CN107630057A - A kind of method and its dedicated engineering bacteria for producing 2- oxygen-α-D- glycopyranosyl ascorbic acid - Google Patents
A kind of method and its dedicated engineering bacteria for producing 2- oxygen-α-D- glycopyranosyl ascorbic acid Download PDFInfo
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- CN107630057A CN107630057A CN201610565717.XA CN201610565717A CN107630057A CN 107630057 A CN107630057 A CN 107630057A CN 201610565717 A CN201610565717 A CN 201610565717A CN 107630057 A CN107630057 A CN 107630057A
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- ascorbic acid
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 title claims abstract description 201
- 238000000034 method Methods 0.000 title claims abstract description 66
- 241000894006 Bacteria Species 0.000 title claims abstract description 62
- 235000010323 ascorbic acid Nutrition 0.000 title claims abstract description 54
- 239000011668 ascorbic acid Substances 0.000 title claims abstract description 54
- 229960005070 ascorbic acid Drugs 0.000 title claims abstract description 46
- 238000006243 chemical reaction Methods 0.000 claims abstract description 86
- 229930006000 Sucrose Natural products 0.000 claims abstract description 51
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 51
- 239000005720 sucrose Substances 0.000 claims abstract description 51
- 239000002253 acid Substances 0.000 claims abstract description 48
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims abstract description 46
- 229930003268 Vitamin C Natural products 0.000 claims abstract description 46
- 235000019154 vitamin C Nutrition 0.000 claims abstract description 46
- 239000011718 vitamin C Substances 0.000 claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 40
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 30
- 230000035784 germination Effects 0.000 claims abstract description 12
- 229940072107 ascorbate Drugs 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 8
- 239000007791 liquid phase Substances 0.000 claims description 24
- 239000013612 plasmid Substances 0.000 claims description 17
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 16
- 241001013691 Escherichia coli BW25113 Species 0.000 claims description 7
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 125000000539 amino acid group Chemical group 0.000 claims description 4
- 230000008676 import Effects 0.000 claims description 3
- 150000003700 vitamin C derivatives Chemical class 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 10
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract 3
- 229910052760 oxygen Inorganic materials 0.000 abstract 3
- 239000001301 oxygen Substances 0.000 abstract 3
- MLSJBGYKDYSOAE-DCWMUDTNSA-N L-Ascorbic acid-2-glucoside Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)=C1O MLSJBGYKDYSOAE-DCWMUDTNSA-N 0.000 description 43
- 108020004414 DNA Proteins 0.000 description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 16
- 239000000047 product Substances 0.000 description 14
- 239000008363 phosphate buffer Substances 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 9
- 102000053602 DNA Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 241000254173 Coleoptera Species 0.000 description 5
- 241000233866 Fungi Species 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 4
- 108010072454 CTGCAG-specific type II deoxyribonucleases Proteins 0.000 description 4
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 3
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- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 3
- 102100022624 Glucoamylase Human genes 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000004260 Potassium ascorbate Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000000348 glycosyl donor Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 235000019275 potassium ascorbate Nutrition 0.000 description 3
- 229940017794 potassium ascorbate Drugs 0.000 description 3
- CONVKSGEGAVTMB-RXSVEWSESA-M potassium-L-ascorbate Chemical compound [K+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] CONVKSGEGAVTMB-RXSVEWSESA-M 0.000 description 3
- 235000010378 sodium ascorbate Nutrition 0.000 description 3
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 3
- 229960005055 sodium ascorbate Drugs 0.000 description 3
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical group [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
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- 235000005979 Citrus limon Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000051366 Glycosyltransferases Human genes 0.000 description 2
- 108700023372 Glycosyltransferases Proteins 0.000 description 2
- 229920002774 Maltodextrin Polymers 0.000 description 2
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- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 2
- 229940043377 alpha-cyclodextrin Drugs 0.000 description 2
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 229940035034 maltodextrin Drugs 0.000 description 2
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- 239000012488 sample solution Substances 0.000 description 2
