CN107058257A - One kind synthesis alcoholic aroma glucosides highly effective biological zyme and preparation method thereof - Google Patents

One kind synthesis alcoholic aroma glucosides highly effective biological zyme and preparation method thereof Download PDF

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CN107058257A
CN107058257A CN201710311140.4A CN201710311140A CN107058257A CN 107058257 A CN107058257 A CN 107058257A CN 201710311140 A CN201710311140 A CN 201710311140A CN 107058257 A CN107058257 A CN 107058257A
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leu
ser
glu
glucosides
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CN107058257B (en
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宋传奎
荆婷婷
高婷
张娜
董绪平
宛晓春
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Anhui Agricultural University AHAU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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Abstract

The invention discloses one kind synthesis alcoholic aroma glucosides highly effective biological zyme and preparation method thereof, belong to biological technical field, including CsGT1, CsGT2, CsGT3, the nucleotides such as SEQ ID NO of the CsGT1:Shown in 1, amino acid such as SEQ ID NO:Shown in 2；Described CsGT2 nucleotides such as SEQ ID NO:Shown in 3, amino acid such as SEQ ID NO:Shown in 4；Described CsGT2 nucleotides such as SEQ ID NO:Shown in 5, amino acid such as SEQ ID NO:Shown in 6.The present invention albumen that it is encoded by prokaryotic expression can be substrate using a variety of alcoholic aromas, and synthesize corresponding glucosides, biology enzyme is provided for industrial high efficiency production fragrance glucosides, environmentally friendly enzyme system method synthesized aroma glycoside compounds synthetic method of the invention has very big application value.

Description

One kind synthesis alcoholic aroma glucosides highly effective biological zyme and preparation method thereof
Technical field
The invention belongs to biological technical field, it is related to a kind of synthesis alcoholic aroma glucosides highly effective biological zyme and its preparation side Method.
Background technology
Alcoholic aroma has tempting flowers and fruits fragrant, and such as phenmethylol, benzyl carbinol have tonifying kidney and strengthening yang, Liver protection, anti-oxidant Bioactivity and the flowers and fruits taste for possessing pleasure, have a wide range of applications in food, cosmetics, tobacco and daily chemical product；Peppermint Alcohol has peppermint fragrance, can be used as the odorant of toothpaste, perfume, beverage and candy etc., pharmaceutically as excitants, acts on skin Skin or mucous membrane, there is refrigerant itching-relieving action, take orally available for headache and nose, pharynx, laryngitis etc.；That is discharged every year in the whole world is most normal With in the spices maximum with consumption, linalool almost ranks the first every year, the fragrant fragrance of a flower with rose of existing lilac, the lily of the valley, again There are the banksia rose, fruity breath, but its Free aroma stability is poor, easily occurs isomerization, not very persistently；Eugenol has dense Strongly fragrant China pink muskiness, is the blending basis of health and Qing systems essence, has in the blending of the essence such as cosmetic, soap use, edible Use, while having very strong sterilizing power, can be used for carious tooth as Bangesic, and have local antisepsis, its shortcoming concurrently It is volatile to go bad, be difficult to long storage；Geraniol geraniol and nerol stereoisomer each other, with linalool meta each other Isomers, is colourless to pale yellow oily liquid, has the savory gas of rose, be the host of rose system essence, be various floral perfumes In indispensable blending raw material, can also regard sweetener as, it may also be used for synthetic food, perfumed soap, daily cosmetics essence, but its Easily it is oxidized in atmosphere, limits its application in bigger atmosphere.
Glucosides state aroma substance is that a class does not have volatility, in glucosides state form after being combined with glucide by glycosidic bond The class Aroma precursor existed, with important biology and pharmacological function.With the research of plant glucosides state Aroma precursor increasingly Deeply, a large amount of glucosides state aroma substances are separated and identified, some are also proven to have important bioactivity, such as antibacterial, resist Protective effect scorching and to nerve.Glucosides state fragrance improves the water solubility and stability of free aroma substance, in specific bar Under part, the aroma substance of people's needs is discharged, there is extensive commercial value in cosmetics, food and course of drug development (such as the price of every gram of geraniol glucosides is 200,000 RMB in the market), the popular class as natural product field research Topic.In nature, glycosylation is one of mostly important modification reaction of Secondary Metabolism of Plant, and glycoside matter is by UDP- glycosyls Shift enzymatic to complete, glycosyl can be transferred on small molecule fragrance (aglycon) by glycosyl transferase from the donor of activation, so as to adjust Acceptor molecule is saved in the bioactivity of matrix, solubility, Subcellular Localization and stability.Plant alcoholic aroma glycosyl transferase is ground Study carefully and make slow progress, limit fragrance glucosides efficiently synthesizing in vitro, constrain fragrance glycoside matter in food, cosmetics and doctor The application of medicine industry.
