CN103923959B - A kind of D Tagatose production methods based on enzymatic isomerization reaction and continuous chromatography separation coupling in situ - Google Patents
A kind of D Tagatose production methods based on enzymatic isomerization reaction and continuous chromatography separation coupling in situ Download PDFInfo
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- 238000000926 separation method Methods 0.000 title claims abstract description 16
- 238000006317 isomerization reaction Methods 0.000 title claims abstract description 15
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 14
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 13
- 238000010168 coupling process Methods 0.000 title claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 title claims description 24
- 238000004587 chromatography analysis Methods 0.000 title claims description 10
- 230000008878 coupling Effects 0.000 title claims description 9
- 238000005859 coupling reaction Methods 0.000 title claims description 9
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 title abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 55
- 238000006243 chemical reaction Methods 0.000 claims abstract description 46
- 238000004088 simulation Methods 0.000 claims abstract description 43
- 108090000790 Enzymes Proteins 0.000 claims abstract description 29
- 102000004190 Enzymes Human genes 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000000463 material Substances 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108010018080 L-arabinose isomerase Proteins 0.000 claims abstract description 8
- LKDRXBCSQODPBY-OEXCPVAWSA-N D-tagatose Chemical compound OCC1(O)OC[C@@H](O)[C@H](O)[C@@H]1O LKDRXBCSQODPBY-OEXCPVAWSA-N 0.000 claims description 70
- 239000000243 solution Substances 0.000 claims description 22
- 230000000694 effects Effects 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 108090000769 Isomerases Proteins 0.000 claims description 4
- 102000004195 Isomerases Human genes 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 4
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 4
- 239000001488 sodium phosphate Substances 0.000 claims description 4
- 235000011008 sodium phosphates Nutrition 0.000 claims description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 230000033228 biological regulation Effects 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 claims 1
- 230000003139 buffering effect Effects 0.000 claims 1
- 229930182830 galactose Natural products 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 8
- 238000007445 Chromatographic isolation Methods 0.000 abstract description 4
- 238000010924 continuous production Methods 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- XDBZPHDFHYZHNG-UHFFFAOYSA-L disodium 3-[(5-chloro-2-phenoxyphenyl)diazenyl]-4-hydroxy-5-[(4-methylphenyl)sulfonylamino]naphthalene-2,7-disulfonate Chemical compound [Na+].[Na+].C1=CC(C)=CC=C1S(=O)(=O)NC(C1=C2O)=CC(S([O-])(=O)=O)=CC1=CC(S([O-])(=O)=O)=C2N=NC1=CC(Cl)=CC=C1OC1=CC=CC=C1 XDBZPHDFHYZHNG-UHFFFAOYSA-L 0.000 abstract 2
- 150000003474 tagatoses Chemical class 0.000 abstract 1
- 238000009826 distribution Methods 0.000 description 8
- GZCGUPFRVQAUEE-KCDKBNATSA-N aldehydo-D-galactose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 description 7
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 235000013305 food Nutrition 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
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- FBPFZTCFMRRESA-GUCUJZIJSA-N galactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-GUCUJZIJSA-N 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- -1 alkali metal salt Chemical class 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 238000011953 bioanalysis Methods 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
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- 239000011734 sodium Substances 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- GMWTXQKKRDUVQG-WOPPDYDQSA-N 4-amino-5-bromo-1-[(2r,3s,4s,5r)-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-yl]pyrimidin-2-one Chemical compound C[C@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C(Br)=C1 GMWTXQKKRDUVQG-WOPPDYDQSA-N 0.000 description 1
- 101100264195 Caenorhabditis elegans app-1 gene Proteins 0.000 description 1
- LKDRXBCSQODPBY-VRPWFDPXSA-N D-fructopyranose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-VRPWFDPXSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 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 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
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- 150000001299 aldehydes Chemical class 0.000 description 1
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- JSAIENUMNDAGTD-UHFFFAOYSA-N benzene ethene styrene Chemical compound C1=CC=CC=C1.C=C.C=C.C=CC1=CC=CC=C1 JSAIENUMNDAGTD-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
