CN102094052B - 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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- CN102094052B CN102094052B CN 201010584745 CN201010584745A CN102094052B CN 102094052 B CN102094052 B CN 102094052B CN 201010584745 CN201010584745 CN 201010584745 CN 201010584745 A CN201010584745 A CN 201010584745A CN 102094052 B CN102094052 B CN 102094052B
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- glycosylase
- salting
- reaction
- catalysis
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- 238000005185 salting out Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 14
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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 salting-out extraction and the coupling of Glycosylase hydrolysis reaction prepare medicine or pharmaceutical intermediate.
Background technology
Natural product becomes the important sources of medicine, food and feeds gradually, has at present sizable market take natural product as main protective foods and medicine.Glucosides class natural product occupies critical role in numerous natural products, a series of glucosides with biological activity and pharmaceutical use come into operation in succession, such as Gastrodine, arbutin, cardigin, platycodin, ginsenoside etc.The activity of glycoside is normal relevant with 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 subject to people's common concern.Than chemistry (acid or alkali) hydrolysis, biology or enzymatic hydrolysis have the characteristics such as selectivity is high, mild condition, activity preservation rate is high, environmental pollution is 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 immobilized β-glucosidase hydrolysis Gingko yellow ketoside to prepare Flavone aglycone (Harbin Engineering University's journal, 2009,30 (5): 584-588); Yang Yishun etc. utilize immobilized β-glucosidase conversion Geniposide to prepare genipin (East China science and engineering master thesis, 2009); Hu Yingjie etc. prepare aglycon (Traditional Chinese Medicine University Of Guangzhou's journal, 2004,21 (6): 473-475) with helicase hydrolysis arctinin; 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); , the usefulness cellulases such as Li Mengqing the polydatin in the giant knotweed change into trans-resveratrol (fine chemistry industry, 2008,25 (5): 467-470), etc.
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) 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 the continuous accumulation along with product, the activity of enzyme often is subject to the inhibition of high density product, and the monose that especially is hydrolyzed has obvious restraining effect to the activity of enzyme, has a strong impact on speed of response and transformation efficiency.Studies show that such as Yu Huilei etc. the monose of high density can make the glycoside hydrolysis reaction of glycoside hydrolysis enzyme catalysis carry out (organic chemistry, 2006,26 (6): 1052-1058) to contrary direction.If product in time can be removed, will remove so undoubtedly product to the enzymeinhibition effect, 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 there is no bibliographical information aspect the enzymatic hydrolysis reaction of glucosides.Salting-out extraction 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 upper phase, and three liquid-phase systems that perhaps form with inorganic salt solution, hydrophilic low molecule organic matter and hydrophobic organic compound separate the effective constituent (Chinese patent: CN101637667) of opposed polarity.Glycosylase catalysis and salting-out extraction separation coupling are also needed to solve the stability problem of enzyme in organic phase, investigate 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
Transform and extract the problems that exist in the sepn process for solving active skull cap components in enzyme catalysis, low in aqueous phase solubleness such as effective composition, 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 salting-out extraction 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 up and down two-phase, then Glycosylase is added upper phase, carries out conversion reaction.Monose under the hydrolysis is allocated in lower phase (inorganic salt face), and upper is the extraction liquid that is rich in target product mutually; Perhaps in biphasic system, add hydrophobic organic solvent, form three liquid phases, phase in the middle of then Glycosylase being added, the monose that reaction produces is allocated in inorganic salt face, target product is enriched in phase, unreacted substrate with in the middle of enzyme then is retained in mutually.
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., the Chinese medicinal materialss such as medicinal material such as Rhizome of Peltate Yam, giant knotweed, cape jasmine, ginseng, Radix Glycyrrhizae, soybean.Target product is medicine or the pharmaceutical intermediate that the glycosyl number is less than conversion of substrate, such as diosgenin, trans-resveratrol, genipin, ginsenoside Rg3, ginsenoside Rh2, GF2, Ginsenoside compound K, single glucuronic acid-glycyrrhetinic acid, soyasterol B etc.
Glycosylase is one or more 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, in order to improve Enzymic stability and reusing.Glycosylase can be hydrolyzed several glycosidic links that exist in the 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 type, alkylene oxides or its mixture more than two kinds, and the mass fraction that hydrophilic organic solvent accounts for system is 5~40%.
