CN1844405A - A method for reaction and separation coupled enzymatic synthesis of salidroside - Google Patents
A method for reaction and separation coupled enzymatic synthesis of salidroside Download PDFInfo
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- CN1844405A CN1844405A CNA2006100240941A CN200610024094A CN1844405A CN 1844405 A CN1844405 A CN 1844405A CN A2006100240941 A CNA2006100240941 A CN A2006100240941A CN 200610024094 A CN200610024094 A CN 200610024094A CN 1844405 A CN1844405 A CN 1844405A
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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000000926 separation method Methods 0.000 title claims abstract description 4
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 title claims abstract 7
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 title claims abstract 7
- 230000002255 enzymatic effect Effects 0.000 title claims description 6
- 230000015572 biosynthetic process Effects 0.000 title claims 2
- 238000003786 synthesis reaction Methods 0.000 title claims 2
- 239000000758 substrate Substances 0.000 claims abstract description 38
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 37
- 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 claims abstract description 17
- 239000008103 glucose Substances 0.000 claims abstract description 17
- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-hydroxyphenyl)ethanol Chemical compound OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000003463 adsorbent Substances 0.000 claims abstract description 13
- 238000001179 sorption measurement Methods 0.000 claims abstract description 7
- 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 claims abstract description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims abstract 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 30
- 239000000047 product Substances 0.000 claims description 20
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 18
- 239000000843 powder Substances 0.000 claims description 12
- 239000012065 filter cake Substances 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 9
- 229920005989 resin Polymers 0.000 claims description 8
- 239000011347 resin Substances 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 229920001429 chelating resin Polymers 0.000 claims description 5
- 229930182470 glycoside Natural products 0.000 claims description 5
- 150000002338 glycosides Chemical class 0.000 claims description 5
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
- 229920001467 poly(styrenesulfonates) Polymers 0.000 claims description 3
- 238000004064 recycling Methods 0.000 claims description 2
- 230000003068 static effect Effects 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 7
- 108090000790 Enzymes Proteins 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 239000003480 eluent Substances 0.000 abstract description 3
- 235000013399 edible fruits Nutrition 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 239000011942 biocatalyst Substances 0.000 abstract 1
- 238000003795 desorption Methods 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 239000011541 reaction mixture Substances 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 229930182478 glucoside Natural products 0.000 description 15
- 150000008131 glucosides Chemical class 0.000 description 14
- 238000010521 absorption reaction Methods 0.000 description 12
- 239000002594 sorbent Substances 0.000 description 8
- 241001165494 Rhodiola Species 0.000 description 5
- 238000002390 rotary evaporation Methods 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- ILRCGYURZSFMEG-RKQHYHRCSA-N Salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RKQHYHRCSA-N 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
- 239000008057 potassium phosphate buffer Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 229930014626 natural product Natural products 0.000 description 3
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 244000042430 Rhodiola rosea Species 0.000 description 2
- 235000003713 Rhodiola rosea Nutrition 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 102000006995 beta-Glucosidase Human genes 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- RNHDAKUGFHSZEV-UHFFFAOYSA-N 1,4-dioxane;hydrate Chemical compound O.C1COCCO1 RNHDAKUGFHSZEV-UHFFFAOYSA-N 0.000 description 1
- 244000144725 Amygdalus communis Species 0.000 description 1
- 235000011437 Amygdalus communis Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001411320 Eriogonum inflatum Species 0.000 description 1
- 102000030595 Glucokinase Human genes 0.000 description 1
- 108010021582 Glucokinase Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 101100412856 Mus musculus Rhod gene Proteins 0.000 description 1
- 241000083902 Rhodiola sachalinensis Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 235000020224 almond Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000000498 cooling water Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003534 oscillatory effect Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- -1 p-nitrophenyl glucoside Chemical class 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 150000008495 β-glucosides Chemical class 0.000 description 1
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
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- Saccharide Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种反应与分离耦合酶促合成红景天苷的方法。该方法以果脯厂废弃的苹果籽为廉价生物催化剂，以葡萄糖和对羟基苯乙醇为底物，在有机溶剂－水体系中酶促合成了珍贵的天然保健品红景天苷，反应混合液经过氧化铝等吸附剂吸附后，通过洗脱剂的选择性解吸作用，使底物对羟基苯乙醇与产物红景天苷相分离，简化了产物的纯化过程，并使未反应的醇能够循环使用，是一种极具应用前景的批量生产红景天苷的新方法。The invention discloses a method for synthesizing salidroside coupled with reaction and separation enzymatically. In this method, the precious natural health product salidroside was enzymatically synthesized in an organic solvent-water system by using the discarded apple seeds from the preserved fruit factory as a cheap biocatalyst, glucose and p-hydroxyphenylethanol as substrates, and the reaction mixture was passed through After the adsorption of alumina and other adsorbents, the substrate p-hydroxyphenylethanol is separated from the product salidroside through the selective desorption of the eluent, which simplifies the purification process of the product and enables the unreacted alcohol to be recycled , is a new method for mass production of salidroside with great application prospects.