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
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- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- GDSOZVZXVXTJMI-SNAWJCMRSA-N (e)-1-methylbut-1-ene-1,2,4-tricarboxylic acid Chemical compound OC(=O)C(/C)=C(C(O)=O)\CCC(O)=O GDSOZVZXVXTJMI-SNAWJCMRSA-N 0.000 description 1
- 208000030090 Acute Disease Diseases 0.000 description 1
- 244000248349 Citrus limon Species 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 239000001116 FEMA 4028 Substances 0.000 description 1
- -1 Glycosyl ascorbic acid Chemical compound 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 241001468192 Leuconostoc citreum Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 1
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- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of method and its dedicated engineering bacteria for producing 2 oxygen α D glycopyranosyl ascorbic acid.The invention provides a kind of method for producing 2 oxygen α D glycopyranosyl ascorbic acid, comprise the following steps：Using Vitamin C acid and sucrose as raw material, in the presence of engineering bacteria, 2 oxygen α D glycopyranosyl ascorbic acid are produced；The engineering bacteria is the recombinant bacterium of expressive function albumen, is that the functional gene for encoding the functional protein is imported out into what bacterium germination obtained；The functional protein is the protein shown in the sequence 1 of sequence table or the protein shown in the sequence 3 of sequence table；The Vitamin C acid is ascorbic acid or ascorbate.Method provided by the invention realizes that a step enzyme method generates AA 2G using cheap sucrose and ascorbic acid as raw material, has the distinguishing features such as gentle, pollution-free, simple, the suitable large-scale industrial production of process route of reaction condition.
Description
Technical field
The present invention relates to a kind of method and its dedicated engineering bacteria for producing 2- oxygen-α-D- glycopyranosyl ascorbic acid.
Background technology
2- oxygen-α-D- glycopyranosyls ascorbic acid (2-O- α-D-Glucopyranosyl-L-ascorbic acid,
AA-2G) it is a kind of important biologic artifact.
AA-2G is ascorbic acid (vitamin C, L-ascorbic acid, AA) a kind of important derivative, is
The compound formed is modified by glucosyl group by the hydroxyl on AA C2 positions, due to having glucosyl group masking on C2 positions,
AA oxidation reaction is not susceptible to, thus it is particularly stable in aqueous, and without direct-reduction property, effectively protect
Its bioactivity is protected.
AA-2G and AA has identical sol original activity, can be used for the synthesis for strengthening collagen in human skin cell,
The acute disease triggered by ultraviolet irradiation can also be prevented and treated.Under the comprehensive function of human enzymes system, AA-2G
D-Glucose and AA very easily are decomposed into, the AA for decomposing to obtain and in general AA has identical physiological function, D-
Glucose can also be absorbed by the body, so AA-2G securities are high.AA-2G can both keep the stability of AA molecules,
It can be made to be converted into the enediol structure of active function in human body cell again, be optimal AA substitutes, at present
It is widely used in the industries such as cosmetics, food, health care and animal husbandry and aquaculture.
At present, the whole world is applied to AA-2G almost all in cosmetics by one offer of Japanese Lin Yuan companies, its
In the market occupies the status of monopolization.AA-2G price is about 1000 yuan/kg, is probably more than 50 times of ascorbic acid,
Its market value is huge.China is the big producer of ascorbic acid, but the product of ascorbic acid about 3/4ths is used for
Outlet, domestic product are mainly used in medicine, and kind is more dull, for ascorbic acid derivates product (such as AA-2G)
Production be still in the desk study stage.
AA-2G chemical synthesis is very difficult, mainly enzymatic conversion method is used at present, first with glycosyl transferase
(glycosyl-transferase) by glucosyl group donor, (such as starch, cyclodextrin, malt are pasted for transglycosylation
Essence, maltose etc.) on glucoside be transferred on AA C2 positions, according to the difference of the glucose chain length of connection,
It is AA-2Gn (n=1,2,3,4,5,6,7) to obtain product, recycles glucoamylase (glucoamylase)
It is into the AA-2G for only connecting a glucose group by sugar chain degradation longer AA-2Gn.Due to cyclodextrin glucose base
The strong product specificities of transferase (CGTase), become most popular enzyme in current AA-2G biosynthesis
Kind, for example, Chen Jian of Southern Yangtze University et al. utilizes improved CGTase, improve its substrate specificity to maltodextrin
Property, and AA-2G is produced using maltodextrin as glycosyl donor, yield reaches 1.66g/L.Another someone by transforming CGTase,
Its substrate specificity to maltose is improved, and AA-2G is produced by glycosyl donor of maltose, yield reaches 1.01g/L.
