CN104673862A - Methodfor synthesizing prunin from naringin in presence of alpha-L-rhamnosidase - Google Patents
Methodfor synthesizing prunin from naringin in presence of alpha-L-rhamnosidase Download PDFInfo
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- CN104673862A CN104673862A CN201510077358.9A CN201510077358A CN104673862A CN 104673862 A CN104673862 A CN 104673862A CN 201510077358 A CN201510077358 A CN 201510077358A CN 104673862 A CN104673862 A CN 104673862A
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Abstract
The invention discloses a method for synthesizing prunin for synthesizing prunin from naringin in presence of alpha-L-rhamnosidase. The method comprises the following steps: firstly dissolving the naringin in an acetic acid-sodium acetate buffer solution to obtain a naringin substrate solution, then adding macroreticular resin so that naringin is absorbed and fixed in the macroreticular resin to prepare an immobilized naringin solution; secondly carrying out oriented synthesis of prunin through hydrolysis reaction of immobilized naringin under catalytic action of alpha-L-rhamnosidase; and finally separating and purifying hydrolysate to obtain a prunin sample. By utilizing the method, the problems that of the product generated by the existing synthesizing method, the purity is low, yield is low and industrial production is difficult to realize can be solved. By utilizing the method, oriented hydrolysis of alpha-L-rhamnoside bond in naringin molecules can be efficiently realized, and a new process is provided for industrialized application of preparing high purity prunin with an enzymatic method.
Description
Technical field
The invention belongs to natural product biosynthesizing field, be specifically related to a kind of method utilizing alpha-L-Rhamnosidase catalysis naringin to synthesize Pu Luning.
Background technology
Naringin, formal name used at school is naringenin-7-β-D-Glucose (2 → 1)-α-L-rhamnosyl, sloughs a L-rhamanopyranosyl and generate Pu Luning (eriodictyol-7-O-glucoside) under the effect of alpha-L-Rhamnosidase.The bitter taste of Pu Luning is 1/3rd of naringin, and has several functions.Pu Luning has different anti-ultravirus activity and anti-inflammatory activity to DNA/RNA virus, can also as the sweeting agent of diabetic subject, also can be used as medicine material, be used for synthesizing various chemical medical, as the medicine etc. expanded for the synthesis of kapillary, can by the good absorption of human body.
Pu Luning is a kind of flavanone kind composition, and obtained by extraction in the folk medicine mountain peach of South Korea at first, it is mainly distributed in the plants such as Caesalpiniaceae, the Rosaceae, Euphorbiaceae at occurring in nature.Its water-soluble, fat-soluble difference, more difficult acquisition, expensive.The preparation method of current Pu Luning mainly contains two kinds, chemical leaching test and enzymatic hydrolysis naringin method.The mild condition of Pu Luning, transformation efficiency are high, safety and environmental protection to utilize enzymatic hydrolysis naringin to prepare, and therefore studying enzyme legal system has very high using value for Pu Luning.
In the bio-transformation research of enzyme, many substrates solvability in water is poor, therefore needs the touch opportunity being increased enzyme-to-substrate by some method or approach, thus improves the transformation efficiency of substrate or increase the growing amount of product.Method conventional is at present the medium changing enzymic catalytic reaction, wherein most study, most widely used be organic solvent, but organic solvent enzymic catalytic reaction shortcoming is many, can have an effect with substrate or product directly or indirectly and change the structure of zymoprotein molecule, the catalytic activity of destructive enzyme, reduces the stability of enzyme; Organic solvent makes the separation and purification in later stage more difficult.
In view of this, the present inventor studies and devises a kind of method utilizing alpha-L-Rhamnosidase catalysis immobilization naringin to synthesize Pu Luning, and this case produces thus.