Description
Technical field
The present invention relates to the production method of natural product rhodioside.
Background technology
Rhodioside, or claim rhodioloside, be one of effective constituent of rhodiola plant, the quality quality of in practical study, often just coming the comparison Root of Kirilow Rhodiola again with the content of rhodioloside.Root of Kirilow Rhodiola has hypermnesis, improves the cardio-cerebrovascular function, enhancing immunity, antifatigue, hypoxia tolerance, antitumor, hypoglycemic, disturb virus, resisting microwave radiation, armour and avoid many-sided pharmacological actions such as radical damage, have a extensive future.Aspect range of application by " adaptation former state " drug development to Rhodiola root oral liquid, Root of Kirilow Rhodiola food, gadol beverage etc.Rhodiola rosea formulated product can improve trial volunteer's muscle power and intelligence, increases the work capacity index, and the error of performance rate is reduced.Healthy people takes rhodiola rosea formulated product just can improve attention, memory and excited process after one course of treatment intensity, takes the excitement and the process of inhibition that can strengthen the patient after a course of treatment for suffering from neurosal patient, and handiness is recovered normally.
At present, mainly from the Rhodida plant rhizome, separate obtaining rhodioside, comprise immersion, alcohol reflux extraction, organic solvent extraction, polymeric adsorbent and silica gel column chromatography by the method for extracting.The extraction step complexity, general productive rate is 0.4%～0.8%.And the method steps of chemosynthesis is more, must be through hydroxyl protection and deprotection steps.In recent years people to begin to utilize microorganism or enzyme to carry out biotransformation of Natural Products and biocatalysis synthetic.Journal of Biotechnology 1998,61:69-73 discloses a kind of Rhodiola sachalinensis cell biological of artificial culture that adopts and has transformed the method for synthesizing rhodioside, by adding p-hydroxyphenylethanol, the ultimate yield of its product rhodioside is 0.93g/L.Tetrahedron Asymmetry 1999,10:2429-2439 disclose a kind of employing almond beta-glucosidase (Sigma G-0395) catalysis changes glycosylation reaction, and synthetic a series of natural beta-glucosides comprise the method for rhodioside.But this method adopts expensive glucokinase activity glycosides (as the p-nitrophenyl glucoside) as the glucosyl group donor, and cost is very high.Process Biochemistry 2005,40:3143-3147 discloses a kind of Aspergillas of utilization niger beta-glucosidase, with 1.5% p-hydroxyphenylethanol and 6% glucose is substrate, the method of the synthetic rhodioside of catalysis in water solution system, productive rate is 10%, and production concentration only is 1.5g/L.
