CN102094052A - Method for preparing natural medicines by coupling glycosidase catalysis and salting-out extraction - Google Patents
Method for preparing natural medicines by coupling glycosidase catalysis and salting-out extraction Download PDFInfo
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- CN102094052A CN102094052A CN 201010584745 CN201010584745A CN102094052A CN 102094052 A CN102094052 A CN 102094052A CN 201010584745 CN201010584745 CN 201010584745 CN 201010584745 A CN201010584745 A CN 201010584745A CN 102094052 A CN102094052 A CN 102094052A
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- glycosylase
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- catalysis
- enzyme
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- 238000005859 coupling reaction Methods 0.000 title claims abstract description 20
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- 238000005185 salting out Methods 0.000 title abstract 4
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- FVIZARNDLVOMSU-IRFFNABBSA-N ginsenoside C-K Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O FVIZARNDLVOMSU-IRFFNABBSA-N 0.000 description 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 229930189407 platycodin Natural products 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a method for preparing natural medicines by coupling glycosidase catalysis and salting-out extraction, and belongs to the technical field of bioengineering. The method is characterized in that enzymatic hydrolysis reaction is coupled with salting-out extraction of a product. Glycosidase and the natural product are added into a salting-out extraction system, and the glycosidase is utilized to convert the natural product containing glycosyl into an active ingredient of which the glycosyl is removed; meanwhile, the product and a substrate are separated through the extracting capacity of the system. The invention has the advantages of developing a novel reaction and separation coupled method easy for industrial production, and improving production efficiency.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to the method that a kind of salt separation extracting and the coupling of Glycosylase hydrolysis reaction prepare medicine or pharmaceutical intermediate.
Background technology
Natural product becomes the important source of medicine, food and feed gradually, has sizable market at present based on the protective foods and the medicine of natural product.Glucosides class natural product occupies critical role in numerous natural products, the glucosides of a series of biologically actives and pharmaceutical use comes into operation in succession, as Gastrodine, arbutin, cardigin, platycodin, ginsenoside etc.The activity of glycoside is normal relevant with the type and the quantity of glycosyl, and the change of glycosyl can cause active variation, and many aglycons all have the biological activity stronger than glucosides, so glycoside hydrolysis research is subjected to people's common concern.Than chemistry (acid or alkali) hydrolysis, biology or enzymatic hydrolysis have characteristics such as selectivity height, mild condition, activity preservation rate height, environmental pollution are little, thereby the report of Recent study exploitation is also many.For example, Shen Yue etc. utilizes milk-acid bacteria beta-glucoside enzyme hydrolysis of soybean isoflavone, preparation isoflavone genin (Chinese oil, 2009,34 (9): 60-63); Wu Yi etc. utilize immobilization beta-glucosidase hydrolysis ginkgolic flavone glycoside glycosides to prepare Flavone aglycone (Harbin Engineering University's journal, 2009,30 (5): 584-588); Yang Yishun etc. utilize immobilization beta-glucosidase conversion Geniposide to prepare genipin (East China science and engineering master thesis, 2009); Usefulness helicase hydrolysis arctinins such as Hu Yingjie prepare aglycon (Traditional Chinese Medicine University Of Guangzhou's journal, 2004,21 (6): 473-475); Han Fengmei etc. carry out pre-treatment with cellulase to Rhizome of Peltate Yam, and raising diosgenin yield (chemistry and biotechnology, 2004,6:26-27); , usefulness cellulases such as Li Mengqing the polydatin in the giant knotweed change into trans-resveratrol (fine chemistry industry, 2008,25 (5): 467-470), or the like.
Because glucosides class natural product is most of water-soluble relatively poor, the concentration of substrate in the reaction system is lower, so the efficiency ratio of biology or enzymatic hydrolysis is lower.Many scholars have carried out some researchs and have attempted to address the above problem, and the result shows that some organic solvent can increase the solubleness of glucosides, improve the speed of enzymic catalytic reaction.As Zhan Xiuwen find tetrahydrofuran (THF) (≤8%), dimethyl sulfoxide (DMSO) (≤18%), contain the dimethyl sulfoxide (DMSO) (≤15%) of 2% tetrahydrofuran (THF), the tetrahydrofuran (THF) (≤8%) that contains 10% dimethyl sulfoxide (DMSO) all has activation (Agricultural University Of South China's master thesis, 2008) to porcine pancreatic lipase.Although organic phase enzymic catalytic reaction speed is improved, but continuous accumulation along with product, the activity of enzyme often is subjected to the inhibition of high density product, and especially the monose that gets off of hydrolysis has the obvious suppression effect to the activity of enzyme, has a strong impact on speed of response and transformation efficiency.Studies show that as Yu Huilei etc. the monose of high density can make the enzymatic glycoside hydrolysis reaction of glycoside hydrolysis carry out (organic chemistry, 2006,26 (6): 1052-1058) to contrary direction.If product in time can be removed, will remove the restraining effect of product so undoubtedly to enzyme, further improve speed of response.
