CN105238826A - Application of beta-glucosidase and method for preparing baicalein through beta-glucosidase - Google Patents
Application of beta-glucosidase and method for preparing baicalein through beta-glucosidase Download PDFInfo
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- CN105238826A CN105238826A CN201510812991.8A CN201510812991A CN105238826A CN 105238826 A CN105238826 A CN 105238826A CN 201510812991 A CN201510812991 A CN 201510812991A CN 105238826 A CN105238826 A CN 105238826A
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Abstract
The invention relates to the technical field of biology, in particular to an application of beta-glucosidase and a method for preparing baicalein through the beta-glucosidase. The amino acid sequence of the beta-glucosidase is shown as SEQ ID No.1. The invention further provides a baicalin enzyme hydrolysis method. The heat-resisting and sugar-resisting beta-glucosidase is immobilized to hydrolyze the baicalin in a buffer solution, and the baicalein is obtained. The beta-glucosidase has high product tolerance, the baicalin can be completely hydrolyzed without being in a double-phase system, and it is avoided that due to organic solvents in the double-phase system, the activity of the glucosidase is reduced. The immobilized glucosidase can be repeatedly used. By means of the application and the method, the high-purity baicalein is obtained, and the purity is higher than 95%. According to the method, after the baicalein is extracted through ethyl acetate, recrystallization can be carried out through organic solvents, the high-purity baicalein can be obtained, and the purity is higher than 98%.
Description
Technical field
The present invention relates to biological technical field, particularly beta-glucosidase purposes and prepare the method for scutellarin with it.
Background technology
The root of large-flowered skullcap is the dry root of the labiate root of large-flowered skullcap (ScutellariabaicalensisGeorgi), document is recorded the root of large-flowered skullcap and is had the hot eliminating dampness that disappears, eliminating fire and detoxication, hemostasis, antiabortive function, can be used for treatment upper respiratory tract infection, urinary system infection, bacillary dysentery, hepatitis, hypertension etc., also can be widely used in protective foods.Main component in the root of large-flowered skullcap is baicalin (glucoside), and structure is such as formula shown in I, and content, up to 7-19%, only contains the wogonoside of trace, scutellarin (structure is such as formula II Suo Shi) and wogonin.After the oral root of large-flowered skullcap, under the effect of the enzyme in Digestive tract of baicalin wherein, change scutellarin into, its drug effect could be played better.Because baicalin is difficult to absorb in human body, transform low in body in addition; And scutellarin easily absorbs, bioavailability is high, and its pharmacologically active is also far above baicalin, and as the apoptosis of anti AIDS virus, inductive infection duck virocyte, the effect such as inhibiting HIV reversed transcriptive enzyme etc. are all far above baicalin.For this reason, people be devoted to research baicalin is become scutellarin.
Existing people utilizes acid-hydrolysis method at present, baicalin is converted into the root of large-flowered skullcap and usually prepares scutellarin in a large number, but due to baicalin be 7-O-glucuronic acid in conjunction with glycosides, glycosidic link comparatively firmly not easy fracture, therefore part baicalin can only be converted into scutellarin by acid hydrolysis, and the rate of transform is subject to a definite limitation; In addition, the method is comparatively large to the destruction of baicalin, and environmental pollution is serious.
Along with the development of biotechnology, enzyme has specificity good as catalyzer, and catalytic efficiency is high, and the advantages such as reaction conditions gentleness, industrial application also substitutes traditional catalyzer more and more.But enzyme is exactly price comparison costliness as the main drawback that Catalyst is applied, and can not reuse.In addition, because baicalin is the inhibitor of GRD beta-glucuronidase or beta-glucosidase, therefore general beta-glucosidase or GRD beta-glucuronidase can not be hydrolyzed baicalin well.Therefore, need to find the enzyme that the beta-glucuronic acid base that can be hydrolyzed baicalin well obtains scutellarin.