The contriver is at document: Bioorganic ﹠amp; Medicinal Chemistry Letters 2004,14:2095-2097 and application number are 200410016652.0, publication number be disclose in the Chinese patent application book of CN1560268A a kind of in water-dioxane system, utilize the method for the synthetic rhodioside of the reverse hydrolysis reaction of apple seed powder catalysis glucosides, production concentration reaches 10g/L, but the concentration of substrate p-hydroxyphenylethanol is up to 345g/L, and adopt conventional separation method from reaction solution, to collect product-rhodioside, therefore, the efficient of producing has been subjected to influence, needs further to improve.
Summary of the invention
The technical issues that need to address of the present invention are to disclose a kind of reaction and the method for separating the synthetic rhodioside of coupling enzymatic, to overcome the defective of prior art.
Design of the present invention is such:
The contriver finds, the reaction solution that contains substrate, product, water and organic solvent is through after the adsorbents adsorb, most substrate alcohol is not adsorbed, and the product glucosides is adsorbed substantially, then according to different strippant polar differences, with the pure and mild glucosides of absorption on a small quantity respectively desorb get off, to reach separating of substrate alcohol and glucosides, thereby realize the cycling and reutilization of superfluous substrate alcohol, and simplify the purge process of product glucosides.
Method of the present invention comprises the steps:
(1) sorbent material is contacted with reaction solution, adsorb 1～5h under 20～40 ℃ the temperature, decompress filter obtains filter cake;
Said reaction solution is that apple seed powder, substrate glucose and p-hydroxyphenylethanol react the product that is obtained in containing the organic solvent of buffered soln, can be that disclosed method is prepared in the Chinese patent application book of CN1560268A at publication number with reference to the contriver; Wherein:
The concentration of substrate glucose is 2～100g/L, and the concentration of p-hydroxyphenylethanol is 100～500g/L, and the concentration of rhodioside is 5～20g/L;
Said sorbent material is selected from a kind of in aluminum oxide, Amberlite series plastics or the Dowex series plastics, the consumption of sorbent material is 0.01～1kg/L reaction solution, adsorption form can be Static Adsorption or dynamic adsorption, on the shaking table of 40～100rpm, adsorb 1～5h as the reaction solution that will be added with sorbent material, perhaps reaction solution is fed the sorbent material packed column;
Said Amberlite series plastics or Dowex series plastics are a kind of polymer absorption exchange resin, can the commercially available prod, and be the product of Amberlite XAD-7 as Rhom and Hass's trade mark;
(2) with the water-containing organic solvent drip washing filter cake that adopts in the reaction system, to wash the not substrate alcohol of absorption, consumption is 0.1～5ml/g sorbent material;
Preferred volume concentration is 70～95% the trimethyl carbinol aqueous solution;
(3) soak filter cake with substrate alcohol strippant then, soak time is 0.5～2 hour, is attracted to substrate alcohol on the resin with desorb, filters, and obtains filter cake, and the consumption of substrate alcohol strippant is 1～10ml/g sorbent material;
Said substrate alcohol strippant is selected from ethyl acetate, ethanol, normal hexane or the trimethyl carbinol;
(4) filter cake with step (3) is immersed in the glucosides strippant, and soak time is 0.5～2 hour, and the consumption of glucosides strippant is 1～10ml/g sorbent material, is attracted to product glucosides on the resin with desorb, filters, and obtains to contain the filtrate of product glucosides;
Said glucosides strippant is selected from a kind of in the trimethyl carbinol aqueous solution that volumetric concentration is 35～95% aqueous ethanolic solution, water, the trimethyl carbinol or volumetric concentration 40～80%, and preferred volume concentration is 35～95% aqueous ethanolic solution;
(5) from the filtrate of step (4), collect rhodioside then.
According to the present invention, the filtrate that step (3) is collected is promptly contained the substrate alcohol strippant of substrate alcohol, and rotary evaporation is removed substrate alcohol strippant, obtains unreacted substrate alcohol, recycles.