Extraction and fermentation or enzymic catalytic reaction coupling realize limit coronite separation, and this is the focus of studying in recent years, but is not still having bibliographical information aspect the catalytic hydrolysis reaction of Glycosylase.The salt separation extracting has some successful trials in recent years aspect Separation of Natural Products, as the novel double water-phase that utilizes hydrophilic low molecule organic matter and inorganic salt solution the to form effective constituent that polarity is stronger is enriched in mutually, perhaps effective constituent (the Chinese patent: CN101637667) of the three liquid-phase systems separation opposed polarity that forms with inorganic salt solution, hydrophilic low molecule organic matter and hydrophobic organic compound.The Glycosylase catalysis and the extracting and separating coupling of saltouing are also needed to solve the stability problem of enzyme in organic phase, investigate the distribution and the coupling of substrate, product, enzyme, reach and improve substrate solubleness, remove product and suppress, improve the purpose of enzyme catalysis efficient.Purpose of the present invention just is to develop a kind of Reaction Separation coupled method new, that be easy to industrialization, enhances productivity, and reduces production costs.
Summary of the invention
For solve the problems that active skull cap components exists in enzyme catalysis conversion and extraction separation process, low as effective composition in aqueous phase solubleness, product accumulation inhibitory enzyme catalytic activity, effective constituent conversion after product separating step is loaded down with trivial details etc., the invention provides the method that a kind of Glycosylase catalysis and salt separation extracting coupling prepare natural drug or intermediate.
The technical solution used in the present invention comprises the steps:
The substrate that enzyme catalysis is transformed is dissolved in the hydrophilic organic solvent, adds soluble inorganic salt, leaves standstill to be divided into two-phase up and down, Glycosylase is added go up phase then, carries out conversion reaction.Monose under the hydrolysis is allocated in down phase (inorganic salt face), on be the extraction liquid that is rich in target product mutually; Perhaps add hydrophobic organic solvent in biphasic system, form three liquid phases, then Glycosylase is added intermediate phase, the monose that reaction produces is allocated in inorganic salt face, and target product is enriched in phase, and unreacted substrate and enzyme then are retained in intermediate phase.
The substrate of Glycosylase catalyzed conversion is glucosides class monomeric compound (steroidal and terpene saponin(e, anthraquinone terpene and cyclenes ethers saponin(e) or medicinal powder, the extract that contains specific examples of such components, monomeric compound such as the new glycosides of shield leaf, triangle leaf saponin(e, diosgenin-glucose three glucosides, diosgenin-glucose bioside, prolong tinkling of pieces of jade grass time glycosides, polydatin, Geniposide, ginsenoside Re, Rg1, Rd, Rc, Potenlini, soybean saponin I, II, IV etc., Chinese medicinal materialss such as medicinal material such as Rhizome of Peltate Yam, giant knotweed, cape jasmine, genseng, Radix Glycyrrhizae, soybean.Target product is less than the medicine or the pharmaceutical intermediate of conversion of substrate for the glycosyl number, as diosgenin, trans-resveratrol, genipin, ginsenoside Rg3, ginsenoside Rh2, ginseng saponin F 2, ginsenoside CK, single glucuronic acid-glycyrrhetinic acid, soyasterol B etc.
Glycosylase is one or more a mixed enzyme in the commercial enzymes such as amylase, cellulase, β glucuroide, naringinase, tilactase or helicase, also can be the isozyme that can produce the microorganism cells of above enzyme or induce generation, isozyme be induced generation by microorganism through medicinal material or glucosides monomeric compound.These enzymes also can be through chemically modified or immobilized enzyme, so that improve the stability and the reuse of enzyme.Several glycosidic links that Glycosylase exists in can the hydrolysis natural product: beta-glucoside key, rhamnosyl glycosidic bond, semi-lactosi glycosidic bond etc.
Soluble inorganic salt is phosphoric acid salt, vitriol, carbonate, halogenide or its mixture more than two kinds, and the mass fraction that soluble inorganic salt accounts for system is 5～35%.
Hydrophilic organic solvent is alcohols, short chain ethers, furans, sulfoxide class, alkylene oxides or its mixture more than two kinds, and the mass fraction that hydrophilic organic solvent accounts for system is 5～40%.
Three liquid phases are saltoutd, and selected hydrophobic organic solvent is alkanes, ester class, long-chain ethers or its mixture in the extraction system, and the massfraction that hydrophobic organic solvent accounts for system is 10～60%.