Selected hydrophobic organic solvent is alkanes, ester class, long-chain ethers or its mixture in the three liquid phase salting-out extraction systems, 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 salting-out extraction coupling are conducive to remove product to the restraining effect of enzymic activity, improve speed of reaction;
(3) enzyme catalysis conversion and salting-out extraction 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, 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 the BDO preparation BDO saponin(e liquid.Get 3.36g ammonium sulfate and 11.04g water is made into ammoniumsulphate soln.Above-mentioned inorganic salt solution and 3.6g BDO saponin(e liquid are mixed, leave 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 lower 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 upper phase, 50 ℃ of temperature of reaction, mixing speed is 70rpm, reaction 48h, the transformation efficiency of diosgenin reaches 11%.In the aqueous phase reaction and remove the lower enzymic catalytic reaction that only stays mutually phase in contrast, the former diosgenin transformation efficiency only is 2.5% with the enzyme of equivalent and substrate, and 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 the BDO preparation BDO 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 BDO saponin(e liquid and the mixing of 2.0g sherwood oil, 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 lower phase (ammonium sulfate phase).Helicase solution is slowly added middle 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 mutually up and down the centre phase enzymic catalytic reaction in contrast, the former diosgenin transformation efficiency only is 2.5%, the latter is 5%; The reaction efficiency of enzyme catalysis and the coupling of three liquid phases improves 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 Isosorbide-5-Nitrae-dioxane preparation Isosorbide-5-Nitrae-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 Isosorbide-5-Nitrae-dioxane saponin(e liquid and the mixing of 2.0g normal hexane, leave standstill 10min after the stirring, form three liquid phases.The analytical results of various saponin(es shows, 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 (Isosorbide-5-Nitrae-dioxane phase).Cellulase solution is slowly added middle 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 mutually up and down the centre phase 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 the reaction efficiency of three liquid phases coupling, improves 27.6 times than water react.
Claims (5)
1. a Glycosylase catalysis and salting-out extraction 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 up and down two-phase, then Glycosylase is added upper phase, carry out conversion reaction; Monose under the hydrolysis is allocated in lower phase, and upper is the extraction liquid that is rich in target product mutually; Perhaps in biphasic system, add hydrophobic organic solvent, form three liquid phases, phase in the middle of then Glycosylase being added, the monose that reaction produces is allocated in inorganic salt face, target product is enriched in phase, unreacted substrate with in the middle of enzyme then is retained in mutually;
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 mixed enzyme in amylase, cellulase, β glucuroide, naringinase, tilactase or the 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 type, alkylene oxides or its mixture more than two kinds, and the mass fraction that hydrophilic organic solvent accounts for system is 5 ~ 40%;
Selected hydrophobic organic solvent is alkanes, ester class, long-chain ethers or its mixture in the three liquid phase salting-out extraction systems, 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 ~ 250 rpm; The time of Glycosylase hydrolysis reaction is 0.5 ~ 200 h.
2. a kind of Glycosylase catalysis according to claim 1 and salting-out extraction coupling prepare the method for natural drug, and be further characterized in that: Glycosylase is through chemically modified or immobilized.
3. a kind of Glycosylase catalysis according to claim 1 and salting-out extraction coupling prepare the method for natural drug, are further characterized in that: 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 salting-out extraction coupling prepare the method for natural drug, are further characterized in that: glucosides class monomeric compound refers to 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, ginsenoside Rg1, Ginsenoside Rd, Ginsenoside Rc, Potenlini, Sayasaponin Ⅰ, soybean saponin II, soybean saponin IV; Medicinal powder refers to Rhizome of Peltate Yam, giant knotweed, cape jasmine, ginseng, Radix Glycyrrhizae, soybean.
5. according to claim 1,2,3 or 4 described a kind of Glycosylase catalysis and salting-out extraction coupling prepare the method for natural drug, 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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CN103184245B (en) * | 2013-03-01 | 2014-12-31 | 华南理工大学 | Method for producing fatty acid by hydrolyzing lipid through three liquid-phase lipase catalytic systems |
CN104003844B (en) * | 2014-05-23 | 2015-12-30 | 大连理工大学 | Extract the method with 2,3-butanediol in fermentation and coupling separate fermentation liquid |
KR20170047007A (en) * | 2015-10-22 | 2017-05-04 | (주)아모레퍼시픽 | Method for selectively preparing ginsenoside F2, compound Mc and compound O from saponins of ginseng by enzymatic process |
CN106074581B (en) * | 2016-07-01 | 2019-02-26 | 中央民族大学 | Vascular protection pharmaceutical composition and application thereof |
WO2018107376A1 (en) * | 2016-12-14 | 2018-06-21 | 邦泰生物工程(深圳)有限公司 | Purification method for ginsenoside rh2 |
CN110372770A (en) * | 2018-04-12 | 2019-10-25 | 耿兆翔 | The highly effective extraction method of natural hydroxyl or/and carboxyl compound |
CN109082452A (en) * | 2018-08-24 | 2018-12-25 | 江苏科技大学 | A kind of method of liquid-liquid-solid system enzymatic hydrolysis extraction and separation mulberry red pigment |
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