Phenmethylol, benzyl carbinol, geraniol, linalool, linalool oxide, 4 hydroxy coumarin, geraniol, and cis/trans- 3- hexenols, the aroma substance such as eugenol is present in many plants and fruit, fragrant with pleasant fragrance and flowers and fruits, but itself Stability is poor, is easily deformed in atmosphere rotten, have impact on its in food, the application in terms of medicine, and the glycosyl of these materials Its stability can be significantly improved by changing product, water-soluble, reduce its toxicity, greatly widen these aroma substances be applied to it is new Field, with very big commercial promise.
Play an important roll in view of alcoholic aroma in production and life, and the alcohol based compound of glucosides state can be effective The drawbacks of avoiding the presence of free state aglycon, the chemical synthesis inefficiency of alcohol system glycoside compounds is big for environment pollution, and To product without selectivity, thus this environmentally friendly enzyme system method synthesized aroma glycoside compounds synthetic method is with very big Application value.
It is badly in need of a kind of synthesis alcoholic aroma glucosides highly effective biological zyme and preparation method thereof in currently available technology.
The content of the invention
It is an object of the invention to provide one kind synthesis alcoholic aroma glucosides highly effective biological zyme and preparation method thereof, pass through original The albumen of its coding of nuclear expression can be substrate using a variety of alcoholic aromas, and synthesize corresponding glucosides, be industrial high efficiency life Produce fragrance glucosides and provide biology enzyme, environmentally friendly enzyme system method synthesized aroma glycoside compounds synthetic method tool of the invention There is very big application value.Its concrete technical scheme is：
One kind synthesis alcoholic aroma glucosides highly effective biological zyme, including CsGT1, CsGT2, CsGT3, the nucleosides of the CsGT1 Acid such as SEQ ID NO:Shown in 1, amino acid such as SEQ ID NO:Shown in 2；Described CsGT2 nucleotides such as SEQ ID NO:3 It is shown, amino acid such as SEQ ID NO:Shown in 4；Described CsGT2 nucleotides such as SEQ ID NO:Shown in 5, amino acid such as SEQ ID NO:Shown in 6.
Further, the reaction system of the highly effective biological zyme is：Buffer solution：Tris-HCl and phosphate buffer, PH scopes For PH 6.5-8.5, temperature is 20-45 degree.
Further, the PH scopes are 7.0-8.5, and temperature is 30-35 degree.
Further, the CsGT1 is to phenmethylol, benzyl carbinol, geraniol, linalool, linalool oxide, 4- hydroxyl tonka-beans Element, geraniol, and cis/trans -3- hexenols are active.
A kind of preparation method of present invention synthesis alcoholic aroma glucosides highly effective biological zyme, comprises the following steps：
Said gene is connected in pGEX-4T1 carriers, 37 DEG C of trainings in BL21 or Rosetta (DE3) Escherichia coli are transferred to Support to OD600After=0.6-0.8, addition 1mM IPTG, 16-18 DEG C, room temperature or 37 DEG C are induced 20-24 hours, are entered with GST- resins Row purifying, albumen after purification is detected with SDS-Page.
Compared with prior art, beneficial effects of the present invention are：
The present invention albumen that it is encoded by prokaryotic expression can be substrate using a variety of alcoholic aromas, and synthesize corresponding Glucosides, biology enzyme, environmentally friendly enzyme system method synthesized aroma of the invention are provided for industrial high efficiency production fragrance glucosides Glycoside compounds synthetic method has very big application value.