The enzymic catalytic reaction temperature is 20～70 ℃, and mixing speed is 20～250rpm.The time of Glycosylase hydrolysis reaction is 0.5～200h.
When reaction is carried out, can the inorganic salt phase transition of sugar, the inorganic salt face that more renews will be contained, the hydrophobicity organic phase that perhaps will contain product in the three phase extraction catalyzed reaction shifts, the hydrophobicity organic phase that more renews is added substrate and a little enzyme again, and reaction is constantly carried out.
Beneficial effect of the present invention:
(1) organic solvent helps to improve the solubleness of substrate;
(2) Glycosylase catalysis and salt separation extracting coupling are helped removing the restraining effect of product to enzymic activity, improve speed of reaction;
(3) enzyme catalysis conversion and salt separation extracting coupling can realize that the limit coronite separates, and simplify Production Flow Chart, enhance productivity, and reduce production costs.
Embodiment
Be described in detail specific embodiments of the invention below in conjunction with technical scheme.
Embodiment 1: cellulase catalyzed conversion polydatin and aqueous two-phase extraction are coupled
5g ammonium sulfate is dissolved in the 10.8g water, is made into ammoniumsulphate soln.The ethanol that above-mentioned inorganic salt solution and 4.2g is dissolved with polydatin mixes, and stirs, and leaves standstill the 10min phase-splitting.Cellulase is slowly added the ethanol phase, phase reaction 10h in the stirring.25 ℃ of temperature of reaction, mixing speed are 70rpm.Analytical results shows that 100% polydatin is distributed in phase, and is converted into trans-resveratrol fully in 10 hours.Enzymic catalytic reaction with aqueous phase is compared, and the trans-resveratrol transformation efficiency reaches 100% needs 12h under the equal conditions, and the existence of double water-phase has obviously been shortened the reaction times.
Embodiment 2: the coupling of helicase catalyzed conversion Rhizome of Peltate Yam and aqueous two-phase extraction
Thick saponin(e is dissolved in 1, in the 4-butyleneglycol, preparation 1,4-butyleneglycol saponin(e liquid.Get 3.36g ammonium sulfate and 11.04g water is made into ammoniumsulphate soln.With above-mentioned inorganic salt solution and 3.6g 1,4-butyleneglycol saponin(e liquid mixes, and leaves standstill the 10min phase-splitting after the stirring.The distribution of analyzing various saponin(es shows, 20% diosgenin is distributed in phase (1,4-butyleneglycol phase), 80% diosgenin is distributed in down phase (ammonium sulfate phase), and the new glycosides of shield leaf, trigone-leaf dioscorea opposita saponin, glucose three glucosides, glucose bioside, Trillium tschonoskii Maxim time glycosides respectively accounts for half in the distribution of phase up and down.Helicase solution is slowly added upward phase, 50 ℃ of temperature of reaction, mixing speed is 70rpm, reaction 48h, the transformation efficiency of diosgenin reaches 11%.With the enzyme of equivalent and substrate in the aqueous phase reaction and remove only stay phase down mutually enzymic catalytic reaction in contrast, the former diosgenin transformation efficiency only is 2.5%, the latter is 5%, illustrates that enzyme catalysis and double water-phase coupling process have a clear superiority in.
Embodiment 3: the coupling of helicase catalyzed conversion Rhizome of Peltate Yam saponin(e and three liquid-phase extractions
Thick saponin(e is dissolved in 1, in the 4-butyleneglycol, preparation 1,4-butyleneglycol saponin(e liquid.Get 3.36g ammonium sulfate and 11.04g water is made into ammoniumsulphate soln.With above-mentioned inorganic salt solution and 3.6g 1,4-butyleneglycol saponin(e liquid and 2.0g sherwood oil mix, and leave standstill 10min after the stirring, form three liquid phases.The distribution of analyzing various saponin(es shows, 95% diosgenin is distributed in phase (sherwood oil phase), 5% diosgenin is distributed in middle phase (1,4-butyleneglycol phase), 80% glucose three glucosides, glucose bioside, Trillium tschonoskii Maxim time glycosides are distributed in middle phase (1,4-butyleneglycol phase), all the other 20% are allocated in down phase (ammonium sulfate phase).Helicase solution is slowly added intermediate phase, 35 ℃ of temperature of reaction, mixing speed is 100rpm, reaction 48h, the diosgenin transformation efficiency is 30%.With the enzyme of equivalent and substrate in the aqueous phase reaction and remove only keep intermediate phase up and down mutually enzymic catalytic reaction in contrast, the former diosgenin transformation efficiency only is 2.5%, the latter is 5%; Enzyme catalysis and three liquid phase coupled reaction efficiencies improve six times at least.
