acid R=C15:0th, ginkgoic acid R=C15:1st, ginkgoic acid R=
C17:1st, ginkgoic acid R=C17:2.
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| CN201510052113.0A CN104770624B (en) | 2015-02-02 | 2015-02-02 | A kind of application of bigcatkin willow acid decarboxylase degraded ginkgoic acid |
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| CN106916855A (en) * | 2017-03-01 | 2017-07-04 | 天津科技大学 | The method modified aldehydes matter using carbon dioxide bioconversion method and application |
| CN110241102A (en) * | 2019-06-21 | 2019-09-17 | 济宁学院 | A kind of method of enzymic degradation 2,6- dihydroxy-benzoic acid |
| CN110205316A (en) * | 2019-06-21 | 2019-09-06 | 济宁学院 | A kind of method of enzymic degradation 4-ASA |
| CN110839644B (en) * | 2019-11-27 | 2021-03-02 | 中南林业科技大学 | Method for improving effective and economic yield of ginkgolic acid from ginkgo biloba sarcotesta |
| CN114645039B (en) * | 2022-04-26 | 2024-02-23 | 天津科技大学 | Mutant salicylic acid decarboxylase, strain and application thereof in degradation of ginkgolic acid |
| CN116376936B (en) * | 2023-06-01 | 2023-08-22 | 青岛农业大学 | Method for biosynthesis of ginkgolic acid and gene sequence thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103053876A (en) * | 2012-12-17 | 2013-04-24 | 南京林业大学 | Biodegradation method of ginkgolic acid |
| CN103960555A (en) * | 2014-04-11 | 2014-08-06 | 天津科技大学 | Method for removing ginkgolic acid in gingko biloba seeds |
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| JP5126808B2 (en) * | 2007-02-22 | 2013-01-23 | 学校法人早稲田大学 | Novel microorganism having ability to synthesize aromatic hydroxycarboxylic acid and method for producing aromatic hydroxycarboxylic acid using the microorganism or protein produced by the microorganism |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103053876A (en) * | 2012-12-17 | 2013-04-24 | 南京林业大学 | Biodegradation method of ginkgolic acid |
| CN103960555A (en) * | 2014-04-11 | 2014-08-06 | 天津科技大学 | Method for removing ginkgolic acid in gingko biloba seeds |
Non-Patent Citations (1)
| Title |
|---|
| Enzymatic Kolbe-Schmitt reaction to form salicylic acid from phenol:Enzymatic characterization and gene identification of a novel enzyme,Trichosporon moniliiforme salicylic acid decarboxylase;Kohtaro Kirimura et.al;《Biochemical and Biophysical Research Communications》;20100225;第279页摘要 * |
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