The subject matter for being produced AA-2G using CGTase enzymes in the prior art is：Generally with alpha-cyclodextrin or β-
Cyclodextrin is glycosyl donor, but alpha-cyclodextrin cost is too high, and beta-schardinger dextrin solubility is relatively low；(2) pasted for malt
The lower-cost substrate specificity such as essence, maltose is poor, and conversion ratio is low；(3) the product obtained using CGTase is AA-2Gn,
The degraded of glucoamylase is also needed to, adds the cost of enzyme.
In summary, there is an urgent need to develop new efficient production AA-2G method for this area.
The content of the invention
It is an object of the invention to provide a kind of method for producing 2- oxygen-α-D- glycopyranosyl ascorbic acid and its special
Engineering bacteria.
The invention provides a kind of method for producing 2- oxygen-α-D- glycopyranosyl ascorbic acid, comprise the following steps：
Using Vitamin C acid and sucrose as raw material, in the presence of engineering bacteria, production 2- oxygen-α-D- glycopyranosyls resist
Bad hematic acid；The engineering bacteria is the recombinant bacterium of expressive function albumen, is to lead the functional gene for encoding the functional protein
Enter out what bacterium germination obtained；The Vitamin C acid is ascorbic acid or ascorbate.
The functional protein is following (a) or (b) or (c)：
(a) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(b) protein being made up of the amino acid sequence shown in sequence in sequence table 3；
(c) by the amino acid sequence of sequence 1 or the amino acid sequence of sequence 3 by one or several amino acid residues
Substitution and/or missing and/or addition and its derivative protein with identical function.
The ascorbate is sodium ascorbate or potassium ascorbate.
The functional gene is following DNA molecular 1) or 2) or 3)：
1) DNA molecular of the code area as shown in sequence 2 in sequence table；
2) DNA molecular of the code area as shown in sequence 4 in sequence table；
1) or 2) 3) under strict conditions with the DNA sequence dna hybridization that limits and encoding the DNA molecular of the functional protein；
4) 1) or 2) with the DNA sequence dna that limits at least with 70%, at least with 75%, at least with 80%, extremely
Less with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least with
98% or at least there is the DNA molecular of more than 99% homology and the coding functional protein.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution, in DNA or RNA
Hybridize in hybrid experiment at 65 DEG C and wash film.
In methods described, the quality proportioning of Vitamin C acid and sucrose is as follows：1-3：1-3.
In methods described, the quality proportioning of Vitamin C acid and sucrose is as follows：1:1.
In methods described, the quality proportioning of Vitamin C acid and sucrose is as follows：1:3.
In methods described, the quality proportioning of Vitamin C acid and sucrose is as follows：3:1.
In methods described, the quality proportioning of engineering bacteria, Vitamin C acid and sucrose is as follows：10：100-300：
100-300。
In methods described, the quality proportioning of engineering bacteria, Vitamin C acid and sucrose is as follows：10：200：200.
In methods described, the quality proportioning of engineering bacteria, Vitamin C acid and sucrose is as follows：10：100：300.
In methods described, the quality proportioning of engineering bacteria, Vitamin C acid and sucrose is as follows：10：300：100.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
100-300g/L, the concentration of sucrose is 100-300g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
200g/L, the concentration of sucrose is 200g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
100g/L, the concentration of sucrose is 300g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
300g/L, the concentration of sucrose is 100g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of engineering bacteria is 1-100g/L (tools
Body can be 10g/L), the concentration of Vitamin C acid is 100-300g/L, and the concentration of sucrose is 100-300g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of engineering bacteria is 10g/L, anti-bad
The concentration of blood acid is 200g/L, and the concentration of sucrose is 200g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of engineering bacteria is 10g/L, anti-bad
The concentration of blood acid is 100g/L, and the concentration of sucrose is 300g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of engineering bacteria is 10g/L, anti-bad
The concentration of blood acid is 300g/L, and the concentration of sucrose is 100g/L.