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- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
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- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to biology and chemical field, particularly discloses one kind and is based on enzymatic isomerization reaction and chromatographic isolation original position coupling technique, the method using D galactolipins as raw material continuous production D Tagatoses, comprises the following steps：Configure cushioning liquid；Thermophilic L Arabinose isomerases are fully dissolved in water, as enzyme solutions；D galactolipins are fully dissolved in water, as material liquid；After material liquid, cushioning liquid, the blended preheating of enzyme solutions, as simulation moving bed reactor（SMBR）Feeding liquid；Feeding liquid enters simulation moving bed reactor, after reaction in-situ and separation, obtains D Tagatose product solutions.The present invention establishes the suitable operating conditions of SMBR, and D galactolipin conversion ratios reach more than 80%, D Tagatose purity and reach more than 95%, and yield is more than 80%.Method cost that the present invention announces is low, pollution less, high conversion rate, and continuous production can be realized, advantageously ensure that the homogeneity of product quality, there is good commercial applications prospect.
Description
Technical field
The present invention relates to biological chemical field, and in particular to one kind is former based on enzymatic isomerization reaction and continuous chromatography separation
The D-Tag production method of position coupling.
Background technology
Tagatose is that a kind of six-carbon ketone is sugared, molecular formula C6H12O6, be fructose epimer, and the aldehyde of galactolipin
Ketone isomer.Tagatose molecular weight 180g/mol, it is white crystal under normality, fusing point and glass transition temperature are respectively 134
DEG C and 15 DEG C, be dissolved in water, be slightly soluble in ethanol, absolute acid stability is good.D-Tag has mouth compared with the sweeteners such as xylitol
The characteristics of taste is more preferable, processability is stronger, health-care effect is more and security is higher, it is a kind of excellent sweetening agent.D- towers
Lattice sugar 2001 by Food and Drug Administration（US FDA）Official approval is 2005 it is widely recognized that safety food (GRAS)
Listed by European Union's approval in Europe.D-Tag obtains a wide range of applications in fields such as food, beverage, shield tooth products
（Kruger et al., Regul. Toxicol. Pharmacol., 1999 29(2), pS29-35; Levin, J.
Medc. Food, 2002 5(1), p23-26）.
D-Tag belongs to rare saccharide, it is difficult to directly largely be obtained from nature, can only be prepared by artificial means.
Industrialized D-Tag production mainly uses chemical method at present, and galactolipin is changed into Tagatose in aqueous, passed through after
Separating step is purified（Beadle et al., US Patent, 5002612, 1991;Fabrice, WO Patent,
066192, 2005）.The subject matter of chemical method is to use alkali metal salt or alkaline-earth metal salt catalyst, and accessory substance is numerous, gives
Follow-up purification process causes difficulty, while will also result in more serious environmental pollution.
The development trend of D-Tag production is to use bioanalysis, and approach mainly has two kinds.One kind is to utilize Institute of Micro-biology
Galactitol is oxidized to Tagatose, Izumori etc., Munimzzaman etc. and Manzoni etc. and found respectively by caused dehydrogenase
Galactitol can be oxidized to Tagatose by different bacterial strains（App1. Environ. Microbiol, 1984 46:
p1055-1057; ProcessBiochem., 2001, 36 p971-977; Ferment Bioeng., 1995 79
p620-622）.It is but simultaneously little as the galactitol selling at exorbitant prices of raw material, the commercial application value of this method.It is another
Approach is then into Tagatose using isomerase by cheap galactolipin isomery.Cheetham etc. utilizes Lactobacillus
L-arabinose isomerase successful conversion galactolipin generation Tagatose caused by gayonii metabolism, propose to utilize lactic acid bacteria first
Produce the imagination of Tagatose（Enzyme Micro. Techno1., 1993 15 p105-108）.Zhang Hua et al. obtains from pickles
Obtain the lactic acid bacteria SKI.002 of high yield Tagatose（Food and fermentation industries, 2,006 32 (6):5-7）.Weng Weihui etc. is from screening
L-arabinose isomerase is obtained in lactic acid bacteria（Food industry science and technology, 2006 (1):158-162）.At present, D- galactolipins are different
L-arabinose isomerase needed for structure generation D-Tag has been obtained for more sufficiently researching and developing.