According to the present invention, the method for collecting rhodioside from the filtrate of step (4) preferably includes following steps:
With the filtrate evaporate to dryness of step (4), through silica gel column chromatography (eluent is an ethyl acetate: methyl alcohol=13: 1, volume ratio) chromatography, recrystallization (solvent is an ethyl acetate: methyl alcohol=13: 1, volume ratio) obtains the product rhodioside.
Reaction of the present invention can be carried out in the reactor of routine, and its form is very unimportant, can be a kind of in stirring tank, packed column or the bubbling column reactor.
The method that adopts enzymatic disclosed in this invention to synthesize and separate coupling amplification production rhodioside can not only obtain high density, highly purified rare natural product rhodioside, and simplify the recycling of substrate alcohol.Enzyme catalyst is easy to reuse in reactor simultaneously, is a kind of processing method that has the industrial application potentiality, can produce natural health-care products rhodioside high-quality, high added value with lower cost.Below will be further described technology contents of the present invention by embodiment.
Embodiment
Embodiment 1
The preparation of apple seed powder: get apple seed waste 1kg from the preserved fruit factory, grind or smash to pieces into powder, wash (300ml * 3) 3 times with acetoacetic ester earlier, wash 1 time with 300ml acetone again, drain and be placed on the vacuum drying oven drying, altogether apple seed dry powder 350g, it is stand-by to put 4 ℃ of refrigerators.
Embodiment 2
49mg glucose is added the 0.1mL potassium phosphate buffer, and (final concentration of sugar in the 1mL reaction system is 0.25mol/L for 50mmol/L, pH6.5) middle heating for dissolving; The 0.383g p-hydroxyphenylethanol is added heating for dissolving in the 0.9mL trimethyl carbinol, the final concentration of alcohol in the 1mL reaction system is 2.5mol/L, with the thick enzyme powder of apple seed 15mg (1.52U), after 192 hours, analyzing the ultimate yield that records rhodioside is 28.2% 50 ℃ of oscillatory reactions after the cooling.
Embodiment 3
In 50ml ground triangular flask, add 250mg apple seed powder successively, 272mg glucose, 0.5ml phosphoric acid buffer (70mmol/L Na
2HPO
4-KH
2PO
4, pH6.0), the 2.28g p-hydroxyphenylethanol, the 5.0ml dioxane, jam-pack ground bottle stopper, and seal with raw material band, at 45 ℃, react 120h. on the 160rpm shaking table
Reaction finishes the back and adds methyl alcohol 10ml, remove by filter enzyme, the enzyme that leaches is used methanol wash (20ml * 3) again, in filtrate, add anhydrous sodium sulfate drying, rotary evaporation removes and desolvates, (ethyl acetate: methyl alcohol=13: 1), obtain rhodioside 66mg, be 15.8%. with respect to the productive rate of glucose to rapid column chromatography
Embodiment 4
In 250ml ground triangular flask, add 2.45g glucose successively, and the 5ml phosphoric acid buffer (the 50mmol/L potassium phosphate buffer, pH7.0), 8.625g p-hydroxyphenylethanol, the 45ml trimethyl carbinol, 2.0g apple seed powder, in 52 ℃, with the rotating speed machinery stirring reaction 72h of 120rpm.
Stop to stir and leaving standstill 0.5h after question response finishes, at the bottom of apple seed powder is sunken to bottle, waits until down batch reaction and continue use;
The concentration of substrate glucose is 49g/L, and the concentration of p-hydroxyphenylethanol is 172.5g/L, and the concentration of rhodioside is 5.2g/L;
In the reaction supernatant liquor, add 30g polymeric adsorbent Amberlite XAD-7, decompress filter behind the absorption 3h on 30 ℃, 60rpm shaking table, and with 20ml 90% tertiary butanol and water solution drip washing filter cake, to wash the not substrate alcohol of absorption, soak 1h with the 50ml ethyl acetate again, be attracted to substrate alcohol on the resin with desorb, soak resin with 50ml 75% aqueous ethanolic solution at last, be attracted to product glucosides on the resin with desorb.Rotary evaporation is removed ethyl acetate, obtain unreacted substrate alcohol, with its with through absorption and the tertiary butanol and water leacheate of reaction supernatant liquor after filtering and polymeric adsorbent merge, drop into the next round reaction again, before the next batch reaction, add glucose 2.45g again.