Utilize endogenous enzyme process degradation condition gentleer, but the used time is longer, still needs to adopt silicagel column and a large amount of organic solvent to be separated after degraded and obtain scutellarin.Such as utilize endogenous enzymatic hydrolysis baicalin in Scutellaria baicalensis Georgi and wogonoside, hydrolysising condition is gentleer, but still need to adopt silicagel column and a large amount of organic solvent to be separated after hydrolysis and obtain scutellarin, therefore, all there is certain limitation in existing preparation method in the rate of transform, operability, security, cost etc.
Someone utilizes high-temperature aerobic bacterium Bacillussp.JF at present
2, high temperature anaerobic bacterium Clostridiumthermocopriae, black-koji mould, aspergillus oryzae or candiyeast add the enzymic hydrolysis baicalin glucal acidic group that baicalin fermentation culture produces, prepare scutellarin, the method improves to some extent, but the accumulation of reaction product can have an impact to the activity of enzyme, also enzyme is not recycled, and its separation method is more complicated.Somebody have studied the method for baicalin enzyme-squash techniqued scutellarin, but large by enzyme amount, is difficult to be applied to suitability for industrialized production, and does not also carry out recycling to enzyme.
Enzyme immobilizatio is fixed on certain carrier by biological catalyst enzyme.Within 1916, sucrase is adsorbed onto on gac and alumina by Nelson and Griffin first, realize first enzyme immobilizatio (R.F.Taylor, Proteinimmobilization, MarcelDekker, Inc:NewYork, 1991, v).Fixation techniques for enzyme just obtains very fast development from then on, has up to the present had very ripe process for fixation: absorption, covalent cross-linking, embedding etc., also show that immobilized enzyme can also improve the stability of enzyme to a certain extent simultaneously.The eighties of last century enzyme immobilizatio seventies started be applied to industrialization (R.F.Taylor, Proteinimmobilization, MarcelDekker, Inc:NewYork, 1991, v).Someone studies immobilized enzyme hydrolysis baicalin in diphasic system and prepares the method for scutellarin, the method has certain advance, reaction product scutellarin can be extracted into organic phase by diphasic system in time, the reaction be conducive in aqueous phase is carried out completely, but the organic solvent in diphasic system easily makes enzyme deactivation, greatly reduce the recycling number of times of immobilized enzyme.In order to enable scutellarin be applied more widely, the scale operation preparation method for scutellarin needs to innovate and improve.
Summary of the invention
In view of this, the invention provides the purposes of beta-glucosidase and prepare the method for scutellarin with it.The method is hydrolyzed by baicalin in buffered soln by heat-resisting for one resistance to sugared beta-glucosidase, this enzyme has very strong product tolerance, without the need to just can baicalin be hydrolyzed completely in diphasic system, the organic solvent avoided in diphasic system makes enzyme deactivation, be separated by extraction into ethyl acetate after reaction terminates and obtain scutellarin, immobilized enzyme can repeatedly utilize.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides beta-glucosidase prepares scutellarin purposes for being hydrolyzed baicalin; The aminoacid sequence of described beta-glucosidase is as shown in SEQIDNo.1.
Beta-glucosidase of the present invention is that direct fermentation obtains from the extreme thermobacillus of one (ThermotogapetrophilaDSM13995), or is transformed into acquisition recombinant chou high expression acquisition in intestinal bacteria (E.coli) after being cloned by this enzyme gene (Tpebgl3).
Present invention also offers a kind of method preparing scutellarin, adopt beta-glucoside enzymic hydrolysis baicalin to prepare scutellarin; The aminoacid sequence of described beta-glucosidase is as shown in SEQIDNo.1.
Beta-glucosidase of the present invention is a kind of thermostable enzyme, has good thermostability, and more general beta-glucoside enzyme reaction temperature is high, is conducive to the dissolving of baicalin in buffered soln.In specific embodiments more of the present invention, the temperature of described hydrolysis is 60 ~ 120 DEG C, and the time of described catalysis baicalin hydrolysis is 5 ~ 10h.
In specific embodiments more of the present invention, the temperature of described hydrolysis is 70 ~ 100 DEG C.
In specific embodiments more of the present invention, the temperature of described hydrolysis is 75 ~ 90 DEG C.