D- galactose isomerizations generation D-Tag is reversible reaction, can be expressed as：
Wherein A is reactant D- galactolipins, and B is isomery enzyme catalyst, and C is product D-Tag.Conversion ratio, product solution
The definition of purity and yield is：
Traditional biological method uses following technological process by D- galactolipins D-Tag：
D- galactolipins --- ultrafiltration --- counter-infiltration --- micro-filtration --- hydrolysis --- concentration --- fermentation --- isomery
Change --- concentration --- crystallization --- D-Tag finished product
This flow fails to realize that industrialized main restricting factor has at 2 points：When from technological level, D- galas
The conversion ratio of sugar is relatively low.Conventional art is mainly set about in terms of the bacterium source of enzyme, it is intended to improves conversion ratio.However, can be converse
Conversion ratio is answered by Equilibrium limit.In theory, any catalyst（Enzyme）This limitation can not all be broken.Therefore, this method
Conversion ratio is often limited in 40% or so.Second, from the mode of production, bioanalysis uses traditional batch operation.With can
The continuous accumulation of product in back reaction, the activity of enzyme gradually reduce, and back reaction influence is increasing, causes reaction time very long
（Usually 2-3 days）, the problem of production efficiency is low, while be also difficult to ensure that the homogeneous of differential responses cycle products obtained therefrom quality
Property.
To improve the conversion ratio of D- galactolipins, Chinese invention patent 200780045683.0 is by adding borate and product
D-Tag combines, and chemical balance is moved right, D- galactolipin conversion ratios are brought up to 70% level.But this method is still
Railway Project be present.First, borate is poisonous, food additives production is not suitable for；Second, added down after addition borate
Trip isolates and purifies the burden of unit；Third, using batch tank reactor carry out, reaction time still more than 20 hours, conversion ratio
Can not further it improve.
In recent years, SMBC technology starts to be employed the production process with D-Tag.As Chinese invention is special
It is related to extracting Tagatose from complicated carbohydrate admixture in sharp 201080017863.X, the production process is a pure point
From process, it is not related to reacting.It is related to using Simulation moving bed knockout tower lattice in Chinese invention patent 201210329027.6
Sugar and galactolipin.But in this process, Simulation moving bed and batch tank reactor are series relationship, what Simulation moving bed played
It is still pure centrifugation, i.e., separates product D-Tag and unreacted raw material sugar or other accessory substances.It is because anti-
Still it should be carried out in batch tank reactor, this kind separation can not change chemical balance, can not be used to improve D- galactolipins
To the conversion ratio of D-Tag, the yield of D-Tag is only 40%.
The content of the invention
In view of the shortcomings of the prior art, the technical problems to be solved by the invention are to provide one kind with D- galactolipins to D- towers
The Efficient Conversion method of lattice sugar, using simulation moving bed reactor, with reference to suitable operating condition, make enzymatic isomerization reaction and
Continuous chromatography separation coupling in situ；The place in situ that Simulation moving bed is carried out as enzymatic isomerization reaction, product and reaction
Thing separates immediately, so as to overcome the influence of equilibrium conversion and product feedback inhibition, before without using toxicity additive
Put, reach the purpose for improving D- galactolipin conversion ratios, while directly obtain the D-Tag product of high-purity.It is amazing
It is that, using the method for the present invention, D-Tag of the purity more than 95% can directly be made from D- galactolipins, while cause D- half
The conversion ratio of lactose and the yield of D-Tag reach more than 80%, and can be by the conversion ratio and D-Tag of D- galactolipins
Yield be further increased to 95%.