Brief description of the drawings
Fig. 1 is that SDS-Page detects figure；
Fig. 2 is determination of activity result, and wherein Fig. 2 a are CsGT1, and Fig. 2 b are CsGT2, and Fig. 2 c are CsGT3；
Fig. 3 is the temperature applicable range of enzyme, and wherein Fig. 3 a are CsGT2, and Fig. 3 b are CsGT3；
Fig. 4 is the pH value scope of application of enzyme, and wherein Fig. 4 a are CsGT2, and Fig. 4 b are CsGT3；
Fig. 5 is donor seletion result；
Fig. 6 is the reaction effect figure of embodiment 2；
Fig. 7 is the reaction effect figure of embodiment 3.
Embodiment
Technical scheme is described in more detail with reference to the accompanying drawings and detailed description.
(1) sequence information
This experiment early stage finally gives a gene C sGT1 by a series of screening to glycosyl transferases, and (nucleotides is shown in Sequence 1, common 1455bp；Amino acid is shown in sequence 2), find that its albumen encoded can be fragrant using a variety of alcohol systems by prokaryotic expression Gas is substrate, and synthesizes corresponding glucosides.
Then, and to CsGT1 homologous sequence cloned, and its corresponding restructuring egg is obtained by Escherichia coli In vain, CsGT2 and CsGT3 are respectively labeled as, its sequence and CsGT1 are quite similar, and homology is up to more than 95%, CsGT2 nucleosides Acid and amino acid are shown in that sequence 3 and 4, CsGT3 nucleotides and amino acid are shown in sequence 5 and 6 respectively respectively.
(2) enzyme is prepared and detection
The preparation of albumen, is illustrated by example of CsGT1：
Said gene is connected in pGEX-4T1 carriers, 37 DEG C of trainings in BL21 or Rosetta (DE3) Escherichia coli are transferred to Support to OD600After=0.6-0.8, addition 1mM IPTG, 16-18 DEG C induces 20-24 hour (can also room temperature or 37 DEG C induce), Purified with GST- resins, albumen after purification is carried out detecting as shown in Figure 1 with SDS-Page.
(3) determination of activity：
Determine CsGT1 to phenmethylol, benzyl carbinol, geraniol, linalool, linalool oxide, 4- hydroxyls by substrate scanning The alcoholic aromas such as butylcoumariii, geraniol, and cis/trans -3- hexenols activity is very strong, and can be efficiently synthesized a variety of fragrance Corresponding glucosides.CsGT2 is most strong to nerol activity, and it is worth noting that, CsGT3 virtue camphor tree different from CsGT1 and CsGT2 The activity of alcohol is far longer than linalool oxide.As shown in Figure 2.
(4) scope of application
Temperature：As shown in figure 3, being illustrated using CsGT2 and CsGT2 as example.The enzyme is respectively provided with the range of 20-45 degree Higher catalytic activity, optimum temperature is 30-35 degree；
PH：As shown in figure 4, being illustrated using CsGT2 and CsGT23 as example.The enzyme has in the range of PH 6.5-8.5 There is higher catalytic activity, optimal pH is 7.0-8.5；
Reaction solution：The enzyme is respectively provided with higher biological activity in Tris-HCl and phosphate buffer, and main PH is being closed Suitable scope.
Donor seletion：The enzyme is in addition to it can form glucoside, also with synthesis galactolipin glucosides and glucuronic acid The ability of glucosides, but glucose is optimal donor, as shown in the figure.
(5) applicating example
Embodiment 1
Using phenmethylol as substrate, glucose is donor, and 30 degree of reactions 1 are small in Tris-HCl is reaction solution (PH is 7.5) When, product is directly used in LC-MS detections after being extracted with ethyl acetate (mobile phase is methanol, water), it is seen that have phenmethylol glucosides Production, conversion ratio is more than 75%.
Embodiment 2
Using linalool oxide as substrate, glucose is donor, 35 degree of reactions 0.5 in phosphatase reaction liquid (PH is 7.0) Hour, product is directly used in LC-MS detections after being extracted with ethyl acetate (mobile phase is methanol, water), it is seen that have linalool oxidation The production of thing glucosides, the speed of reaction is 19.5nmol/min/ug, and efficiency is very high, as shown in Figure 6.
Embodiment 3
So that along 3 hexenols, as substrate, glucose is donor, reacted under the conditions of PH is 7.0, the speed of reaction is 27.8nmol/min/ug, efficiency is very high, as shown in Figure 7.
