CN102286577B - Enhanced enzymic synthesis method for producing isoquercitrin - Google Patents
Enhanced enzymic synthesis method for producing isoquercitrin Download PDFInfo
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- CN102286577B CN102286577B CN 201110270301 CN201110270301A CN102286577B CN 102286577 B CN102286577 B CN 102286577B CN 201110270301 CN201110270301 CN 201110270301 CN 201110270301 A CN201110270301 A CN 201110270301A CN 102286577 B CN102286577 B CN 102286577B
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Abstract
The invention relates to an enhanced enzymic synthesis method for producing isoquercitrin, which comprises the following steps of: selecting ultrasonic waves with the intensity being 0 to 1.5w/cm<2> in a range being 10 to 150kHz for continuously acting on a reaction system for catalyzed hydrolysis of rutin to synthetize isoquercitrin through alpha-L-RhaR at 1 to 60min/h; using disodium hydrogen phosphate-citric acid buffer solution with the concentration being 0.01 to 1 mol/L for preparing 0.01 to 1g/L rutin mother liquid with pH being 5 to 9; adding 0.1 to 100g/L alpha-L-RhaR liquid in the rutin mother liquid according to the volume ratio being 1 to 50 percent; and carrying out oscillation reaction for 1 to 48h under the conditions of 10 to 60 DEG C and 50 to 200 r/min. The enhanced enzymic synthesis method has the advantages that the conversion rate of the isoquercitrin of the ultrasonic field enhancing enzyme synthesis products is higher than that of a method without ultrasonic enhancing under the same condition, in addition, the operation is simple and convenient, the condition is mild, and the speed and the intensity of the enzymatic reaction can be respectively accelerated and improved.
Description
Technical field
The invention belongs to enzyme process synthesised food additive agent field, be specifically related to a kind of ultrasonic wave field and cause the method for strengthening the synthetic isoquercitrin of enzymatic.
Two, background technology
Prior art: isoquercitrin (Isoquercitrin), it is Quercetin-3-O-β-D-glucoside (Quercetin-3-glucose), very rare at occurring in nature, be a kind of have remarkable oxidation-resistance, bioactive flavonoid gourmet food additive and natural drug effective constituent such as antitumor, also be the synthesis material (Toxicology of new type functional food additive EMIQ (Enzymatically Modified Isoquercitrin) in the world in recent years, 2010,268 (3): 213-218).
The rutin that patent CN 101985639 A are extensive with distributed in nature, the source is abundant is raw material, prepares isoquercitrin through the Glycosylase catalytic hydrolysis reaction, has set up the enzymically hydrolyse technology of the directed synthetic isoquercitrin of bio-transformation rutin; It is higher that acid hydrolysis prepares the hydrolysis temperature of isoquercitrin, acid catalyst is serious to equipment corrosion, and produce a large amount of acid waste water, and Quercetin and isoquercitrin coexistence in the hydrolytic process of rutin, must adopt separation means could obtain isoquercitrin (African Journal of Biotechnology to hydrolysate, 2011,10 (8): 1460-1466).On this basis, the enzymically hydrolyse rutin is generally acknowledged it is the most promising method of preparation isoquercitrin, and wherein the reaction of the enzymatic rutin directionally hydrolyzing of a-L-rhamnoside enzyme catalysis is the key of producing isoquercitrin, also is the important channel of improving Production by Enzymes efficient.Therefore, the speed of raising a-L-rhamnosidase enzymatic reaction and intensity are one of gordian techniquies that improves isoquercitrin transformation efficiency, rutin raw material availability and production efficiency.
A large amount of studies show that: ultrasonic wave field causes effect active influential to enzyme, and the supersound process condition of selecting to suit can improve enzymatic reaction speed (Journal of Cleaner Production, 2008,16 (5): 591-597).Ultrasonication depends on the variation of the activity of enzyme in the reaction for the influence of enzymatic reaction, and the performance of reaction enzymes vigor must rely on the conformation of enzyme molecule.Ultrasonic wave field causes effect and improves the reason that enzyme work mainly contains two aspects: catalytic site and product that the miniflow effect that the one side ultrasonic wave produces can promote substrate to enter enzyme rapidly enter medium, thereby have improved the catalytic efficiency of enzyme; On the other hand, the shockwave that cavitation effect of ultrasonic waves produces has changed the space conformation of enzyme molecule, thereby has changed process (Ultrasonics Sonochemistry, 2011,18 (1): 303-309) of enzymatic reaction.Ultrasonic wave is for strengthening two types of enzymatic reaction part endogenous enzyme and exogenous enzyme: (1) acts on endogenous enzyme, disclose the method that strengthening endogenesis alliin enzymatic reaction by ultrasonic and aqueous enzymatic reaction prepare garlic flavor substance continuously as patent ZL 200610152149.7 and CN101696435 A, effectively improved the output of garlic typical flavor composition; (2) act on exogenous enzyme, disclose the method for the plain enzymolysis process of ultrasonic wave auxiliary fiber as CN 101979642 A, improved conversion of reduced sugar rate behind the stalk cellulose enzymolysis significantly.