Therefore, the technical solution adopted by the present invention is as follows：
One kind is based on enzymatic isomerization reaction and chromatographic isolation coupling in situ, using D- galactolipins as raw material continuous production D- towers
The method of lattice sugar, comprises the following steps：
(1) cushioning liquid of pH value 6~7.4 is configured；
(2) thermophilic L-arabinose isomerase is fully dissolved in partial buffer solution, as enzyme solutions；
(3) D- galactolipins are fully dissolved in water, as material liquid；
(4) by after the blended preheating of cushioning liquid, enzyme solutions and material liquid respectively obtained in step (1), (2), (3),
As feeding liquid, target product D-Tag is free of in described feeding liquid；
(5) feeding liquid obtained in step (4) is entered into simulation moving bed reactor, after reaction in-situ and separation, directly
Connect to obtain D-Tag product solution.
Preferably, contain 8~8.5g of sodium chloride, phosphoric acid hydrogen in every liter of the cushioning liquid configured in the step (1)
0.2~0.5g of 30~50g of sodium and sodium dihydrogen phosphate, regulation pH value to 6~7.4；
Preferably, L-arabinose isomerase is to pass through clone from e. coli k12 genome in the step (2)
Crude enzyme liquid made from method expression Arabinose isomerase；
Preferably, it is 10~150 g/l to match somebody with somebody D- galactose concentrations in feeding liquid in the step (4), enzyme activity list
Position is 0.1~0.28 U/ml, and pH value is 6~7.4；
Preferably, mixing warm is carried out in surge tank in the step (4)；
Preferably, mixing preheating temperature is 60~80 DEG C in the step (4)；
Preferably, the simulation moving bed reactor in the step (5) is four-area simulated moving bed reactor, per area by
1~6 root chromatogram column forms；
Preferably, the chromatographic column filler in the step (5) in simulation moving bed reactor is Sugar SP0810；
Preferably, simulation moving bed reactor is isothermal operation in the step (5), 60~80 DEG C of column temperature；
The operating condition of four-area simulated moving bed reactor is in step (5)：Switching time 60~80 min, I areas flow rate
1.35~1.4, II area flow rate 0.4~0.8, III areas flow rate 0.6~0.93, IV areas flow rate≤0.4；
Wherein, dimensionless flow rate is defined as,QFor fluid flow,t s For switching time,VFor single branch
Chromatographic column inner volume,jEach area is represented,Represent the flow rate in each area.
The schematic flow sheet of D-Tag production method of the present invention is shown in Fig. 1, and shown in figure, flow is mainly by three parts
Composition（Separated by dotted line frame）.
Part I is to feed, including step (1), (2), (3), (4), is related to this method such as feeding liquid composition and temperature
Important parameter：
Contain 8~8.5g of sodium chloride, 30~50g of dibastic sodium phosphate and phosphorus in every liter of the cushioning liquid configured in step (1)
Acid dihydride 0.2~0.5g of sodium, regulation pH value to 6~7.4；
L-arabinose isomerase is to express me by cloning process from e. coli k12 genome in step (2)
Crude enzyme liquid made from the sugared isomerase of uncle, has the activity higher than enzyme powder；
It is 10~150 g/l to match somebody with somebody D- galactose concentrations in feeding liquid in step (4), and enzyme activity unit is 0.1~0.28
U/ml, pH value are 6~7.4, and target product D-Tag is free of in described feeding liquid；
Mixed process in step (4) is carried out in surge tank；
Warm is carried out in surge tank in step (4), and preheating temperature is 60~80 DEG C.