Summary of the invention
A kind of alpha-L-Rhamnosidase catalysis immobilization naringin is the object of the present invention is to provide to synthesize the method for Pu Luning.The method can realize the alpha-L-rhamnoside key in directionally hydrolyzing naringin molecule efficiently, and the industrial applications preparing high purity Pu Luning for enzyme process provides novel process.
For achieving the above object, the present invention solves the technical scheme of its technical problem and is:
Utilize alpha-L-Rhamnosidase catalysis immobilization naringin to synthesize a method of Pu Luning, comprise the following steps:
Step one, preparation immobilization naringin:
Naringin is dissolved in the Acetic acid-sodium acetate damping fluid of pH 4.0, obtain the naringin substrate solution that concentration is 2.0 g/L, macroporous adsorbent resin is added in described naringin substrate solution, be placed in 50 DEG C of water-bath constant temperature oscillations fully fixing, naringin is adsorbed and is fixed in macroporous adsorbent resin, obtained immobilization naringin solution;
Step 2, alpha-L-Rhamnosidase catalysis immobilization naringin synthesis Pu Luning:
The alpha-L-Rhamnosidase enzyme liquid of concentration 12U/mL is added in described immobilization naringin solution, be placed in 60 DEG C of constant temperature oscillation water-baths and carry out enzyme digestion reaction, oscillation rate 80 r/min, reaction times 300 min, alpha-L-Rhamnosidase enzyme liquid catalysis immobilization naringin synthesis Pu Luning, obtain the conversion fluid containing hydrolysate Pu Luning, Pu Luning is adsorbed in macroporous adsorbent resin;
The separation and purification of step 3, Pu Luning:
After described enzyme digestion reaction terminates, collecting by filtration macroporous adsorbent resin also fills post, employing volume ratio is that the acetone-chloroform-water of 80:20:4.8 carries out wash-out as eluent to macroporous adsorbent resin, adopt tlc to carry out tracing detection to elutriant simultaneously, elutriant only containing Pu Luning is merged, Rotary Evaporators reduction vaporization under 60 ° of C is then utilized to concentrate, methyl alcohol recrystallization 2 times repeatedly, get Pu Luning sample.
As the optimal way of embodiment, the material of the macroporous adsorbent resin that immobilization is used is polystyrene, and polarity is nonpolar or low-pole, and specific surface area is 480 ~ 800 m
2/ g, mean pore size is 90 ~ 450A.
As the optimal way of embodiment, also comprise and adopt TLC tlc to detect conversion fluid described in step 2, wherein, developping agent: acetone-chloroform-water (volume ratio is 80:20:4.8); Developer: 3g vanillin food grade,1000.000000ine mesh, 50mL dehydrated alcohol, the 0.5mL vitriol oil.
As the optimal way of embodiment, also comprise and adopt HPLC method to detect conversion fluid described in step 2, testing conditions is: column temperature 35 ° of C of reversed-phase column, ultraviolet detection wavelength 280 nm, sampling volume 20 μ L, flow velocity 0.4 mL/min, lose shape time 20 min, moving phase is water, methyl alcohol and acetonitrile, gradient elution.
First Application alpha-L-Rhamnosidase catalytic hydrolysis immobilization naringin of the present invention, realizes the directed biosynthesizing of Pu Luning.The method mild condition, reaction product is more single, and efficiency of pcr product is high, environmental friendliness; In addition, enzymically hydrolyse product diverse problems in the past can be overcome, be easy to suitability for industrialized production high purity Pu Luning, for the application of the Pu Luning of heavy industrialization biosynthesizing is from now on laid a good foundation, for the widespread use tool promoted in the industry such as medicine, food, makeup is of great significance.Existing immobilization technology, be generally used for and enzyme is fixed on carrier, the present inventor goes it around, substrate is fixed on carrier, to change the microenvironment of enzymic catalytic reaction, be fixed on by substrate in solid-phase media and carry out with enzyme reacting activity and the stability that greatly can improve concentration of substrate and enzyme, the bio-transformation for naringin and other materials provides a new approach.