The pH of the liquid-phase reaction system is 6-8.The pH of the liquid-phase reaction system is 6-7, concretely 6.0,
6.5 or 7.0.
The solvent of the liquid-phase reaction system is phosphate buffer (PBS) or acetate buffer.
The phosphate buffer is 50mM PBS.
The liquid-phase reaction system is made up of engineering bacteria, Vitamin C acid, sucrose and phosphate buffer.
In methods described, reaction condition is：20℃-50℃、40h-90h.
In methods described, reaction condition is：30℃-50℃、40h-90h.
In methods described, reaction condition is：30℃-37℃、40h-60h.
In methods described, reaction condition is：30℃、60h.
In methods described, reaction condition is：37℃、40h.
In methods described, reaction condition is：50℃、90h.
In methods described, reacted under oscillating condition.Concretely 80rpm vibrates for the vibration.
It is described go out bacterium germination can be Escherichia coli.It is described go out bacterium germination can be Escherichia coli BW25113.It is described go out bacterium germination may be used also
For BL21 series, Rosetta series, BW25113 series, Origami series.
The functional gene imports out bacterium germination by recombinant plasmid；The recombinant plasmid is to insert out the functional gene
Hair carrier obtains.The recombinant plasmid is concretely：By carrier pBAD/HisB XhoI and PstI restriction enzyme sites
Between small fragment substitute in order to shown in the double chain DNA molecule shown in the sequence 2 of sequence table or the sequence of sequence table 4
Double chain DNA molecule, obtained recombinant plasmid.The carrier that sets out can also be pET series, pETduet series, pTXB1
Series, pTRB1 series, pTYB1 series plasmids.
The engineering bacteria specifically can first be handled as follows：
(1) by recombinant to OD600nm=0.6-0.8 (concretely 0.7)；
(2) after completing step (1), addition L-arabinose is induced in cultivating system；
(3) after completing step (2), engineering bacteria is collected by centrifugation.
The engineering bacteria specifically can first be handled as follows：
(1) engineering bacteria is seeded to fluid nutrient medium (such as LB culture mediums or M9 culture mediums), shaken cultivation to OD600nm=
0.6-0.8 (concretely 0.7)；
(2) after completing step (1), L-arabinose is added in cultivating system and makes it dense in cultivating system
Spend for 0.2g/100mL, shaken cultivation 12 hours；
(3) after completing step (2), engineering bacteria is collected by centrifugation.
The engineering bacteria specifically can first be handled as follows：
(1) engineering bacteria is seeded to LB fluid nutrient mediums, 37 DEG C, 220rpm shaken cultivations to OD600nm=0.6-0.8
(concretely 0.7)；
(2) after completing step (1), L-arabinose is added in cultivating system and makes it dense in cultivating system
Spend for 0.2g/100mL, 30 DEG C, 200rpm shaken cultivations 12 hours；
(3) after completing step (2), a cultivating system is rounded, 4 DEG C, 6000rpm centrifugation 15min, collects cell
Precipitation, as engineering bacteria.
The present invention also protects imports out the engineering bacteria that bacterium germination obtains by recombinant plasmid；The recombinant plasmid is by the function
Gene insertion sets out what carrier obtained；It is described go out bacterium germination be Escherichia coli BW25113；The carrier that sets out is carrier
pBAD/HisB。
The recombinant plasmid is concretely：By the small pieces between carrier pBAD/HisB XhoI and PstI restriction enzyme sites
Section substitution is for the double chain DNA molecule shown in the sequence 2 of sequence table or the double-stranded DNA shown in the sequence of sequence table 4 point
Son, obtained recombinant plasmid.