Water-soluble very poor due to baicalin, suitable buffered soln can improve the solvability of baicalin, and enzymic catalytic reaction needs the baicalin that can dissolve, and can strengthen the solubleness of baicalin at relatively high temperatures, be conducive to the carrying out reacted.
The feature of described beta-glucosidase is that some metal ion is as Hg
2+can suppress the activity that this enzyme catalysis baicalin is hydrolyzed, some metal ion is (as Na
+, Mg
2+, Ca
2+, Mn
2+, Co
2+deng) can improve this enzyme catalysis baicalin hydrolysis activity.In specific embodiments more of the present invention, described hydrolysis also comprises metal ion Na
+, Mg
2+, Ca
2+, Mn
2+or Co
2+combine with described beta-glucosidase.
In specific embodiments more of the present invention, described beta-glucosidase has good tolerance to product glucuronic acid; When monosaccharide concentration is lower than 0.5mol/L, there is activation to described beta-glucosidase; When monosaccharide concentration is greater than 0.5mol/L, have restraining effect to enzyme, when monosaccharide concentration is 1.2mol/L, enzyme inhibiting rate alive is 50%.Beta-glucosidase of the present invention is a kind of product tolerance enzyme, and better to the product tolerance of hydrolytic process, therefore, reaction does not need to carry out in diphasic system, and the organic solvent avoided in diphasic system makes enzyme deactivation.
In specific embodiments more of the present invention, described hydrolysis is carried out in buffered soln; Described buffered soln is acetate buffer, citrate buffer, carbonate buffer solution, phosphate buffered saline buffer or Tris-hydrochloride buffer; The pH value of described buffered soln is 3-10.
In other specific embodiments of the present invention, the pH value of described buffered soln is 4-8.
In other specific embodiments of the present invention, the pH value of described buffered soln is 4.5-6.
In specific embodiments more of the present invention, buffered soln of the present invention specifically can be as acetate buffer.
In specific embodiments more of the present invention, described acetate buffer solution is the solution be made into by acetic acid and sodium-acetate, and the volumetric molar concentration of described acetic acid and sodium acetate soln is 0.005-1.5M.
In other specific embodiments of the present invention, the volumetric molar concentration of described acetic acid and sodium acetate soln is 0.02-0.4M.
In other specific embodiments of the present invention, the volumetric molar concentration of described acetic acid and sodium acetate soln is 0.05M.
In specific embodiments more of the present invention, the proportioning of described baicalin and beta-glucosidase is 1g:(1 ~ 80) U.
In other specific embodiments of the present invention, the proportioning of described baicalin and beta-glucosidase is 1g:10-40U.
In other specific embodiments of the present invention, the proportioning of described baicalin and beta-glucosidase is 1g:20-30U.
In specific embodiments more of the present invention, also comprise the step of extraction into ethyl acetate, organic solvent recrystallization.
In other specific embodiments of the present invention, also comprise concentrated, dry step.
Concrete, the method preparing scutellarin comprises the steps:
By 1g baicalin (the HPLC spectrogram of the baicalin before reaction as shown in Figure 1,
1h-NMR spectrogram as shown in Figure 2,
13c-NMR and DEPT135 spectrogram as shown in Figure 3, under nuclear magnetic data is shown in) the HAc-NaAc buffered soln of to join the pre-configured pH of 50mL be 3-10 or pH to be 4-8 or pH be 4.5-6, acetate buffer, citrate buffer, carbonate buffer solution, in phosphate buffered saline buffer or Tris-hydrochloride buffer, its volumetric molar concentration is 0.005-1.5M or 0.02-0.4M or 0.05M), be heated to 85 DEG C, activation 10min, then the immobilized β-glucosidase of 30U or 1-80U or 10-40U or 20-30U or beta-glucosidase powder or beta-glucosidase liquid is added, with magnetic stirrer reaction 5h or 5-10h at 90 DEG C or 60-120 DEG C or 70-100 DEG C or 75-90 DEG C, a small amount of reaction solution is drawn under the state shaken up, after filtering with microporous membrane, filtrate is detected with HPLC, transformation efficiency is 92.4%.With 50mL extraction into ethyl acetate twice after reaction solution cooling, merge twice extraction liquid, after concentrating under reduced pressure drying, obtain the higher scutellarin of purity (purity is more than 95%).Then use suitable organic solvent recrystallization, obtain highly purified scutellarin (purity more than 98%, the HPLC spectrogram of the scutellarin of acquisition as shown in Figure 5,
1h-NMR spectrogram as shown in figure 12,
13c-NMR and DEPT135 spectrogram is as shown in figure 13), organic solvent can be a kind of or both the above mixtures in ethanol, ethyl acetate, ether, acetone, DMF.