Through after continuous 10 batches of reaction repeated, glucosides solution evaporate to dryness with 75% aqueous ethanolic solution desorb, (eluent is an ethyl acetate: methyl alcohol=13: 1 through silica gel column chromatography, volume ratio) chromatography, (solvent is methyl alcohol and ethyl acetate to recrystallization, ethyl acetate: methyl alcohol=13: 1, volume ratio) after, gets white solid rhodioside 2.0g.Adopt document Bioorganic ﹠amp; Medicinal Chemistry Letters 2004, the described method of 14:2095-2097 is analyzed, and the productivity of product rhodioside is 1.3g/Ld.
Embodiment 5
In having the glass column reactor of thermostat(t)ed water chuck, insert 10g apple seed powder; The about 200ml of reactant ligand liquid in addition, comprising 20g glucose, 20ml potassium phosphate buffer (50mmol/L, pH 7.0), the 34.5g p-hydroxyphenylethanol and the 180ml trimethyl carbinol; With peristaltic pump reaction solution is cycled through filling enzyme post and begin the enzymatic building-up reactions, the temperature of controlling reactor is 48 ± 2 ℃, and the circulation fluid flow velocity is 0.3ml/min, reaction times 72h.In the reaction solution, the concentration of substrate glucose is 100g/L, and the concentration of p-hydroxyphenylethanol is 172.5g/L, and the concentration of rhodioside is 10.3g/L;
After reaction solution fed aluminum oxide (75g) packed column absorption, with 50ml90% tertiary butanol and water solution drip washing pipeline and not absorption substrate alcohol, the a small amount of substrate alcohol that is adsorbed with trimethyl carbinol 100ml desorb, the product glucosides that is adsorbed with 100ml 50% aqueous ethanolic solution wash-out then again.Rotary evaporation is removed ethyl acetate, and substrate alcohol and reaction solution after the absorption and leacheate merging with obtaining drop into the next round reaction again, add glucose 20g again before the next batch reaction.
Through adopting embodiment 4 identical methods to collect product after continuous 3 batches of reaction repeated, obtain rhodioside white solid 5.3g.The productivity of rhodioside is 2.94g/Ld.
Embodiment 6
In having the glass column reactor of thermostat(t)ed water chuck, add apple seed powder 20g and reaction solution 500ml, comprising glucose 25g, potassium phosphate buffer (50mM, pH7.0) 75ml, p-hydroxyphenylethanol 100g and dioxane 425ml.The glass column top of thermostat(t)ed water chuck is connected to cooling water circulating device, and the air compressor pump air-blowing is arranged below, and Ventilation Rate is 0.5L/min, and controlled temperature is at 40 ℃, and the reaction times is 60h.In the reaction solution, the concentration of substrate glucose is 50g/L, and the concentration of p-hydroxyphenylethanol is 200g/L, and the concentration of rhodioside is 4.8g/L;
After reaction finishes, after reaction solution fed aluminum oxide (150g) packed column absorption, with 100ml 90% dioxane-aqueous solution drip washing pipeline and not absorption substrate alcohol, the a small amount of substrate alcohol that is adsorbed with normal hexane 200ml desorb, the product glucosides that is adsorbed with 200ml 70% trimethyl carbinol aqueous solution wash-out then again.Rotary evaporation is removed ethyl acetate, and substrate alcohol and reaction solution after the absorption and leacheate merging with obtaining drop into the next round reaction again, add glucose 25g again before the next batch reaction.
Through after continuous 3 batches of reaction repeated, adopt embodiment 4 identical methods to collect product, obtain rhodioside white solid 6.1g.The productivity of rhodioside is 1.36g/Ld.
Claims (7)
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