At present the biosynthesizing isoquercitrin large-scale industrialization that still is unrealized is at present used, and reason is that tradition is in the reaction system of solvent with water, most natural glucosides substrate such as rutin poorly soluble, and the catalytic efficiency of alpha-L-Rhamnosidase is lower.Therefore, this patent adopts ultrasonic wave field to cause reinforcement in the synthetic isoquercitrin process of enzymatic, this method is easy and simple to handle, the reaction conditions gentleness, the efficiency of pcr product height, environmentally friendly, thus can overcome the low and lower shortcoming of substrate solubleness of traditional glucosides enzyme catalysis combined coefficient, be easy to suitability for industrialized production high purity isoquercitrin, have very important meaning to promoting its widespread use in industry such as medicine, food, makeup.
Summary of the invention
Technical problem: at deficiency described in the prior art, the invention provides a kind of ultrasonic wave field and cause the method for strengthening the synthetic isoquercitrin of enzymatic.This method adopts ultrasonic wave field to cause reinforcement in the synthetic isoquercitrin process of enzymatic, can improve the combined coefficient of isoquercitrin greatly.
Technical scheme:
Selected intensity is for being no more than 1.5w/cm in 10～150kHz scope
2Ultrasonic wave, 1～60min/h continuous action is in the reaction system of the synthetic isoquercitrin of alpha-L-Rhamnosidase catalytic hydrolysis rutin.Be 0.01～1mol/L Sodium phosphate dibasic-citrate buffer solution with concentration, the rutin mother liquor of 0.01～1g/L of preparation pH=5～9.In the rutin mother liquor, 1～50% add 0.1～100g/L alpha-L-Rhamnosidase liquid by volume, oscillatory reaction 1～48h under 10～60 ℃, 50～200r/min condition.Reaction finishes back HPLC and detects and show, ultrasonic wave field causes the transformation efficiency of strengthening enzymatic synthetic product isoquercitrin and will be higher than under the same conditions but do not pass through ultrasound-enhanced method.
Beneficial effect:
In the system of the synthetic isoquercitrin of enzymatic, adopt ultrasonic wave field to cause reinforcement, the efficiency of pcr product of isoquercitrin is than without ultrasound-enhanced method height, so ultrasonic wave field causes the enzymatic reaction technology of strengthening alpha-L-Rhamnosidase and provides new approach for the production of isoquercitrin.This method reaction conditions gentleness, environmentally friendly, be easy to suitability for industrialized production high purity isoquercitrin.In addition, the essentially no generation of waste materials of whole enzymatic reaction technology, non-environmental-pollution has very favorable industrial application prospect.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Use the measuring method that detects rutin and isoquercitrin simultaneously to be high performance liquid chromatography, chromatographic condition in the embodiment of the invention: Alltima C
18(250mm * 4.6mm, 5 μ m), moving phase: 0.02% phosphoric acid-acetonitrile (80: 20, v/v); Detect wavelength: 360nm; Flow velocity: 1.0mL/min; Sample size: 20 μ L.
Wherein, the transformation efficiency method of calculation of product isoquercitrin are:
The preparation of alpha-L-Rhamnosidase liquid is referring to patent CN 101985639 A.
Embodiment 1
Selected frequency is that 10kHz, intensity are 0.1w/cm
2Ultrasonic wave, 1min/h acts on the reaction system of the synthetic isoquercitrin of alpha-L-Rhamnosidase catalytic hydrolysis rutin.With 0.01mol/L Sodium phosphate dibasic-citrate buffer solution, the rutin mother liquor of the 0.01g/L of preparation pH=5.In the rutin mother liquor, 1% add 0.1g/L alpha-L-Rhamnosidase liquid, 50r/min oscillatory reaction 1h under 10 ℃ of conditions by volume.Reaction finishes back HPLC and detects, and the transformation efficiency of product isoquercitrin is than under the same conditions but do not improved 0.2% through ultrasound-enhanced method.
Embodiment 2
Selected frequency is that 150kHz, intensity are 1.5w/cm
2Ultrasonic wave, 60min/h acts on the reaction system of the synthetic isoquercitrin of alpha-L-Rhamnosidase catalytic hydrolysis rutin.With 1mol/L Sodium phosphate dibasic-citrate buffer solution, the rutin mother liquor of the 1g/L of preparation pH=9.In the rutin mother liquor, 50% add 100g/L alpha-L-Rhamnosidase liquid by volume, oscillatory reaction 48h under 60 ℃, 200r/min condition.Reaction finishes back HPLC and detects, and the transformation efficiency of product isoquercitrin is than under the same conditions but do not improved 2% through ultrasound-enhanced method.