Baicalin, faint yellow needle-like crystal, molecular formula is C
21h
18o
11.
1hNMR (400MHz, DMSO-d
6) δ 12.60 (active H, 5-OH), 8.69 (active H, 6-OH), 8.07 (2H, dd, J=8.0Hz, 1.8Hz, H-2 ', 6 '), 7.60 (3H, m, H-3 ', 4 ', 5 '), 7.06 (1H, s, H-3), 7.01 (1H, s, H-8), 4.08 (1H, d, J=9.5Hz, H-G1, the H on sugared end group).
13c-NMR (101MHz, DMSO-d
6) δ: 163.6 (C-2), 106.2 (C-3), 182.6 (C-4), 146.8 (C-5), 130.6 (C-6), 151.3 (C-7), 93.7 (C-8), 149.2 (C-9), 104.8 (C-10), 130.9 (C-1 '), 126.4 (C-2 ', C-6 '), 129.2 (C-3 ', C-5 '), 132.1 (C-4 '), 99.9 (C-G1, carbon on sugar end group), 72.8 (C-G2), 75.3 (C-G3), 71.4 (C-G4), 75.5 (C-G5) 170.1 (C-G6).
Scutellarin, yellow needle-like crystals, molecular formula is C
15h
10o
5.
1H-NMR(400MHz，DMSO-d
6)δ:6.63(1H,s,H-8)，6.93(1H,s,H-3)，7.57(3H,m,H-3’,4’,5’)，8.06(2H,d,J＝8.0Hz,H-2’,6’)，9.66(1H,s,H-7)，12.65(1H,s,H-5)。
13C-NMR(100MHz,DMSO-d
6)δ:153.65(C-2)，104.49(C-3)，182.13(C-4)，149.85(C-5)，129.13(C-6)，162.90(C-7)，94.00(C-8)，146.94(C-9)，104.26(C-10)，131.84(C-1’)，126.32(C-2’)，129.13(C-3’)，130.98(C-4’)。
In specific embodiments more of the present invention, described beta-glucosidase is on-fixed enzyme (the enzyme liquid obtained as direct fermentation or dried enzyme powder) or immobilized enzyme, is preferably immobilized enzyme.
In specific embodiments more of the present invention, beta-glucosidase of the present invention specifically can be immobilized enzyme, and this immobilized enzyme can repeatedly be recycled.
The invention provides beta-glucosidase prepares scutellarin purposes for being hydrolyzed baicalin; The aminoacid sequence of described beta-glucosidase is as shown in SEQIDNo.1.Present invention also offers the method for enzymic hydrolysis baicalin, baicalin is hydrolyzed after heat-resisting for one resistance to sugared beta-glucoside enzyme immobilization in buffered soln, this enzyme has very strong product tolerance, without the need to just can baicalin be hydrolyzed completely in diphasic system, the organic solvent avoided in diphasic system makes enzymic activity reduce, obtained the scutellarin of higher degree after reaction terminates by extraction into ethyl acetate, immobilized enzyme can repeatedly utilize.
