CN107058257B - Synthetic alcohol-series aroma glucoside biological enzyme and preparation method thereof - Google Patents

Synthetic alcohol-series aroma glucoside biological enzyme and preparation method thereof Download PDF

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CN107058257B
CN107058257B CN201710311140.4A CN201710311140A CN107058257B CN 107058257 B CN107058257 B CN 107058257B CN 201710311140 A CN201710311140 A CN 201710311140A CN 107058257 B CN107058257 B CN 107058257B
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CN107058257A (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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Abstract

The invention discloses a synthetic alcohol-series aroma glucoside biological enzyme and a preparation method thereof, belonging to the technical field of biology, and comprising CsGT1, CsGT2 and CsGT3, wherein the nucleotide of CsGT1 is shown as SEQ ID NO. 1, and the amino acid is shown as SEQ ID NO. 2; the nucleotide of the CsGT2 is shown as SEQ ID NO. 3, and the amino acid is shown as SEQ ID NO. 4; the nucleotide of the CsGT3 is shown in SEQ ID NO. 5, and the amino acid is shown in SEQ ID NO. 6. The protein encoded by prokaryotic expression can utilize various alcohol series aromas as substrates and synthesize corresponding glucoside, so that the biological enzyme is provided for the industrial high-efficiency production of aroma glucoside.