Part II is that enzymatic isomerization reaction couples with chromatographic isolation original position, i.e. step (5), is reacted in Simulation moving bed
Carried out on device, this is the core of D-Tag production method of the present invention：
Chromatographic column filler in step (5) in simulation moving bed reactor is Sugar SP0810, and the chromatographic column can be with water
The separation in situ of D- galactolipins and D-Tag is realized for mobile phase, avoids introducing the organic reagents such as acetonitrile；
Simulation moving bed reactor is isothermal operation in step (5), and 60~80 DEG C of column temperature, the temperature range not only improves D-
Galactolipin is advantageous to the separation of D- galactolipins and D-Tag on Sugar SP0810 again to the conversion of D-Tag；
Simulation moving bed reactor in step (5) is four-area simulated moving bed reactor, each area's post number can with identical or
Difference, II areas and III areas are main reaction and Disengagement zone, can use more post number, consider Simulation moving bed reaction
The performance and equipment cost of device, it is made up of per area 1~6 root chromatogram column, preferable total post number is 8 ~ 16；
The schematic diagram of heretofore described simulation moving bed reactor is shown in accompanying drawing 2 in step (5).As shown in Fig. 2 simulation moves
Dynamic bed reactor has two imports, respectively charging aperture and elution mouth, respectively two outlets, extract port and raffinate mouth.This four
Simulation moving bed reactor is divided into four areas by individual inlet and outlet, respectively with different functions, is acted synergistically between each other, jointly
D- galactolipins are completed to the high conversion production process of D-Tag.Wherein：It is I areas to elute between mouth and extract port, main work(
Can be eluted product D-Tag and regeneration stationary phase；It is II areas between extract port and charging aperture, major function is reaction and richness
Collect product D-Tag；It is III areas between charging aperture and raffinate mouth, major function is reaction and enrichment reactant D- galactolipins；
It is IV areas between raffinate mouth and elution mouth, major function is the unreacted D- galactolipins of recovery section and regenerated solvent.
The simulation moving bed reactor being related in the present invention is used to produce D-Tag by D- galactolipins, each fully to realize
The function in area, reaches the purpose for improving D- galactolipins conversion ratio, D-Tag yield and D-Tag purity, and Simulation moving bed is anti-
Device is answered to be operated within the specific limits.The present invention establishes the operating condition of simulation moving bed reactor：Switching time
60~80 min, I area flow rate 1.35~1.4, II areas flow rate 0.4~0.8, III areas flow rate 0.6~0.93, IV areas flow rate≤
0.4。
Wherein, nondimensional flow rate is defined as,QFor fluid flow,t s For switching time,VFor
Single branch chromatographic column inner volume,j(=I, II, III, IV) each area is represented,、、RespectivelyjFluid flow, the post number in area
And flow rate.
The present invention is different from the important symbol that Simulation moving bed is applied in existing correlation technique：Described simulation
In moving-burden bed reactor, D-Tag is free of in the feeding liquid entered from charging aperture, and in the extract port of one of two discharging openings
It can obtain purity>95% D-Tag product solution, this is that there occurs the result of enzymatic isomerization reaction in Simulation moving bed.
And in Chinese invention patent 201210329027.6, two steps use Simulation moving bed：Simulation moving bed in step (2)
It is used for the separation of galactolipin and glucose, Tagatose is not contained in charging and discharging；In step (4) Simulation moving bed by with
In the separation of galactolipin and Tagatose, contain Tagatose in charging and discharging.Simulation moving bed does not relate in the two steps
And it is pure separation process to reaction.
Through simulation moving bed reactor, under preferable operating condition, D- galactolipin conversion ratios reach more than 80%, product
D-Tag purity reaches more than 95% in solution, and D-Tag yield reaches more than 80%.
Part III is conventional last handling process, and the product solution that simulation moving bed reactor extract port obtains is through conventional de-
D-Tag crystal product can be made after color, desalination, concentration, crystallization, centrifugation, drying.