The present invention also protects application of the recombinant bacterium in 2- oxygen-α-D- glycopyranosyl ascorbic acid is prepared.
In the application, using Vitamin C acid and sucrose as raw material.
The Vitamin C acid is ascorbic acid or ascorbate.
The ascorbate is sodium ascorbate or potassium ascorbate.
The quality proportioning of Vitamin C acid and sucrose is as follows：1-3：1-3.
The quality proportioning of Vitamin C acid and sucrose is as follows：1:1.
The quality proportioning of Vitamin C acid and sucrose is as follows：1:3.
The quality proportioning of Vitamin C acid and sucrose is as follows：3:1.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
100-300g/L, the concentration of sucrose is 100-300g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
200g/L, the concentration of sucrose is 200g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
100g/L, the concentration of sucrose is 300g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
300g/L, the concentration of sucrose is 100g/L.
The present invention also protects a kind of method for producing 2- oxygen-α-D- glycopyranosyl ascorbic acid, comprises the following steps：
Using Vitamin C acid and sucrose as raw material, in the presence of the functional protein, 2- oxygen-α-D- pyrans Portugal is produced
Glycosyl ascorbic acid；The Vitamin C acid is ascorbic acid or ascorbate.
The ascorbate is sodium ascorbate or potassium ascorbate.
In methods described, the quality proportioning of Vitamin C acid and sucrose is as follows：1-3：1-3.
In methods described, the quality proportioning of Vitamin C acid and sucrose is as follows：1:1.
In methods described, the quality proportioning of Vitamin C acid and sucrose is as follows：1:3.
In methods described, the quality proportioning of Vitamin C acid and sucrose is as follows：3:1.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
100-300g/L, the concentration of sucrose is 100-300g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
200g/L, the concentration of sucrose is 200g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
100g/L, the concentration of sucrose is 300g/L.
Liquid-phase reaction system is used in methods described；In initial reaction system, the concentration of Vitamin C acid is
300g/L, the concentration of sucrose is 100g/L.
The pH of the liquid-phase reaction system is 6-7, concretely 6.0,6.5 or 7.0.
The solvent of the liquid-phase reaction system is phosphate buffer.
The phosphate buffer is 50mM phosphate buffer.
In methods described, reaction condition is：30℃-50℃、40h-90h.
In methods described, reaction condition is：30℃、60h.
In methods described, reaction condition is：37℃、40h.
In methods described, reaction condition is：50℃、90h.
In methods described, reacted under oscillating condition.Concretely 80rpm vibrates for the vibration.
The existence form of the functional protein can be：Complete microbial cell, clasmatosis liquid, thick enzyme or pure enzyme.
The present invention is provided using cheap sucrose and ascorbic acid as raw material, realizes that a step enzyme method generates AA-2G, tool
There are the distinguishing features such as gentle, pollution-free, simple, the suitable large-scale industrial production of process route of reaction condition.
Brief description of the drawings
Fig. 1 is the structural representation of 2- oxygen-α-D- glycopyranosyl ascorbic acid.
Fig. 2 is the structural representation of ascorbic acid.
Fig. 3 is the chromatogram of AA Standard product and AA-2G standard items.
In the step of Fig. 4 is embodiment 1 two, the reaction system I for completing step 4 carries out the chromatogram of step 5.
Fig. 5 is 6 result of step 2 in embodiment 1.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, unless otherwise specified,
It is to be commercially available from routine biochemistry reagent shop.Quantitative test in following examples, is respectively provided with and repeats reality three times
Test, results averaged.Unless otherwise specified, the phosphate buffer in embodiment is 50mM PBS.
For the chromatogram of the different secondary responses under identical conditions parameter, target peak retention time has certain error model
Enclose, general differ in 0.1min can be considered as error.
Lemon yellow leukonid (Leuconostoc citreum)：CGMCC1.2461.Bifidobacterium adolescentis
(Bifidobacterium adolescentis)：CGMCC 1.2190.Carrier pBAD/HisB：Invitrogen is public
Department, catalog number V430-01.Escherichia coli BW25113：Biovector NTCC INC, article No. 355297.