Embodiment 4
Using geraniol as substrate, glucose is donor, is reacted under the conditions of PH is 8.5, product is extracted with ethyl acetate LC-MS detections are directly used in after taking (mobile phase is methanol, water), it is seen that have the synthesis of geraniol glucosides, and carry out Mass Spectrometer Method.
Embodiment 5
Using n-octyl alcohol as substrate, glucose is donor, is reacted under the conditions of PH is 8.5, product is extracted with ethyl acetate LC-MS detections are directly used in after taking (mobile phase is methanol, water), it is seen that have the synthesis of n-octyl alcohol glucosides, and carry out Mass Spectrometer Method.
Preferably embodiment only of the invention, protection scope of the present invention not limited to this is any to be familiar with this technology The technical staff in field in the technical scope of present disclosure, the simple change for the technical scheme that can be become apparent to or Equivalence replacement is each fallen within protection scope of the present invention.
SEQUENCE LISTING
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<120>One kind synthesis alcoholic aroma glucosides highly effective biological zyme and preparation method thereof
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atgggttcga tgggcaagtt agaaaaacct catgcagttt gtataccata cccagctcaa 60
ggccacatca accccatgct caaactctcc aaactcctcc accaaagagg ctttcacatc 120
acctttgtca acactgagtt caaccacaaa cgcctcctca aatctcgggg ccccgactcc 180
ctcaacgggc tctcttcctt caggttcgaa accatacccg atgggctccc cgagtccgat 240
gccgatgcaa cccaacacat cccatcactg tgcgagtcca ccaggaaaca ctgcttggcc 300
ccttttaaag accttctttc gaagctaaac gacaccgctt cgtcgaacgt cccgccggtg 360
acttgcatag tctccgatgg ggttatgagc ttcactgtgg atgctgctga agaattggac 420
attcctgaag ttcttttctg gacaactagt gcttgtgggt tcatgggtta tgaacagtat 480
cgtaatctta tcgataagag ttatattcca cttgaagata agagttgcat aacaaatggg 540
tatttggata cggttatcga ttggatacct ggtatgaaag gtatacgttt gaaggattta 600
ccaagttttc ttcgaaccac agacctaagc gattttatga ttgattttgt gtgtggggaa 660
actgaaagag ctcgcagagg ctttgccatt attttcaata catttgagaa attggaacat 720
aacgttttgg aggctctttc atctatgttc cctccaatct acaccattgg acctttacac 780
ttattaatga atcaaatcaa cgatgatagt ctaaagttga taggatcaaa tctgtggaaa 840
gaagaacccg agtgcctcga atggctcgat acaaaaggac ccaactctgt tgtttatgtg 900
aactttggaa gcatcactgt catgacacca aaccaaatgg ttgagtttgc ttggggactt 960
gctaatagca accagacctt tttgtgggta ataagacctg atcttgtcac tggcgacttg 1020
gctgttcttc caccagaatt catggaagca acaaaagaaa ggggtttatt ggcaagttgg 1080
tgcccccaag agcaagttct tgaccacccg tctattggag ggtttttaac tcactctggg 1140
tggaactcta cacttgaaag tatttcgagc ggagttccaa tggtctgttg gccatttttc 1200
gcagagcaac aaacaaattg ttggcactgt tgcacccaat gggggatagg gatggagata 1260
gataatgatg ttaagaaaga tgaagttgag agccttgtga gagagttgat ggttggagag 1320
aaagggaagg agatgaagaa aaaggctatg gagtggaaga gattggctca agagaccatt 1380