The present invention has advantages below：
(1) coupling in situ of enzymatic isomerization reaction and continuous chromatography separation is realized using simulation moving bed reactor, gram
Reversible reaction Equilibrium limit and product have been taken in feedback inhibition kinetically, can under preferable operating condition
D- galactolipin conversion ratios is reached more than 80%, D-Tag yield more than 80%, D-Tag product solution purity reach 95% with
On；
(2) coupling in situ of enzymatic isomerization reaction and continuous chromatography separation is realized using simulation moving bed reactor, can
With simple flow, operating unit is reduced, reduces equipment cost；
(3) simulation moving bed reactor is continuous operation, it is possible to achieve continuous production, so as to ensure that product quality
Homogeneity.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of D-Tag production method of the present invention.
Fig. 2 is the four-area simulated moving bed reactor schematic diagram that the present invention uses, and shown in figure, is flowed clockwise for liquid
With inlet and outlet switching direction, black arrow is current inlet and outlet position, including elutes mouth, extract port, charging aperture, raffinate mouth.Ash
Color arrow is the inlet and outlet position after once switching.A is reactant D- galactolipins, and C is product D-Tag, and S is aqueous solvent.Color
It is I areas to compose 1~J1 of post sequence number, and (J1+1)~J2 is II areas, and (J2+1)~J3 is III areas, and (J3+1)~J4 is IV areas.
Embodiment
Describe the present invention in detail with reference to the accompanying drawings and examples.In addition, it is to be understood that reading in of the invention tell about
Rong Hou, those skilled in the art can make various changes and modification to the present invention, and these equivalent form of values equally fall within the application institute
Attached claims limited range.
1. operating process, as shown in Figure 1.
(1) Part I is charging：In every liter of cushioning liquid containing 8~8.5g of sodium chloride, 30~50g of dibastic sodium phosphate and
0.2~0.5g of sodium dihydrogen phosphate, adjust pH value 6~7.4；Crude enzyme liquid is substantially soluble in partial buffer solution, it is molten that enzyme is made
Liquid；D- galactolipins are substantially soluble in water, material liquid is made；The material liquid, enzyme solutions, cushioning liquid of configuration are respectively placed in height
In the groove of position, through being sufficiently mixed and preheating in the surge tank with stirring and heat exchange jacket, feeding liquid is obtained, D- galas in feeding liquid
Sugared concentration is 10~150 g/l, and the U/ml of enzyme activity unit 0.1~0.28, pH value 6~7.4, temperature is 60~80 DEG C.
(2) Part II is that enzymatic isomerization reaction couples with chromatographic isolation original position：Using four area's operation simulation moving beds
Reactor, per area 1~6 root chromatogram column, totally 8~16 root chromatogram column.Operating condition is 60~80 DEG C of column temperature, switching time 60~
80 min, I area flow rate 1.35~1.4, II areas flow rate 0.4~0.8, III areas flow rate 0.6~0.93.Reacted in Simulation moving bed
Device extract port obtains D-Tag product solution, and purity reaches more than 95%, and yield reaches more than 80%, D- galactolipin conversion ratios and reached
To more than 80%.
(3) Part III is conventional last handling process：D-Tag product solution is refined using conventional meanses,
D-Tag crystal product can be made after decolouring, desalination, concentration, crystallization, centrifugation, drying.
2. simulation moving bed reactor.As shown in Figure 2, outlet port is regularly switched into along liquid flow direction,
Under suitable conditions, opposite direction of the liquid with solid on the basis of importing and exporting position can be formed to move, inhaled so as to realize
The continuous operation of attached process.Flowed clockwise for liquid in figure and import and export switching direction.Black arrow is current disengaging in figure
Mouth position, including elution mouth, extract port, charging aperture, raffinate mouth.Grey arrow is the inlet and outlet position after once switching.A is anti-
Thing D- galactolipins are answered, C is product D-Tag, and S is aqueous solvent.1~J1 of chromatographic column sequence number is I areas, and (J1+1)~J2 is II areas,
(J2+1)~J3 is III areas, and (J3+1)~J4 is IV areas.Enzymatic isomerization reaction can be realized using simulation moving bed reactor
Coupling in situ is separated with continuous chromatography, while D- galactolipin high conversions are realized, obtains the D-Tag product of high-purity
Solution.
