CN102286577B - Enhanced enzymic synthesis method for producing isoquercitrin - Google Patents

Enhanced enzymic synthesis method for producing isoquercitrin Download PDF

Info

Publication number
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
Authority
CN
China
Prior art keywords
isoquercitrin
rutin
reaction
alpha
intensity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN 201110270301
Other languages
Chinese (zh)
Other versions
CN102286577A (en
Inventor
王俊
吴福安
许晏
张旭东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Noneng Biotechnology Co., Ltd.
Original Assignee
Jiangsu University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu University of Science and Technology filed Critical Jiangsu University of Science and Technology
Priority to CN 201110270301 priority Critical patent/CN102286577B/en
Publication of CN102286577A publication Critical patent/CN102286577A/en
Application granted granted Critical
Publication of CN102286577B publication Critical patent/CN102286577B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

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

Strengthen the method for the synthetic isoquercitrin of enzymatic
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:
Figure BDA0000090913700000031
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.
CN 201110270301 2011-09-14 2011-09-14 Enhanced enzymic synthesis method for producing isoquercitrin Expired - Fee Related CN102286577B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 201110270301 CN102286577B (en) 2011-09-14 2011-09-14 Enhanced enzymic synthesis method for producing isoquercitrin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201110270301 CN102286577B (en) 2011-09-14 2011-09-14 Enhanced enzymic synthesis method for producing isoquercitrin

Publications (2)

Publication Number Publication Date
CN102286577A CN102286577A (en) 2011-12-21
CN102286577B true CN102286577B (en) 2013-08-07

Family

ID=45333331

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201110270301 Expired - Fee Related CN102286577B (en) 2011-09-14 2011-09-14 Enhanced enzymic synthesis method for producing isoquercitrin

Country Status (1)

Country Link
CN (1) CN102286577B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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)

* Cited by examiner, † Cited by third party
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

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101880698B (en) * 2010-05-19 2012-10-24 江苏科技大学 Application of helicase in directionally hydrolyzing rutin for biosynthesizing Quereetin

Patent Citations (2)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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 *

Also Published As

Publication number Publication date
CN102286577A (en) 2011-12-21

Similar Documents

Publication Publication Date Title
CN102876746A (en) Method of ionic liquid cosolvent effect reinforced enzymatic synthesis of isoquercitrin
CN101985639B (en) Application of alpha-L-rhamnoside enzyme in directional synthesis of isoquercitrin by biological conversion of rutin
CN102533717B (en) Method for immobilizing beta-glucosidase and hydrolyzing straw cellulose by cooperating beta-glucosidase with cellulase
CN101255479A (en) Pre-treatment method for highly-effective saccharification of lignocellulose
CN101693910A (en) New process for producing cellooligosaccharides by microbe enzyme method
CN102827891A (en) Method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase
CN102286577B (en) Enhanced enzymic synthesis method for producing isoquercitrin
CN101603067A (en) A kind of method of oriented biosynthesis of GAMG
CN103484512B (en) Method for producing high-functional-trisaccharide-content isomaltooligosaccharide by using immobilized cells
CN106520580A (en) Yeast and application of yeast in catalyzing synthesis of 2,5-dihydroxymethyl furan
CN103320480A (en) Method for preparing beta-alanine by coupled enzymatic reaction
CN102286576B (en) Medium engineering method for synthesizing isoquercitrin by enhanced enzyme method
CN103627740B (en) A kind of microorganism cells conversion method produces the method for 2-KDG
CN103113425A (en) Method for separating mycose from glucose
CN104450833A (en) Low-temperature microwave field intensified enzymatic synthesis method for isoquercitrin
CN104762360A (en) High-content nicotinamide synthesis induced by new-feature nitrile hydratase
CN101851646B (en) Method for producing L-ornithine hydrochloride through immobilized enzyme process
CN103436560B (en) Preparation method for plant cirrus fermentation liquid glucose
CN111705096A (en) Method for producing beta-nicotinamide mononucleotide by enzyme conversion method
CN108285908B (en) Method for catalytic synthesis of (S) -1- (2, 6-dichloro-3-fluoro-phenyl) ethanol by using immobilized double enzymes
CN102286575B (en) Method for synthesizing isoquercitrin by metal ion-reinforced enzyme method
CN113004348B (en) Method for preparing oligosaccharide by mechanical self-assembly
CN101624612B (en) Method for catalytic synthesis of glycoside esters compound by immobilized penicillium expansum lipase
CN104531820A (en) Method for preparing DL-alanine with fumaric acid as raw material through multi-enzyme coupling
CN114292890A (en) Novel method for synthesizing 5&#39; -cytidylic acid by enzyme method

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C56 Change in the name or address of the patentee
CP02 Change in the address of a patent holder

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

TR01 Transfer of patent right

Effective date of registration: 20190428

Address after: 2002 A604, 521 Wanping South Road, Xuhui District, Shanghai

Patentee after: Shanghai Noneng Biotechnology Co., Ltd.

Address before: 212028 No. 9 Ruishan Road, Dantu New Town Industrial Park, Zhenjiang City, Jiangsu Province

Patentee before: Jiangsu University of Science and Technology

TR01 Transfer of patent right
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20130807

Termination date: 20190914

CF01 Termination of patent right due to non-payment of annual fee