Ascorbic acid (also known as AA, ascorbic acid standard items or AA Standard product)：Aladdin companies, catalog number A103537.
2- oxygen-α-D- glycopyranosyls ascorbic acid (also known as AA-2G, 2- oxygen-α-D- glycopyranosyl ascorbic acid standard items
Or AA-2G standard items)：Sigma companies, catalog number SMB00390.The implication of ascorbic acid in the present invention is equal
For L-AA." g " in cell concentration " g/L " in embodiment represents wet cell weight.2- oxygen-α-D-
The structural representation of glycopyranosyl ascorbic acid is shown in Fig. 1.The structural representation of ascorbic acid is shown in Fig. 2.
Embodiment 1, structure recombinant bacterium and application recombinant bacterium prepare AA-2G
First, recombinant bacterium is built
1st, the genomic DNA of lemon yellow leukonid is extracted.
2nd, using the genomic DNA that step 1 obtains as template, performing PCR amplification is entered using the F1 and R1 primer pairs formed,
Reclaim pcr amplification product.
F1：5’-GCCTGGTGCCGCGCGGCAGCCTCGAGATGGAAATTCAAAACAAAGCAATGC-3’；
R1：5’-CAGCTGCAGACCGAGCTCACCCTGCAGTTATTTGTTTTGTAAGACTGTCTTG-3’。
3rd, the pcr amplification product for taking step 2 to obtain, double digestion is carried out using restriction enzyme XhoI and PstI,
Reclaim digestion products.
4th, with restriction enzyme XhoI and PstI double digestion carrier pBAD/HisB, recovery about 4000bp carrier
Skeleton.
5th, the digestion products of step 3 are connected with the carrier framework of step 4, obtains recombinant plasmid first.
According to sequencing result, structure is carried out to recombinant plasmid first and is described as follows：By carrier pBAD/HisB XhoI and
Small fragment between PstI restriction enzyme sites substitutes the double chain DNA molecule shown in for the sequence 2 of sequence table.Sequence table
Protein shown in the sequence 1 of DNA molecular polynucleotide shown in sequence 2.
6th, recombinant plasmid first is imported into Escherichia coli BW25113, obtains recombinating fungus beetle.
Recombinate in fungus beetle, the protein shown in intracellular expression sequence 1.
7th, carrier pBAD/HisB is imported into Escherichia coli BW25113, obtains recombinant bacterium second.
2nd, AA-2G (step enzyme method) is prepared using recombinant bacterium
1st, the monoclonal of restructuring fungus beetle is taken, is seeded to LB fluid nutrient mediums, 37 DEG C, 220rpm shaken cultivations to OD600nm=
0.7 (in practical application, OD600nm=0.6-0.8).
2nd, after completing step 1, L-arabinose is added in cultivating system and makes its concentration in cultivating system be
0.2g/100mL, 30 DEG C, 200rpm shaken cultivations 12 hours.
3rd, after completing step 2, a cultivating system is rounded, 4 DEG C, 6000rpm centrifugation 15min, collects cell precipitation.
4th, prepare different reaction systems and reacted using different reaction conditions.
Cell precipitation, ascorbic acid, sucrose and the phosphate buffer that reaction system is obtained by step 3 form.
In reaction system I, the initial concentration of each component is as follows：Cell 10g/L, ascorbic acid 200g/L, sucrose 200g/L.
In reaction system I, using pH6.0 phosphate buffer.Reaction condition：30 DEG C, 80rpm vibrations 60h.
In reaction system II, the initial concentration of each component is as follows：Cell 10g/L, ascorbic acid 100g/L, sucrose 300g/L.
In reaction system II, using pH6.5 phosphate buffer.Reaction condition：37 DEG C, 80rpm vibrations 40h.
In reaction system III, the initial concentration of each component is as follows：Cell 10g/L, ascorbic acid 300g/L, sucrose 100g/L.
In reaction system III, using pH7.0 phosphate buffer.Reaction condition：50 DEG C, 80rpm vibrations 90h.