3. material and medium
Chromatographic column filler is Sugar SP0810, using hard styrene diethylene benzene copoly mer as matrix, bond wire
Ion, 7 μm, internal diameter 20mm, length 300mm of particle diameter.Crude enzyme liquid comes from e. coli k12 genome, is made through clonal expression.D-
Galactolipin is technical grade, and water is deionized water, and dibastic sodium phosphate, sodium dihydrogen phosphate, the sodium chloride that cushioning liquid configuration is related to are
Analyze pure, heating medium is vapor.
Embodiment 1
Operating condition：The g/l of D- galactose concentrations 10 in feeding liquid, the U/ml of unit of enzyme activity 0.1, pH value 6；Preheating temperature
Degree, 60 DEG C；Column temperature, 60 DEG C；Post number and distribution, 8,2/2/2/2 distribution；Switching time, 60 min；I areas flow rate 1.4, II areas
Flow rate 0.8, III areas flow rate 0.807, IV areas flow rate 0.4.
As a result：Feeding liquid treating capacity 0.59 ml/min, D- galactolipin conversion ratio 82.9%, D-Tag yield 82.0%, production
Product solution D-Tagatose purity 99.5%.
Embodiment 2
Operating condition：The g/l of D- galactose concentrations 10 in feeding liquid, the U/ml of unit of enzyme activity 0.18, pH value 7.4；Preheating
Temperature, 70 DEG C；Column temperature, 70 DEG C；Post number and distribution, 8,1/3/3/1 distribution；Switching time, 80 min；I areas flow rate 1.35,
II areas flow rate 0.4, III areas flow rate 0.6, IV areas flow rate 0.4.
As a result：The % of feeding liquid treating capacity 12.7 ml/min, D- galactolipin conversion ratio 94.8, D-Tag yield 94.8%,
Product solution D-Tag purity 95.0%.
Embodiment 3
Operating condition：The g/l of D- galactose concentrations 100 in feeding liquid, the U/ml of unit of enzyme activity 0.28, pH value 7；Preheating temperature
Degree, 80 DEG C；Column temperature, 80 DEG C；Post number and distribution, 16,2/6/6/2 distribution；Switching time, 80 min；I areas flow rate 1.35, II
Area's flow rate 0.5, III areas flow rate 0.93, IV areas flow rate 0.35.
As a result：Feeding liquid treating capacity 27.3 ml/min, D- galactolipin conversion ratio 81.6%, D-Tag yield 80.9%, production
Product solution D-Tagatose purity 95.0%.
Embodiment 4
Operating condition：The g/l of D- galactose concentrations 150 in feeding liquid, the U/ml of unit of enzyme activity 0.28, pH value 7；Preheating temperature
Degree, 80 DEG C；Column temperature, 80 DEG C；Post number and distribution, 16,2/6/6/2 distribution；Switching time, 74 min；I areas flow rate 1.35, II
Area's flow rate 0.43, III areas flow rate 0.73, IV areas flow rate 0.4.
As a result：Feeding liquid treating capacity 20.6 ml/min, D- galactolipin conversion ratio 94.8%, D-Tag yield 94.8%, production
Product solution D-Tagatose purity 95.0%.
As a comparison, using identical crude enzyme liquid and conventional batch tank reactor, under the operating condition of optimization, 32 is small
When interior D- galactolipins conversion ratio be only 25.8%, D-Tag is removed in product solution, also have more unreacted galactolipins, it is necessary to
Split in subsequent treatment using means such as chromatography, batch-wise chromatography, Simulation moving beds.