Accompanying drawing explanation
Fig. 1 20mL reaction system enzymolysis process;
Fig. 2 1L iodine system enzymolysis process;
Fig. 3 enzymolysis product separation and purification TLC schemes; Wherein, 1 is naringenin, Pu Luning, the mixed mark of naringin; 2 is rhamnosyl mark product; 3 is enzyme-added front reaction solution; 4 is reaction 3h reaction solution; 5 is reaction 6h reaction solution;
The interpretation of result figure of Fig. 4 HPLC method detection reaction product; Wherein, a) enzyme-added front reaction solution; B) 2h reaction solution is reacted; C) 4h reaction solution is reacted; D) reaction solution after reacting completely;
Fig. 5 detects collection of illustrative plates to the TLC that the elutriant only containing Pu Luning does.
Embodiment
the detection method adopted in the following example:
The mensuration of alpha-L-Rhamnosidase enzyme activity: add the naringin standardized solution that 1.0 mL concentration are 300 μ g/mL in the centrifuge tube of 10 mL, as substrate, add 950 μ L citrate-phosphate disodium hydrogens damping fluid (pH 5.0) again, whirlpool mixes, be placed in 50 ° of C water-baths and be incubated 10 min, after making the temperature of the substrate in centrifuge tube and buffer solution mixture reach 50 ° of C, add rapidly 50 μ L enzyme solution, mixing, thermal treatment 30 min in 100 ° of C boiling water baths is immediately reacted after 15 min in 50 ° of C thermostat water baths, make the thorough inactivation of enzyme liquid, rapid cooling, in 0.22 μm of membrane filtration after centrifugal 10 min of 12000 rpm room temperature, HPLC is utilized to measure the content of naringin and Pu Luning.Blank group replaces protoenzyme solution with the enzyme liquid of deactivation, and all the other steps are the same.
TLC tlc: developping agent: acetone-chloroform-water (volume ratio is 80:20:4.8);
Developer: 3g vanillin food grade,1000.000000ine mesh, 50mL dehydrated alcohol, the 0.5mL vitriol oil.
HPLC adopts Waters e2695(Waters e2695 Separations Module, Waters 2489 UV/Visible Detector), the content of naringin and Pu Luning in assaying reaction liquid.Column temperature 35 ° of C of Symmetry C18 reversed-phase column, ultraviolet detection wavelength 280 nm, sampling volume 20 μ L, flow velocity 0.4 mL/min, lose shape time 20 min, and moving phase is water, methyl alcohol, acetonitrile, gradient elution.
Alpha-L-Rhamnosidase enzyme activity unit definition (U): react 1 min under the condition of 50 ° of C, pH 5.0, the amount consuming the alpha-L-Rhamnosidase needed for 1 μ g naringin is defined as an alpha-L-Rhamnosidase unit of activity.
The mensuration of reducing sugar growing amount: adopt 2,4-dinitrosalicylic acid (DNS) method measures the reducing sugar growing amount of enzymolysis process: get 0.2 mL reaction solution and mix with 0.6 mL DNS solution, shake up, 100 ° of C water-bath 15 min, taking-up tap water cools immediately, be settled to 5 mL with distilled water again, under 520 nm wavelength, measure light absorption value, establishing criteria curve calculates the growing amount of reducing sugar in reaction solution.
The wherein growing amount (mg) of reducing sugar during growing amount (mg) × 100/ naringin complete hydrolysis of reducing sugar in naringin transformation efficiency (%)=enzyme digestion reaction.