After in the method for the invention, reaction terminates, be extracted with ethyl acetate, then revolve and steam the described ethyl acetate of removing, obtain the scutellarin that purity is higher, purity is more than 95%.After in method of the present invention, extraction into ethyl acetate obtains scutellarin, organic solvents available recrystallization, obtain highly purified scutellarin, purity is more than 98%.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 shows the HPLC spectrogram of the baicalin before reaction, the purity 95.7% of the baicalin before reaction;
Fig. 2 shows the baicalin before reaction
1h-NMR spectrogram;
Fig. 3 shows the baicalin before reaction
13c-NMR and DEPT135 spectrogram;
Fig. 4 shows the HPLC spectrogram of detection reaction liquid after enzymic catalytic reaction in embodiment 1, and the transformation efficiency of baicalin is about 92%;
Fig. 5 shows the HPLC spectrogram of the scutellarin obtained by recrystallization in embodiment 1, and the purity of scutellarin is 98.6%;
Fig. 6 shows the HPLC spectrogram of the scutellarin obtained by recrystallization in embodiment 2, and the purity of scutellarin is 98.3%;
Fig. 7 shows the HPLC spectrogram of the scutellarin obtained by recrystallization in embodiment 3, and the purity of scutellarin is 99.2%;
Fig. 8 shows the HPLC spectrogram of the scutellarin obtained by recrystallization in embodiment 4, and the purity of scutellarin is 98.3%;
Fig. 9 shows the HPLC spectrogram of the scutellarin obtained by recrystallization in embodiment 5, and the purity of scutellarin is 98.2%;
Figure 10 shows the HPLC spectrogram of the scutellarin obtained by recrystallization in embodiment 6, and the purity of scutellarin is 99.1%;
Figure 11 shows the HPLC spectrogram of the scutellarin obtained by recrystallization in embodiment 7, and the purity of scutellarin is 98.7%;
Figure 12 shows the scutellarin of acquisition
1h-NMR spectrogram;
Figure 13 shows the scutellarin of acquisition
13c-NMR and DEPT135 spectrogram.
Embodiment
The invention discloses the purposes of beta-glucosidase and prepare the method for scutellarin with it, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
The purposes of beta-glucosidase provided by the invention and prepare raw materials used in the method for scutellarin and reagent with it and all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1
By 1g baicalin (the HPLC spectrogram of the baicalin before reaction as shown in Figure 1,
1h-NMR spectrogram as shown in Figure 2,
13c-NMR and DEPT135 spectrogram is as shown in Figure 3) join in the HAc-NaAc buffered soln of the pre-configured pH5.5 of 50mL, be heated to 80 DEG C, activation 10min, then the immobilized β-glucosidase that vigor is 60U is added, at 80 DEG C, react 5h by magnetic stirrer, under the state shaken up, draw a small amount of reaction solution, after filtering with microporous membrane, filtrate detects (as shown in Figure 4) with HPLC, and transformation efficiency is 93.6%.With 50mL extraction into ethyl acetate twice after reaction solution cooling, merge twice extraction liquid, after concentrate drying, add 50mL re-crystallizing in ethyl acetate again, obtain scutellarin 528.2mg, Powdered, color cadmium yellow, HPLC detects, and scutellarin purity was 98.3% (as shown in Figure 5).
Scutellarin, yellow needle-like crystals, molecular formula is C
15h
10o
5.
1H-NMR(400MHz，DMSO-d
6)δ:6.63(1H,s,H-8)，6.93(1H,s,H-3)，7.57(3H,m,H-3’,4’,5’)，8.06(2H,d,J＝8.0Hz,H-2’,6’)，9.66(1H,s,H-7)，12.65(1H,s,H-5)。
13C-NMR(100MHz,DMSO-d
6)δ:153.65(C-2)，104.49(C-3)，182.13(C-4)，149.85(C-5)，129.13(C-6)，162.90(C-7)，94.00(C-8)，146.94(C-9)，104.26(C-10)，131.84(C-1’)，126.32(C-2’)，129.13(C-3’)，130.98(C-4’)。
Embodiment 2
1g baicalin is joined (volumetric molar concentration is 0.05M) in the citrate buffer of the pre-configured pH3 of 50mL, be heated to 85 DEG C, activation 10min, then 2mL beta-glucosidase liquid (enzyme is lived as 300U) is added, at 85 DEG C, react 8h by magnetic stirrer, under the state shaken up, draw a small amount of reaction solution, after filtering with microporous membrane, filtrate is detected with HPLC, and transformation efficiency is 91.3%.With 50mL extraction into ethyl acetate twice after reaction solution cooling, merge twice extraction liquid, after concentrate drying, add 50mL95% ethyl alcohol recrystallization again, finally obtain scutellarin 512.7mg, Powdered, color cadmium yellow, HPLC detects, and scutellarin content was 98.3% (as shown in Figure 6).