And in embodiment 1, while ensuring that D- galactolipins conversion ratio and D-Tag yield are more than 80%, it can obtain
Purity reaches 99.5% D-Tag product solution.In embodiment 2, D- galactolipins conversion ratio, D-Tag yield and purity are equal
It can reach about 95%.In embodiment 3, ensure D- galactolipins conversion ratio, D-Tag yield reach 80% and D-Tag it is pure
While degree reaches 95%, larger equipment treating capacity can be obtained.In embodiment 4, D- galactolipins conversion ratio, D-Tag are pure
Degree and yield can obtain the equipment treating capacity of higher level simultaneously more than 95%.
Claims (7)
1. a kind of D-Tag production method based on enzymatic isomerization reaction and continuous chromatography separation coupling in situ, including it is following
Step：
(1) cushioning liquid of pH value 6~7.4 is configured；
(2) thermophilic L-arabinose isomerase is fully dissolved in partial buffer solution, as enzyme solutions；
(3) D- galactolipins are fully dissolved in water, as material liquid；
(4) by after the blended preheating of cushioning liquid, enzyme solutions and material liquid respectively obtained in step (1), (2), (3), as
Feeding liquid, target product D-Tag is free of in described feeding liquid；
(5) feeding liquid obtained in step (4) is entered into simulation moving bed reactor, after reaction in-situ and separation, directly
To D-Tag product solution；The simulation moving bed reactor is isothermal operation, 60~80 DEG C of column temperature；The Simulation moving bed
Reactor is four-area simulated moving bed reactor, is made up of per area 1~6 root chromatogram column；The four-area simulated moving bed reactor
Operating condition be：60~80min of switching time, I areas flow rate 1.35~1.4, II areas flow rate 0.4~0.8, III areas flow rate 0.6
~0.93, IV area flow rate≤0.4.
2. D-Tag production method according to claim 1, it is characterised in that：The buffering configured in step (1) is molten
Contain 0.2~0.5g of 8~8.5g of sodium chloride, 30~50g of dibastic sodium phosphate and sodium dihydrogen phosphate in every liter of liquid, regulation pH value to 6~
7.4。
3. D-Tag production method according to claim 1, it is characterised in that：L-arabinose isomery in step (2)
Enzyme is to express crude enzyme liquid made from Arabinose isomerase by cloning process from e. coli k12 genome.
4. D-Tag production method according to claim 1, it is characterised in that：Match somebody with somebody D- in feeding liquid in step (4)
Galactose concentration is 10~150g/l, and enzyme activity unit is 0.1~0.28U/ml, and pH value is 6~7.4.
5. D-Tag production method according to claim 1, it is characterised in that：Mixing warm exists in step (4)
Carried out in surge tank.
6. D-Tag production method according to claim 1, it is characterised in that：Mixing preheating temperature is in step (4)
60~80 DEG C.
7. D-Tag production method according to claim 1, it is characterised in that：Simulation moving bed is reacted in step (5)
Chromatographic column filler in device is Sugar SP0810.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6057135A (en) * | 1992-01-16 | 2000-05-02 | Kraft Foods, Inc. | Process for manufacturing D-tagatose |
| CN101925609A (en) * | 2008-01-28 | 2010-12-22 | Cj第一制糖株式会社 | Process for manufacturing tagatose using soy oligosaccharide |
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| US8802843B2 (en) * | 2012-05-22 | 2014-08-12 | Orochem Technologies, Inc. | Tagatose production using simulated moving bed separation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6057135A (en) * | 1992-01-16 | 2000-05-02 | Kraft Foods, Inc. | Process for manufacturing D-tagatose |
| CN101925609A (en) * | 2008-01-28 | 2010-12-22 | Cj第一制糖株式会社 | Process for manufacturing tagatose using soy oligosaccharide |
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