Embodiment 1: the enzymolysis of immobilization naringin transforms produces Pu Luning technique
The material of the macroporous adsorbent resin that immobilization is used is polystyrene, and polarity is nonpolar or low-pole, and specific surface area is 480 ~ 800 m
2/ g, mean pore size is 90 ~ 450A.Use macroporous adsorbent resin will through pre-treatment, treatment process is roughly: resin first screens through floating method, then uses 95% alcohol immersion 24 h, fully swelling, keeps resin to be submerged completely in immersion process.Then wet method upper prop, with 95% ethanol by resin layer, till effluent liquid is not in white opacity, effluent liquid is washed till without ethanol taste with same flow velocity again with distilled water, then pass through resin layer with 2% hydrochloric acid soln, and soak 3 h, it is neutral for being then washed till with distilled water the pH value flowing out water; Finally pass through resin layer with 2% sodium hydroxide solution, and soak 3 h, it is neutral, stand-by for being then washed till with distilled water the pH value flowing out water.
Embodiment 2: the enzymolysis of immobilization naringin transforms produces Pu Luning technique
The present invention use enzyme be in the early-stage Study of laboratory, by by L-rha gene recombination in aspergillus niger JMU-TS528 and utilize pichia yeast expression system carry out alpha-L-Rhamnosidase fermentation obtain restructuring alpha-L-Rhamnosidase.
In 20ml reaction system, the resin of 0.4g weight in wet base is joined in citric acid (0.01 mol/L)-Sodium phosphate dibasic (0.02 mol/L) damping fluid that naringin concentration is the pH5.0 of 2.0g/L, be placed in 50 DEG C of water-bath constant temperature oscillations fully fixing after, add the enzyme liquid of 240 U containing alpha-L-Rhamnosidase, alpha-L-Rhamnosidase concentration in solution is made to reach 12 U/mL, continue in the constant temperature oscillation tank of 50 DEG C and react 300min, oscillation rate is 80r/min, reaction terminates, by reducing sugar amount in DNS method assaying reaction liquid, calculating naringin transformation efficiency is 93.99%, as shown in Figure 1.
Embodiment 3: immobilization naringin enzymolysis transforms generation Pu Luning and is amplified to 1L technique
The present invention use enzyme be in the early-stage Study of laboratory, by by L-rha gene recombination in aspergillus niger JMU-TS528 and utilize pichia yeast expression system carry out alpha-L-Rhamnosidase fermentation obtain restructuring alpha-L-Rhamnosidase.
The resin of 4.0g weight in wet base joined in citric acid (0.01 mol/L)-Sodium phosphate dibasic (0.02 mol/L) damping fluid that naringin concentration is the pH5.0 of 2.0g/L in 1L reaction system, be placed in 50 DEG C of water-bath constant temperature oscillations fully fixing after, add the enzyme liquid containing 240 U alpha-L-Rhamnosidases, alpha-L-Rhamnosidase concentration in reaction solution is made to reach 12 U/mL, the constant temperature oscillation tank continuing to be placed in 50 DEG C reacts 300min, oscillation rate is 80r/min, reaction terminates, by reducing sugar amount in DNS method assaying reaction liquid, calculating naringin transformation efficiency is 91%, as shown in Figure 2.
Embodiment 4: thin-layer chromatography (TLC) detection reaction product
The reaction product that naringin obtains under the katalysis of alpha-L-Rhamnosidase is general Shandong peace rhamnosyl, can go out the changing conditions of product in 5 h reaction process by tracing detection by tlc.After enzyme digestion reaction terminates, collecting by filtration macroporous adsorbent resin also fills post, and employing volume ratio is that the acetone-chloroform-water of 80:20:4.8 carries out wash-out as eluent to macroporous adsorbent resin, adopts tlc to carry out tracing detection to elutriant simultaneously.The developping agent of TLC thin-layer chromatography: acetone-chloroform-water (volume ratio is 80:20:4.8); Developer: 3g vanillin food grade,1000.000000ine mesh, 50mL dehydrated alcohol, the 0.5mL vitriol oil.The TLC figure obtained, as shown in Figure 3.