Embodiment 4: the coupling of cellulase catalyzed conversion Rhizome of Peltate Yam saponin(e and three liquid-phase extractions
Thick saponin(e is dissolved in 1, in the 4-dioxane, preparation 1,4-dioxane saponin(e liquid.Get 3.6g ammonium sulfate and 10.8g water is made into ammoniumsulphate soln.With above-mentioned inorganic salt solution and 3.6g 1,4-dioxane saponin(e liquid and 2.0g normal hexane mix, and leave standstill 10min after the stirring, form three liquid phases.The analytical results of various saponin(es shows that 100% diosgenin is distributed in phase (normal hexane phase), and 100% glucose three glucosides, glucose bioside, Trillium tschonoskii Maxim time glycosides are distributed in middle phase (1,4-dioxane phase).Cellulase solution is slowly added intermediate phase, 35 ℃ of temperature of reaction, mixing speed is 100rpm.Reaction 72h, the diosgenin transformation efficiency is 69%.With the enzyme of equivalent and substrate in the aqueous phase reaction and remove only keep intermediate phase up and down mutually enzymic catalytic reaction in contrast, the former diosgenin transformation efficiency only is 2.5%, the latter is 34%; Enzyme catalysis doubles than the organic phase reaction with three liquid phase coupled reaction efficiencies, improves 27.6 times than water react.
Claims (5)
1. Glycosylase catalysis and salt separation extracting coupling prepare the method for natural drug, it is characterized in that: the substrate that enzyme catalysis is transformed is dissolved in the hydrophilic organic solvent, adds soluble inorganic salt, leaves standstill to be divided into two-phase up and down, then Glycosylase is added and go up phase, carry out conversion reaction; Monose under the hydrolysis is allocated in down phase, on be the extraction liquid that is rich in target product mutually; Perhaps add hydrophobic organic solvent in biphasic system, form three liquid phases, then Glycosylase is added intermediate phase, the monose that reaction produces is allocated in inorganic salt face, and target product is enriched in phase, and unreacted substrate and enzyme then are retained in intermediate phase;
The substrate of Glycosylase catalyzed conversion is glucosides class monomeric compound or medicinal powder, the extract that contains specific examples of such components;
Glycosylase is one or more a mixed enzyme in the commercial enzymes such as amylase, cellulase, β glucuroide, naringinase, tilactase or helicase; Or can produce the microorganism cells of above enzyme or induce the isozyme of generation, isozyme is induced generation by microorganism through medicinal material or glucosides monomeric compound;
Soluble inorganic salt is phosphoric acid salt, vitriol, carbonate, halogenide or its mixture more than two kinds, and the mass fraction that soluble inorganic salt accounts for system is 5～35%;
Hydrophilic organic solvent is alcohols, short chain ethers, furans, sulfoxide class, alkylene oxides or its mixture more than two kinds, and the mass fraction that hydrophilic organic solvent accounts for system is 5～40%;
Three liquid phases are saltoutd, and selected hydrophobic organic solvent is alkanes, ester class, long-chain ethers or its mixture in the extraction system, and the massfraction that hydrophobic organic solvent accounts for system is 10～60%;
The enzymic catalytic reaction temperature is 20～70 ℃, and mixing speed is 20～250rpm; The time of Glycosylase hydrolysis reaction is 0.5～200h.
2. a kind of Glycosylase catalysis according to claim 1 and salt separation extracting coupling prepare the method for natural drug, and its feature also is: Glycosylase is through chemically modified or immobilized enzyme.
3. a kind of Glycosylase catalysis according to claim 1 and salt separation extracting coupling prepare the method for natural drug, and its feature also is: glucosides class monomeric compound comprises steroidal and terpene saponin(e, anthraquinone terpene and cyclenes ethers saponin(e.
4. a kind of Glycosylase catalysis according to claim 3 and salt separation extracting coupling prepare the method for natural drug, and its feature also is: glucosides class monomeric compound is meant the new glycosides of shield leaf, triangle leaf saponin(e, diosgenin-glucose three glucosides, diosgenin-glucose bioside, prolongs tinkling of pieces of jade grass time glycosides, polydatin, Geniposide, ginsenoside Re, Rg1, Rd, Rc, Potenlini, soybean saponin I, II, IV;
Medicinal powder is meant Rhizome of Peltate Yam, giant knotweed, cape jasmine, genseng, Radix Glycyrrhizae, soybean.
5. the method for preparing natural drug according to claim 1,2,3 or 4 described a kind of Glycosylase catalysis and salt separation extracting coupling, it is characterized in that: when reaction is carried out, to contain the inorganic salt phase transition of sugar, the inorganic salt face that more renews, the hydrophobicity organic phase that perhaps will contain product in the three phase extraction catalyzed reaction shifts, the hydrophobicity organic phase that more renews is added substrate and a little enzyme again, and reaction is constantly carried out.
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