Scutellarin, yellow needle-like crystals, molecular formula is C
15h
10o
5.
1H-NMR(400MHz，DMSO-d
6)δ:6.63(1H,s,H-8)，6.93(1H,s,H-3)，7.57(3H,m,H-3’,4’,5’)，8.06(2H,d,J＝8.0Hz,H-2’,6’)，9.66(1H,s,H-7)，12.65(1H,s,H-5)。
13C-NMR(100MHz,DMSO-d
6)δ:153.65(C-2)，104.49(C-3)，182.13(C-4)，149.85(C-5)，129.13(C-6)，162.90(C-7)，94.00(C-8)，146.94(C-9)，104.26(C-10)，131.84(C-1’)，126.32(C-2’)，129.13(C-3’)，130.98(C-4’)。
Embodiment 3
1g baicalin is joined (volumetric molar concentration is 1.5M) in the HAc-NaAc buffered soln of the pre-configured pH8 of 50mL, be heated to 85 DEG C, activation 10min, then 2mL beta-glucosidase liquid (enzyme is lived as 460U) is added, at 120 DEG C, react 5h by magnetic stirrer, under the state shaken up, draw a small amount of reaction solution, after filtering with microporous membrane, filtrate is detected with HPLC, and transformation efficiency is 89.6%.With 50mL extraction into ethyl acetate twice after reaction solution cooling, merge twice extraction liquid, after concentrate drying, add 50mL recrystallizing methanol again, obtain scutellarin 503.1mg, Powdered, color cadmium yellow, HPLC detects, and scutellarin content was 99.2% (as shown in Figure 7).
Scutellarin, yellow needle-like crystals, molecular formula is C
15h
10o
5.
1H-NMR(400MHz，DMSO-d
6)δ:6.63(1H,s,H-8)，6.93(1H,s,H-3)，7.57(3H,m,H-3’,4’,5’)，8.06(2H,d,J＝8.0Hz,H-2’,6’)，9.66(1H,s,H-7)，12.65(1H,s,H-5)。
13C-NMR(100MHz,DMSO-d
6)δ:153.65(C-2)，104.49(C-3)，182.13(C-4)，149.85(C-5)，129.13(C-6)，162.90(C-7)，94.00(C-8)，146.94(C-9)，104.26(C-10)，131.84(C-1’)，126.32(C-2’)，129.13(C-3’)，130.98(C-4’)。
Embodiment 4
1g baicalin is joined (volumetric molar concentration is 0.02M) in the Tris-hydrochloride buffer of the pre-configured pH10 of 50mL, be heated to 85 DEG C, activation 10min, then 2mL beta-glucosidase liquid (enzyme is lived as 360U) is added, at 70 DEG C, react 5h by magnetic stirrer, under the state shaken up, draw a small amount of reaction solution, after filtering with microporous membrane, filtrate is detected with HPLC, and transformation efficiency is 90.4%.With 50mL extraction into ethyl acetate twice after reaction solution cooling, merge twice extraction liquid, after concentrate drying, add 30mL acetone and 20mL water recrystallization again, obtain scutellarin 517.4mg, Powdered, color cadmium yellow, HPLC detects, and scutellarin content was 98.3% (as shown in Figure 8).
Scutellarin, yellow needle-like crystals, molecular formula is C
15h
10o
5.