gagagttcat ctggttcatc tttcttgaat ctagacaagt tggtcaacca agttcttctc 1440
tctccaagac attag 1455
<210> 6
<211> 484
<212> PRT
<213>Artificial sequence
<400> 6
Met Gly Ser Met Gly Lys Leu Glu Lys Pro His Ala Val Cys Ile Pro
1 5 10 15
Tyr Pro Ala Gln Gly His Ile Asn Pro Met Leu Lys Leu Ser Lys Leu
20 25 30
Leu His Gln Arg Gly Phe His Ile Thr Phe Val Asn Thr Glu Phe Asn
35 40 45
His Lys Arg Leu Leu Lys Ser Arg Gly Pro Asp Ser Leu Asn Gly Leu
50 55 60
Ser Ser Phe Arg Phe Glu Thr Ile Pro Asp Gly Leu Pro Glu Ser Asp
65 70 75 80
Ala Asp Ala Thr Gln His Ile Pro Ser Leu Cys Glu Ser Thr Arg Lys
85 90 95
His Cys Leu Ala Pro Phe Lys Asp Leu Leu Ser Lys Leu Asn Asp Thr
100 105 110
Ala Ser Ser Asn Val Pro Pro Val Thr Cys Ile Val Ser Asp Gly Val
115 120 125
Met Ser Phe Thr Val Asp Ala Ala Glu Glu Leu Asp Ile Pro Glu Val
130 135 140
Leu Phe Trp Thr Thr Ser Ala Cys Gly Phe Met Gly Tyr Glu Gln Tyr
145 150 155 160
Arg Asn Leu Ile Asp Lys Ser Tyr Ile Pro Leu Glu Asp Lys Ser Cys
165 170 175
Ile Thr Asn Gly Tyr Leu Asp Thr Val Ile Asp Trp Ile Pro Gly Met
180 185 190
Lys Gly Ile Arg Leu Lys Asp Leu Pro Ser Phe Leu Arg Thr Thr Asp
195 200 205
Leu Ser Asp Phe Met Ile Asp Phe Val Cys Gly Glu Thr Glu Arg Ala
210 215 220
Arg Arg Gly Phe Ala Ile Ile Phe Asn Thr Phe Glu Lys Leu Glu His
225 230 235 240
Asn Val Leu Glu Ala Leu Ser Ser Met Phe Pro Pro Ile Tyr Thr Ile
245 250 255
Gly Pro Leu His Leu Leu Met Asn Gln Ile Asn Asp Asp Ser Leu Lys
260 265 270
Leu Ile Gly Ser Asn Leu Trp Lys Glu Glu Pro Glu Cys Leu Glu Trp
275 280 285
Leu Asp Thr Lys Gly Pro Asn Ser Val Val Tyr Val Asn Phe Gly Ser
290 295 300
Ile Thr Val Met Thr Pro Asn Gln Met Val Glu Phe Ala Trp Gly Leu
305 310 315 320
Ala Asn Ser Asn Gln Thr Phe Leu Trp Val Ile Arg Pro Asp Leu Val
325 330 335
Thr Gly Asp Leu Ala Val Leu Pro Pro Glu Phe Met Glu Ala Thr Lys
340 345 350
Glu Arg Gly Leu Leu Ala Ser Trp Cys Pro Gln Glu Gln Val Leu Asp
355 360 365
His Pro Ser Ile Gly Gly Phe Leu Thr His Ser Gly Trp Asn Ser Thr
370 375 380
Leu Glu Ser Ile Ser Ser Gly Val Pro Met Val Cys Trp Pro Phe Phe
385 390 395 400
Ala Glu Gln Gln Thr Asn Cys Trp His Cys Cys Thr Gln Trp Gly Ile
405 410 415
Gly Met Glu Ile Asp Asn Asp Val Lys Lys Asp Glu Val Glu Ser Leu
420 425 430
Val Arg Glu Leu Met Val Gly Glu Lys Gly Lys Glu Met Lys Lys Lys
435 440 445
Ala Met Glu Trp Lys Arg Leu Ala Gln Glu Thr Ile Glu Ser Ser Ser
450 455 460
Gly Ser Ser Phe Leu Asn Leu Asp Lys Leu Val Asn Gln Val Leu Leu
465 470 475 480
Ser Pro Arg His

Claims (5)

1. one kind synthesis alcoholic aroma glucosides highly effective biological zyme, it is characterised in that described including CsGT1, CsGT2, CsGT3 CsGT1 nucleotides such as SEQ ID NO:Shown in 1, amino acid such as SEQ ID NO:Shown in 2；Described CsGT2 nucleotides is such as SEQ ID NO:Shown in 3, amino acid such as SEQ ID NO:Shown in 4；Described CsGT2 nucleotides such as SEQ ID NO:Shown in 5, Amino acid such as SEQ ID NO:Shown in 6.