Can be found out to only have naringin in reaction starting stage reaction solution, naringin hydrolysis Cheng Pulu peace rhamnosyl after reaction for some time by thin-layer chromatogram, after reacting 6 h, naringin transforms completely, only has general Shandong peace rhamnosyl in reaction solution.Illustrate that the alpha-L-Rhamnosidase of this restructuring and naringin specificity enzyme digestion reaction can get rid of the puzzlement of naringenin, save the process of product separation purifying.
The interpretation of result of embodiment 5:HPLC method detection reaction product
In order to determine the changing conditions of product in enzyme digestion reaction further, HPLC being carried out to the product of each step of reaction and detects analysis.Liquid Detection condition is: column temperature 35 ° of C of reversed-phase column, and ultraviolet detection wavelength 280 nm, sampling volume 20 μ L, flow velocity 0.4 mL/min, lose shape time 20 min, and moving phase is water, methyl alcohol and acetonitrile, gradient elution.Acquired results, as shown in Figure 4.
Result as can be seen from figure, only naringin is contained in reaction solution before enzyme-added liquid, after adding the reaction of enzyme liquid, naringin reduces gradually, change into Pu Luning, along with the prolongation in reaction times, the amount that naringin generates Pu Luning under the effect of alpha-L-Rhamnosidase increases gradually, and after reacting completely, in reaction solution, naringin transforms substantially completely.
Embodiment 6: the separation and purification of reaction product
The product Pu Luning of this enzyme digestion reaction belongs to Flavonoid substances, can by absorption with macroporous adsorbent resin, and carbohydrate rhamnosyl can not by absorption with macroporous adsorbent resin, therefore after reacting completely, collecting by filtration resin carrier, the ethanolic soln of different concns gradient is adopted to carry out wash-out to resin, adopt tlc to carry out tracing detection to elutriant simultaneously, elutriant only containing Pu Luning is merged, Rotary Evaporators reduction vaporization under 60 ° of C is then utilized to concentrate, methyl alcohol recrystallization 2 times repeatedly, obtains highly purified Pu Luning sample.Make TLC collection of illustrative plates to the elutriant only containing Pu Luning to detect, the collection of illustrative plates obtained showing elutriant is single slice, as shown in Figure 5, namely only containing Pu Luning.
First Application alpha-L-Rhamnosidase catalytic hydrolysis immobilization naringin of the present invention, realizes the directed biosynthesizing of Pu Luning.The method mild condition, reaction product is more single, and efficiency of pcr product is high, environmental friendliness; In addition, enzymically hydrolyse product diverse problems in the past can be overcome, be easy to suitability for industrialized production high purity Pu Luning, for the application of the Pu Luning of heavy industrialization biosynthesizing is from now on laid a good foundation, for the widespread use tool promoted in the industry such as medicine, food, makeup is of great significance.
All distortion that those of ordinary skill in the art can directly derive from the disclosure of invention or associate, all should think protection scope of the present invention.
Claims (4)
1. utilize alpha-L-Rhamnosidase catalysis immobilization naringin to synthesize a method of Pu Luning, it is characterized in that: comprise the following steps:
Step one, preparation immobilization naringin:
Naringin is dissolved in the Acetic acid-sodium acetate damping fluid of pH 4.0, obtain the naringin substrate solution that concentration is 2.0 g/L, macroporous adsorbent resin is added in described naringin substrate solution, be placed in 50 DEG C of water-bath constant temperature oscillations fully fixing, naringin is adsorbed and is fixed in macroporous adsorbent resin, obtained immobilization naringin solution;
Step 2, alpha-L-Rhamnosidase catalysis immobilization naringin synthesis Pu Luning:
Alpha-L-Rhamnosidase enzyme liquid is added in described immobilization naringin solution, the alpha-L-Rhamnosidase concentration in solution is made to reach 12U/mL, be placed in 60 DEG C of constant temperature oscillation water-baths and carry out enzyme digestion reaction, oscillation rate 80 r/min, reaction times 300 min, alpha-L-Rhamnosidase enzyme liquid catalysis immobilization naringin synthesis Pu Luning, obtain the conversion fluid containing hydrolysate Pu Luning, Pu Luning is adsorbed in macroporous adsorbent resin;
The separation and purification of step 3, Pu Luning:
After described enzyme digestion reaction terminates, collecting by filtration macroporous adsorbent resin also fills post, employing volume ratio is that the acetone-chloroform-water of 80:20:4.8 carries out wash-out as eluent to macroporous adsorbent resin, adopt tlc to carry out tracing detection to elutriant simultaneously, elutriant only containing Pu Luning is merged, Rotary Evaporators reduction vaporization under 60 ° of C is then utilized to concentrate, methyl alcohol recrystallization 2 times repeatedly, get Pu Luning sample.