5th, after completing step 4, reaction system is taken, AA-2G contents therein is detected, comprises the following steps that：
(1) reaction system is taken, 12000rpm centrifugation 2min, collects supernatant.
(2) supernatant obtained with distilled water diluting step (1), obtains sample solution.
(3) sample solution for taking step (2) to obtain, AA-2G contents are detected using high performance liquid chromatography.
HPLC system：Agilent 1260；Chromatographic column：Agilent ZORBAX SB-C18；
Mobile phase：KH2PO4-H3PO4Buffer solution (pH2.0,20mM)；Flow velocity：0.4mL/min；
Temperature：25℃；
Detector：DAD；Detection wavelength：254nm.
The chromatogram of AA Standard product and AA-2G standard items is shown in Fig. 3.The peak position that goes out of AA Standard product is 2.348min.
The peak position that goes out of AA-2G standard items is 3.048min.
The calibration curve equation that AA-2G contents and peak area are established with AA-2G standard items is as follows：Y=3380.8x+18.13
(R2=0.9999)；Wherein x is the peak area in HPLC chromatogram, and y is AA-2G concentration, unit g/L.
Complete step 4 reaction system I carry out step 5 chromatogram see Fig. 4 (respectively in 2.348min and
3.048min show appearance).The concentration for completing AA-2G in the reaction system I of step 4 is 140g/L (10 repetitions
The average value of experiment).
The concentration for completing AA-2G in the reaction system II of step 4 is 120g/L (average value of 10 repetition experiments).
The concentration for completing AA-2G in the reaction system III of step 4 is 100g/L (average value of 10 repetition experiments).
6th, AA-2G is prepared using recombinant bacterium
Cell precipitation, ascorbic acid, sucrose and the phosphate buffer that reaction system is obtained by step 3 form.
In reaction system, the initial concentration of each component is as follows：Cell 10g/L, ascorbic acid 300g/L, sucrose 100g/L.
In reaction system, using pH7.0 phosphate buffer.Reaction condition：50 DEG C, 80rpm vibrations.
Sampled at interval of 5 hours, AA-2G contents are detected according to the method for step 5.
As a result Fig. 5 (average value of 10 repetition experiments) is seen.
Embodiment 2, structure recombinant bacterium and application recombinant bacterium prepare AA-2G
First, recombinant bacterium is built
Recombinant plasmid third is prepared.According to sequencing result, structure is carried out to recombinant plasmid third and is described as follows：By carrier
Small fragment between pBAD/HisB XhoI and PstI restriction enzyme sites substitutes in order to double shown in the sequence 4 of sequence table
Ssdna molecule.Protein shown in the sequence 3 of DNA molecular polynucleotide shown in the sequence 4 of sequence table.Sequence
Double chain DNA molecule shown in the sequence 4 of list is to expand to obtain from the genomic DNA of bifidobacterium adolescentis.
Recombinant plasmid third is imported into Escherichia coli BW25113, obtains recombinant bacterium third.
2nd, AA-2G (step enzyme method) is prepared using recombinant bacterium
Restructuring fungus beetle is replaced with recombinant bacterium third, successively the 1 to 5 of the step of progress embodiment 1 two.
The concentration for completing AA-2G in the reaction system I of step 4 is 100g/L (average value of 10 repetition experiments).
The concentration for completing AA-2G in the reaction system II of step 4 is 95g/L (average value of 10 repetition experiments).
The concentration for completing AA-2G in the reaction system III of step 4 is 80g/L (average value of 10 repetition experiments).
Comparative example,
Replaced recombinating fungus beetle with recombinant bacterium second, successively the 1 to 5 of the step of progress embodiment 1 two.
The concentration for completing AA-2G in the reaction system I of step 4 is 0g/L.
The concentration for completing AA-2G in the reaction system II of step 4 is 0g/L.
The concentration for completing AA-2G in the reaction system III of step 4 is 0g/L.