1H-NMR(400MHz，DMSO-d
6)δ:6.63(1H,s,H-8)，6.93(1H,s,H-3)，7.57(3H,m,H-3’,4’,5’)，8.06(2H,d,J＝8.0Hz,H-2’,6’)，9.66(1H,s,H-7)，12.65(1H,s,H-5)。
13C-NMR(100MHz,DMSO-d
6)δ:153.65(C-2)，104.49(C-3)，182.13(C-4)，149.85(C-5)，129.13(C-6)，162.90(C-7)，94.00(C-8)，146.94(C-9)，104.26(C-10)，131.84(C-1’)，126.32(C-2’)，129.13(C-3’)，130.98(C-4’)。
Embodiment 5
1g baicalin is joined (volumetric molar concentration is 0.05M) in the phosphate buffered saline buffer of the pre-configured pH4.5 of 50mL, be heated to 85 DEG C, activation 10min, then 2mL beta-glucosidase liquid (enzyme is lived as 400U) is added, at 100 DEG C, react 5h by magnetic stirrer, under the state shaken up, draw a small amount of reaction solution, after filtering with microporous membrane, filtrate is detected with HPLC, and transformation efficiency is 88.7%.With 50mL extraction into ethyl acetate twice after reaction solution cooling, merge twice extraction liquid, after concentrate drying, add 50mL Diethyl ether recrystallization again, obtain scutellarin 503.4mg, Powdered, color cadmium yellow, HPLC detects, and scutellarin content was 98.2% (as shown in Figure 9).
Scutellarin, yellow needle-like crystals, molecular formula is C
15h
10o
5.
1H-NMR(400MHz，DMSO-d
6)δ:6.63(1H,s,H-8)，6.93(1H,s,H-3)，7.57(3H,m,H-3’,4’,5’)，8.06(2H,d,J＝8.0Hz,H-2’,6’)，9.66(1H,s,H-7)，12.65(1H,s,H-5)。
13C-NMR(100MHz,DMSO-d
6)δ:153.65(C-2)，104.49(C-3)，182.13(C-4)，149.85(C-5)，129.13(C-6)，162.90(C-7)，94.00(C-8)，146.94(C-9)，104.26(C-10)，131.84(C-1’)，126.32(C-2’)，129.13(C-3’)，130.98(C-4’)。
Embodiment 6
1g baicalin is joined (volumetric molar concentration is 0.4M) in the carbonate buffer solution of the pre-configured pH6 of 50mL, be heated to 85 DEG C, activation 10min, then 2mL beta-glucosidase liquid (enzyme is lived as 100U) is added, at 75 DEG C, react 10h by magnetic stirrer, under the state shaken up, draw a small amount of reaction solution, after filtering with microporous membrane, filtrate is detected with HPLC, and transformation efficiency is 91.5%.With 50mL extraction into ethyl acetate twice after reaction solution cooling, merge twice extraction liquid, after concentrate drying, add 40mL methyl alcohol and 10mL water recrystallization again, obtain scutellarin 498.7mg, Powdered, color cadmium yellow, HPLC detects, and scutellarin content was 99.1% (as shown in Figure 10).
Scutellarin, yellow needle-like crystals, molecular formula is C
15h
10o
5.
1H-NMR(400MHz，DMSO-d
6)δ:6.63(1H,s,H-8)，6.93(1H,s,H-3)，7.57(3H,m,H-3’,4’,5’)，8.06(2H,d,J＝8.0Hz,H-2’,6’)，9.66(1H,s,H-7)，12.65(1H,s,H-5)。
13C-NMR(100MHz,DMSO-d
6)δ:153.65(C-2)，104.49(C-3)，182.13(C-4)，149.85(C-5)，129.13(C-6)，162.90(C-7)，94.00(C-8)，146.94(C-9)，104.26(C-10)，131.84(C-1’)，126.32(C-2’)，129.13(C-3’)，130.98(C-4’)。
Embodiment 7
1g baicalin is joined (volumetric molar concentration is 1M) in the citrate buffer of the pre-configured pH4 of 50mL, be heated to 85 DEG C, activation 10min, then 2mL beta-glucosidase liquid (enzyme is lived as 80U) is added, at 80 DEG C, react 5h by magnetic stirrer, under the state shaken up, draw a small amount of reaction solution, after filtering with microporous membrane, filtrate is detected with HPLC, and transformation efficiency is 90.7%.With 50mL extraction into ethyl acetate twice after reaction solution cooling, merge twice extraction liquid, after concentrate drying, add 50mL acetone recrystallization again, obtain scutellarin 518.5mg, Powdered, color cadmium yellow, HPLC detects, and scutellarin content was 98.7% (as shown in figure 11).