2. synthesis alcoholic aroma glucosides highly effective biological zyme according to claim 1, it is characterised in that the highly effective biological zyme Reaction system be：Buffer solution：Tris-HCl and phosphate buffer, PH scopes are PH 6.5-8.5, and temperature is 20-45 degree.
3. synthesis alcoholic aroma glucosides highly effective biological zyme according to claim 2, it is characterised in that the PH scopes are 7.0-8.5, temperature is 30-35 degree.
4. synthesis alcoholic aroma glucosides highly effective biological zyme according to claim 1, it is characterised in that the CsGT1 is to benzene Methanol, benzyl carbinol, geraniol, linalool, linalool oxide, 4 hydroxy coumarin, geraniol, and cis/trans -3- hexenols are equal It is active.
5. a kind of preparation method of the synthesis alcoholic aroma glucosides highly effective biological zyme described in claim 1, it is characterised in that including Following steps：CsGT1, CsGT2, CsGT3 described in claim 1 is connected in pGEX-4T1 carriers, be transferred to BL21 or Cultivated in Rosetta Escherichia coli to OD for 37 DEG C600After=0.6-0.8, addition 1mM IPTG, 16-18 DEG C, room temperature or 37 DEG C are lured Lead 20-24 hours, purified with GST- resins, albumen after purification is detected with SDS-Page.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108070576A (en) * 2018-02-05 2018-05-25 安徽农业大学 A kind of tea tree glycosyl transferase mutant and its application in plant insect defence
CN108342410A (en) * 2018-02-05 2018-07-31 安徽农业大学 A method of improving tobacco flowers and fruits perfume (or spice) fragrance glycosidic precursors
CN109628421A (en) * 2019-01-11 2019-04-16 安徽农业大学 It is a kind of it is special synthesis furanone glucoside glycosyl transferase and its application
CN110317765A (en) * 2018-03-29 2019-10-11 中国科学院天津工业生物技术研究所 A kind of E. coli expression strains of high yield geraniol glucoside and its application

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1415017A (en) * 1999-12-01 2003-04-30 阿德雷德研究创新股份有限公司 UDP-glucose: aglycon-glacosyltransferase
US6780980B1 (en) * 1999-06-30 2004-08-24 Centre Suisse D'electronique Et De Microtechnique S.A. Process for the modification of surface
CN103298946A (en) * 2010-12-28 2013-09-11 三得利控股株式会社 Method for utilizing monoterpene glycosyltransferase
CN104774816A (en) * 2015-04-07 2015-07-15 中国科学院天津工业生物技术研究所 Glycosyl transferase synthesized by catalytic gastrodine as well as gene of encoding enzyme and application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6780980B1 (en) * 1999-06-30 2004-08-24 Centre Suisse D'electronique Et De Microtechnique S.A. Process for the modification of surface
CN1415017A (en) * 1999-12-01 2003-04-30 阿德雷德研究创新股份有限公司 UDP-glucose: aglycon-glacosyltransferase
CN103298946A (en) * 2010-12-28 2013-09-11 三得利控股株式会社 Method for utilizing monoterpene glycosyltransferase
CN104774816A (en) * 2015-04-07 2015-07-15 中国科学院天津工业生物技术研究所 Glycosyl transferase synthesized by catalytic gastrodine as well as gene of encoding enzyme and application

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108070576A (en) * 2018-02-05 2018-05-25 安徽农业大学 A kind of tea tree glycosyl transferase mutant and its application in plant insect defence
CN108342410A (en) * 2018-02-05 2018-07-31 安徽农业大学 A method of improving tobacco flowers and fruits perfume (or spice) fragrance glycosidic precursors
CN110317765A (en) * 2018-03-29 2019-10-11 中国科学院天津工业生物技术研究所 A kind of E. coli expression strains of high yield geraniol glucoside and its application
CN110317765B (en) * 2018-03-29 2020-11-20 中国科学院天津工业生物技术研究所 Escherichia coli expression strain for high-yield geraniol glucoside and application thereof
CN109628421A (en) * 2019-01-11 2019-04-16 安徽农业大学 It is a kind of it is special synthesis furanone glucoside glycosyl transferase and its application
CN109628421B (en) * 2019-01-11 2022-11-01 安徽农业大学 Glycosyl transferase for specifically synthesizing furanone glucoside and application thereof

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