Claims (10)
1. a kind of method for producing 2- oxygen-α-D- glycopyranosyl ascorbic acid, comprises the following steps：With Vitamin C
Acid and sucrose are raw material, in the presence of engineering bacteria, produce 2- oxygen-α-D- glycopyranosyl ascorbic acid；
The engineering bacteria is the recombinant bacterium of expressive function albumen, is to import out the functional gene for encoding the functional protein
What bacterium germination obtained；
The functional protein is following (a) or (b) or (c)：
(a) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(b) protein being made up of the amino acid sequence shown in sequence in sequence table 3；
(c) by the amino acid sequence of sequence 1 or the amino acid sequence of sequence 3 by one or several amino acid residues
Substitution and/or missing and/or addition and its derivative protein with identical function；
The Vitamin C acid is ascorbic acid or ascorbate.
2. the method as described in claim 1, it is characterised in that：In methods described, Vitamin C acid and sucrose
Quality proportioning it is as follows：1-3：1-3.
3. the method as described in claim 1, it is characterised in that：In methods described, engineering bacteria, Vitamin C acids thing
Matter and the quality proportioning of sucrose are as follows：10：100-300：100-300.
4. the method as described in claim 1, it is characterised in that：Liquid-phase reaction system is used in methods described；Initially
In reaction system, the concentration of Vitamin C acid is 100-300g/L, and the concentration of sucrose is 100-300g/L.
5. the method as described in claim 1, it is characterised in that：Liquid-phase reaction system is used in methods described；Initially
In reaction system, the concentration of engineering bacteria is 1-100g/L, and the concentration of Vitamin C acid is 100-300g/L, sucrose
Concentration be 100-300g/L.
6. the method as described in any in claim 1 to 5, it is characterised in that：In methods described, reaction condition is：
30℃-50℃、40h-90h。
7. the method as described in any in claim 1 to 6, it is characterised in that：It is described go out bacterium germination be Escherichia coli
BW25113。
8. recombinant plasmid is imported into out the recombinant bacterium that bacterium germination obtains；The recombinant plasmid is that functional gene is inserted into load of setting out
What body obtained；It is described go out bacterium germination be BW25113；The carrier that sets out is carrier pBAD/HisB；The functional gene is
The gene of encoding function albumen；
The functional protein is following (a) or (b) or (c)：
(a) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(b) protein being made up of the amino acid sequence shown in sequence in sequence table 3；
(c) by the amino acid sequence of sequence 1 or the amino acid sequence of sequence 3 by one or several amino acid residues
Substitution and/or missing and/or addition and its derivative protein with identical function.
9. application of the recombinant bacterium described in claim 8 in 2- oxygen-α-D- glycopyranosyl ascorbic acid is prepared.
10. a kind of method for producing 2- oxygen-α-D- glycopyranosyl ascorbic acid, comprises the following steps：With Vitamin C
Acid and sucrose are raw material, in the presence of functional protein, produce 2- oxygen-α-D- glycopyranosyl ascorbic acid；
The functional protein is following (a) or (b) or (c)：
(a) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(b) protein being made up of the amino acid sequence shown in sequence in sequence table 3；
(c) by the amino acid sequence of sequence 1 or the amino acid sequence of sequence 3 by one or several amino acid residues
Substitution and/or missing and/or addition and its derivative protein with identical function；
The Vitamin C acid is ascorbic acid or ascorbate.
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| WO2014060452A1 (en) * | 2012-10-18 | 2014-04-24 | Universiteit Gent | A thermostable sucrose and sucrose-6'-phosphate phosphorylase |
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| WO2014060452A1 (en) * | 2012-10-18 | 2014-04-24 | Universiteit Gent | A thermostable sucrose and sucrose-6'-phosphate phosphorylase |
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| TAEYEON KWON等: "Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase from Bifidobacterium longum", 《BIOTECHNOLOGY LETTERS》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913641A (en) * | 2018-06-29 | 2018-11-30 | 浙江工业大学 | A kind of recombination bacillus coli and its application |
| CN108913641B (en) * | 2018-06-29 | 2021-02-02 | 浙江工业大学 | Recombinant escherichia coli and application thereof |
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