Scutellarin, yellow needle-like crystals, molecular formula is C
15h
10o
5.
1H-NMR(400MHz，DMSO-d
6)δ:6.63(1H,s,H-8)，6.93(1H,s,H-3)，7.57(3H,m,H-3’,4’,5’)，8.06(2H,d,J＝8.0Hz,H-2’,6’)，9.66(1H,s,H-7)，12.65(1H,s,H-5)。
13C-NMR(100MHz,DMSO-d
6)δ:153.65(C-2)，104.49(C-3)，182.13(C-4)，149.85(C-5)，129.13(C-6)，162.90(C-7)，94.00(C-8)，146.94(C-9)，104.26(C-10)，131.84(C-1’)，126.32(C-2’)，129.13(C-3’)，130.98(C-4’)。
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. beta-glucosidase prepares the purposes of scutellarin for being hydrolyzed baicalin; The aminoacid sequence of described beta-glucosidase is as shown in SEQIDNo.1.
2. prepare a method for scutellarin, it is characterized in that, adopt beta-glucoside enzymic hydrolysis baicalin to prepare scutellarin; The aminoacid sequence of described beta-glucosidase is as shown in SEQIDNo.1.
3. method according to claim 2, is characterized in that, the temperature of described hydrolysis is 60 ~ 120 DEG C, and the time of described hydrolysis is 5 ~ 10h.
4. method according to claim 2, is characterized in that, the temperature of described hydrolysis is 70 ~ 100 DEG C.
5. method according to claim 2, is characterized in that, the temperature of described hydrolysis is 75 ~ 90 DEG C.
6. the method according to any one of claim 2 to 5, is characterized in that, described hydrolysis also comprises metal ion Na
+, Mg
2+, Ca
2+, Mn
2+or Co
2+combine with described beta-glucosidase.
7. the method according to any one of claim 2 to 6, is characterized in that, the resistance to sugar of described beta-glucosidase;
When monosaccharide concentration is lower than 0.5mol/L, there is activation to described beta-glucosidase;
When monosaccharide concentration is greater than 0.5mol/L, have restraining effect to enzyme, when monosaccharide concentration is 1.2mol/L, enzyme inhibiting rate alive is 50%.
8. the method according to any one of claim 2 to 7, is characterized in that, described hydrolysis is carried out in buffered soln; Described buffered soln is acetate buffer, citrate buffer, carbonate buffer solution, phosphate buffered saline buffer or Tris-hydrochloride buffer; The pH value of described buffered soln is 3-10.
9. the method according to any one of claim 2 to 8, is characterized in that, the proportioning of described baicalin and beta-glucosidase is 1g:(1 ~ 80) U.
10. the method according to any one of claim 2 to 7, is characterized in that, also comprises the step of extraction into ethyl acetate, organic solvent recrystallization.
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| EP3913063A1 (en) | 2020-05-20 | 2021-11-24 | Synbionik GmbH | Non-naturally occurring microorganism for the biosynthetic production of baicalein |
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| CN107354143A (en) * | 2017-08-11 | 2017-11-17 | 南京林业大学 | A kind of immobilization beta glucuroide and its preparation method and application |
| CN108398392A (en) * | 2018-03-01 | 2018-08-14 | 江西师范大学 | Research method for inhibiting reaction rate of salt ion pair β -glucosidase |
| CN109266701A (en) * | 2018-11-06 | 2019-01-25 | 西安雨田农业科技有限公司 | A method of scutelloside in radix scutellariae medicinal materials is hydrolyzed to baicalein |
| EP3913063A1 (en) | 2020-05-20 | 2021-11-24 | Synbionik GmbH | Non-naturally occurring microorganism for the biosynthetic production of baicalein |
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