2. a kind of alpha-L-Rhamnosidase catalysis immobilization naringin that utilizes synthesizes the method for Pu Luning as claimed in claim 1, it is characterized in that: the material of the macroporous adsorbent resin that immobilization is used is polystyrene, polarity is nonpolar or low-pole, and specific surface area is 480 ~ 800 m
2/ g, mean pore size is 90 ~ 450A.
3. a kind of alpha-L-Rhamnosidase catalysis immobilization naringin that utilizes synthesizes the method for Pu Luning as claimed in claim 1, it is characterized in that: also comprise and adopt TLC tlc to detect conversion fluid described in step 2, wherein, developping agent: acetone-chloroform-water (volume ratio is 80:20:4.8); Developer: 3g vanillin food grade,1000.000000ine mesh, 50mL dehydrated alcohol, the 0.5mL vitriol oil.
4. a kind of alpha-L-Rhamnosidase catalysis immobilization naringin that utilizes synthesizes the method for Pu Luning as claimed in claim 1, it is characterized in that: also comprise and adopt HPLC method to detect conversion fluid described in step 2, testing conditions is: column temperature 35 ° of C of reversed-phase column, ultraviolet detection wavelength 280 nm, sampling volume 20 μ L, flow velocity 0.4 mL/min, lose shape time 20 min, moving phase is water, methyl alcohol and acetonitrile, gradient elution.
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| CN107254503A (en) * | 2017-08-04 | 2017-10-17 | 齐鲁工业大学 | A kind of enzymatic conversion preparation method of ginsenoside Rd |
| CN112210548A (en) * | 2020-09-07 | 2021-01-12 | 佛山市汇腾生物技术有限公司 | Pichia pastoris for expressing alpha-L-rhamnosidase and preparation method and application thereof |
| CN115094106A (en) * | 2022-06-29 | 2022-09-23 | 四川大学 | Preparation method of pulutinine |
| CN116103354A (en) * | 2021-11-10 | 2023-05-12 | 桂林莱茵生物科技股份有限公司 | Method for preparing trilobatin by using naringin as raw material |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107254503A (en) * | 2017-08-04 | 2017-10-17 | 齐鲁工业大学 | A kind of enzymatic conversion preparation method of ginsenoside Rd |
| CN107254503B (en) * | 2017-08-04 | 2021-04-09 | 齐鲁工业大学 | Enzymatic conversion preparation method of ginsenoside Rd |
| CN112210548A (en) * | 2020-09-07 | 2021-01-12 | 佛山市汇腾生物技术有限公司 | Pichia pastoris for expressing alpha-L-rhamnosidase and preparation method and application thereof |
| CN116103354A (en) * | 2021-11-10 | 2023-05-12 | 桂林莱茵生物科技股份有限公司 | Method for preparing trilobatin by using naringin as raw material |
| CN115094106A (en) * | 2022-06-29 | 2022-09-23 | 四川大学 | Preparation method of pulutinine |
| CN115094106B (en) * | 2022-06-29 | 2023-07-04 | 四川大学 | Preparation method of pullulan |
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Application publication date: 20150603 |