Embodiment 3
Selected frequency is that 40kHz, intensity are 0.28w/cm
2Ultrasonic wave, 10min/h acts on the reaction system of the synthetic isoquercitrin of alpha-L-Rhamnosidase catalytic hydrolysis rutin.With 0.2mol/L Sodium phosphate dibasic-0.1mol/L citrate buffer solution, the rutin mother liquor of the 0.3g/L of preparation pH=7.In the rutin mother liquor, 5% add 10g/L alpha-L-Rhamnosidase liquid, 120r/min oscillatory reaction 20h under 40 ℃ of conditions by volume.Reaction finishes back HPLC and detects, and the transformation efficiency of product isoquercitrin is than under the same conditions but do not improved 31% through ultrasound-enhanced method.
Embodiment 4
Selected frequency is that 40kHz, intensity are 0.28w/cm
2Ultrasonic wave, 20min/h acts on the reaction system of the synthetic isoquercitrin of alpha-L-Rhamnosidase catalytic hydrolysis rutin.With 0.2mol/L Sodium phosphate dibasic-0.1mol/L citrate buffer solution, the rutin mother liquor of the 0.3g/L of preparation pH=7.In the rutin mother liquor, 5% add 10g/L alpha-L-Rhamnosidase liquid, 120r/min oscillatory reaction 20h under 40 ℃ of conditions by volume.Reaction finishes back HPLC and detects, and the transformation efficiency of product isoquercitrin is than under the same conditions but do not improved 40% through ultrasound-enhanced method.
Embodiment 5
Selected frequency is that 40kHz, intensity are 0.28w/cm
2Ultrasonic wave, 30min/h acts on the reaction system of the synthetic isoquercitrin of alpha-L-Rhamnosidase catalytic hydrolysis rutin.With 0.2mol/L Sodium phosphate dibasic-0.1mol/L citrate buffer solution, the rutin mother liquor of the 0.3g/L of preparation pH=7.In the rutin mother liquor, 5% add 10g/L alpha-L-Rhamnosidase liquid, 120r/min oscillatory reaction 20h under 40 ℃ of conditions by volume.Reaction finishes back HPLC and detects, and the transformation efficiency of product isoquercitrin is than under the same conditions but do not improved 53% through ultrasound-enhanced method.
Claims (1)
1. strengthen the method for the synthetic isoquercitrin of enzymatic, it is characterized in that selected frequency is that 40kHz, intensity are 0.28w/cm
2Ultrasonic wave, 30min/h acts on the reaction system of the synthetic isoquercitrin of alpha-L-Rhamnosidase catalytic hydrolysis rutin; Wherein with 0.2mol/L Sodium phosphate dibasic-0.1mol/L citrate buffer solution, the rutin mother liquor of the 0.3g/L of preparation pH=7 5% adds 10g/L alpha-L-Rhamnosidase liquid, 120r/min oscillatory reaction 20h under 40 ℃ of conditions by volume in the rutin mother liquor.
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| CN102876746A (en) * | 2012-10-11 | 2013-01-16 | 江苏科技大学 | Method of ionic liquid cosolvent effect reinforced enzymatic synthesis of isoquercitrin |
| CN108467419A (en) * | 2018-03-23 | 2018-08-31 | 四川天添生物科技应用有限公司 | A method of synthesizing isoquercitrin with rutin |
| CN109438536A (en) * | 2018-10-29 | 2019-03-08 | 广东金骏康生物技术有限公司 | The application and preparation method of a kind of isoquercitin and its derivative |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101787361A (en) * | 2010-01-11 | 2010-07-28 | 山西大学 | Rutin hydrolase, preparation method and application thereof |
| CN101985639A (en) * | 2010-11-16 | 2011-03-16 | 江苏科技大学 | Application of alpha-L-rhamnoside enzyme in directional synthesis of isoquercitrin by biological conversion of rutin |
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| CN101880698B (en) * | 2010-05-19 | 2012-10-24 | 江苏科技大学 | Application of helicase in directionally hydrolyzing rutin for biosynthesizing Quereetin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101787361A (en) * | 2010-01-11 | 2010-07-28 | 山西大学 | Rutin hydrolase, preparation method and application thereof |
| CN101985639A (en) * | 2010-11-16 | 2011-03-16 | 江苏科技大学 | Application of alpha-L-rhamnoside enzyme in directional synthesis of isoquercitrin by biological conversion of rutin |
Non-Patent Citations (2)
| Title |
|---|
| 曾霖霖 等.超声波及热处理对柚皮苷酶稳定性及其二级结构的影响.《食品工业科技》.2011,第32卷(第8期),101-104. |
| 超声波及热处理对柚皮苷酶稳定性及其二级结构的影响;曾霖霖 等;《食品工业科技》;20110801;第32卷(第8期);102-103 * |
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Address after: 212028 Zhenjiang, Dantu Metro Industrial Park Rui East Road, No. 9 Patentee after: Jiangsu University of Science and Technology Address before: 212003 Zhenjiang City, Jiangsu province dream Creek Road, No. 2 Patentee before: Jiangsu University of Science and Technology |
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