Description

Synthetic alcohol-series aroma glucoside biological enzyme and preparation method thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a synthetic alcohol-series aroma glucoside biological enzyme and a preparation method thereof.
Background
The alcohol fragrance has attractive flower and fruit fragrance, such as benzyl alcohol and phenethyl alcohol, has biological activities of invigorating kidney and strengthening yang, protecting liver, resisting oxidation and the like, has joyful flower and fruit taste, and is widely applied to food, cosmetics, tobacco and daily chemical products; menthol has peppermint fragrance, can be used as flavoring agent for toothpaste, perfume, beverage and candy, can be used as irritant in medicine, has refreshing and antipruritic effects by acting on skin or mucosa, and can be used for treating headache, and inflammation of nose, pharynx, and larynx by oral administration; among the most commonly used spices and the most used spices, linalool is arranged at the head of the spices all the year round, has the fragrance of lilac, lily of the valley and rose, and the fragrance of costus root and fruit, but has poor stability of free fragrance, easy isomerization and short duration; eugenol has strong odor of dianthus chinensis and musk, is the blending basis of essence of KANG and series, is used in the blending of essence for cosmetics, soaps, edible products and the like, has strong bactericidal power, can be used for dental caries as local analgesic, has local antiseptic effect, and has the defects of easy volatilization, deterioration and difficult long-term storage; geraniol and nerol are stereoisomers and meta-isomers, are colorless to faint yellow oily liquid, have rose fragrance, are main agents of rose essence, are indispensable fragrance blending raw materials in various flower fragrance essences, can be also used as sweeteners, can be used for preparing food, perfumed soap and daily cosmetic essences, but are easily oxidized in the air, and limit the application of the essence in a larger atmosphere.
The glucoside-state aroma substances are aroma precursors which have no volatility and exist in a glucoside state after being combined with the saccharide substances through glucoside bonds, and have important biological and pharmacological functions. With the intensive research on plant glucoside aroma precursors, a large number of glucoside aroma substances are separated and identified, and some of the glucoside aroma substances are proved to have important biological activities such as antibacterial, anti-inflammatory and neuroprotective effects. The glucoside-state aroma improves the water solubility and stability of free aroma substances, releases the aroma substances required by people under specific conditions, has wide commercial value in the fields of cosmetics, foods and drug development (for example, the price of 20 ten thousand RMB per gram of geraniol glycoside in the current market is 20 ten thousand RMB), and becomes a hot topic for the research in the field of natural products. Glycosylation is one of the most important modification reactions in plant secondary metabolism in nature, and the glycosidic substance is catalyzed by UDP-glycosyltransferase which can transfer glycosyl from an activated donor to a small molecule fragrance (aglycone), thereby regulating the bioactivity, solubility, subcellular localization and stability of an acceptor molecule in a matrix. The research of the plant alcohol-series aroma glycosyltransferase is slow, the high-efficiency synthesis of the aroma glucoside in vitro is limited, and the application of the aroma glucoside in the industries of food, cosmetics and medicines is limited.
Fragrant substances such as benzyl alcohol, phenethyl alcohol, geraniol, linalool oxide, 4-hydroxycoumarin, cis/trans-3-hexenol, eugenol and the like exist in a plurality of plants and fruits, have pleasant fragrance and flower and fruit fragrance, but have poor stability, are easy to deform and deteriorate in the air, and influence the application of the fragrant substances in the aspects of food and medicine.
Considering that the alcohol series aroma has important function in production and life, and the alcohol series compound in the glucoside state can effectively avoid the defect of existence of free aglycone, the chemical synthesis efficiency of the alcohol series glucoside compound is low, the environmental pollution is large, and the product has no selectivity, so the synthesis method of the aroma glucoside compound by the environment-friendly enzyme series method has great application value.
At present, a synthetic alcohol-series aroma glucoside biological enzyme and a preparation method thereof are urgently needed in the prior art.
Disclosure of Invention
The invention aims to provide a synthetic alcohol-series aroma glycoside biological enzyme and a preparation method thereof, wherein the protein coded by the enzyme can utilize various alcohol-series aromas as a substrate through prokaryotic expression, and can synthesize corresponding glycoside, so that the biological enzyme is provided for the industrial high-efficiency production of aroma glycoside. The specific technical scheme is as follows:
a synthetic alcohol-series aromaculoside biological enzyme comprises CsGT1, CsGT2 and CsGT3, wherein the nucleotide of the CsGT1 is shown as SEQ ID NO. 1, and the amino acid is shown as SEQ ID NO. 2; the nucleotide of the CsGT2 is shown as SEQ ID NO. 3, and the amino acid is shown as SEQ ID NO. 4; the nucleotide of the CsGT3 is shown in SEQ ID NO. 5, and the amino acid is shown in SEQ ID NO. 6.
Further, the reaction system of the biological enzyme is as follows: buffer solution: Tris-HCl and phosphate buffer, pH6.5-8.5, temperature 20-45 deg.
Further, the pH range is 7.0-8.5, and the temperature is 30-35 ℃.
Further, the CsGT1 is active on benzyl alcohol, phenethyl alcohol, geraniol, linalool oxide, 4-hydroxycoumarin, and cis/trans-3-hexenol.
The invention relates to a preparation method of synthetic alcohol-series aroma glucoside biological enzyme, which comprises the following steps:
the gene is connected to pGEX-4T1 vector, transferred into BL21 or Rosetta (DE3) escherichia coli and cultured to OD at 37 DEG C600After addition of 1mM IPTG (0.6-0.8), the mixture was induced at 16-18 ℃ or 37 ℃ for 20-24 hours, purified with GST-resin, and the purified protein was detected with SDS-Page.
Compared with the prior art, the invention has the beneficial effects that:
the protein encoded by prokaryotic expression can utilize various alcohol series aromas as substrates and synthesize corresponding glucoside, so that the biological enzyme is provided for the industrial high-efficiency production of aroma glucoside.
Drawings
FIG. 1 is an SDS-Page view;
FIG. 2 shows the results of activity assays, where FIG. 2a is CsGT1, FIG. 2b is CsGT2, and FIG. 2c is CsGT 3;
FIG. 3 shows the temperature range of the enzyme, wherein CsGT2 is shown in FIG. 3a and CsGT3 is shown in FIG. 3 b;
FIG. 4 shows the pH range of the enzyme, wherein CsGT2 is shown in FIG. 4a and CsGT3 is shown in FIG. 4 b;
FIG. 5 shows donor selection results;
FIG. 6 is a graph showing the effect of the reaction in example 2;
FIG. 7 is a graph showing the effect of the reaction in example 3.
Detailed Description
The technical solutions of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.
(1) Sequence information
In the early stage of the experiment, a gene CsGT1 (the nucleotide is shown as a sequence 1, 1455bp totally, and the amino acid is shown as a sequence 2) is finally obtained by screening a series of glycosyltransferases, and the coded protein is found to be capable of utilizing various alcohol fragrances as substrates and synthesizing corresponding glucoside through prokaryotic expression.
Subsequently, the homologous sequence of the CsGT1 is cloned, and corresponding recombinant proteins are obtained through escherichia coli and are respectively marked as CsGT2 and CsGT3, the sequences of the recombinant proteins are quite similar to CsGT1, the homology is over 95 percent, the nucleotide and the amino acid of the CsGT2 are respectively shown in sequences 3 and 4, and the nucleotide and the amino acid of the CsGT3 are respectively shown in sequences 5 and 6.
(2) Enzyme preparation and detection
Preparation of the protein, exemplified by CsGT 1:
the gene is connected to pGEX-4T1 vector, transferred into BL21 or Rosetta (DE3) escherichia coli and cultured to OD at 37 DEG C600After addition of 1mM IPTG (0.6-0.8), the protein was induced at 16-18 ℃ for 20-24 hours (which may be induced at room temperature or 37 ℃), purified with GST-resin, and the purified protein was detected with SDS-Page as shown in FIG. 1.
(3) And (3) activity determination:
the substrate scanning determines that CsGT1 has strong activity on the aroma of alcohol systems such as benzyl alcohol, phenethyl alcohol, geraniol, linalool oxide, 4-hydroxycoumarin, cis/trans-3-hexenol and the like, and can efficiently synthesize glycosides corresponding to various aromas. CsGT2 is most active on nerol, and notably, unlike CsGT1 and CsGT2, CsGT3 linalool is much more active than linalool oxide. As shown in fig. 2.
(4) Application scope
Temperature: as shown in fig. 3, the CsGT2 and CsGT3 are used as examples for explanation. The enzyme has high catalytic activity within the range of 20-45 ℃, and the optimal temperature is 30-35 ℃;
pH: as shown in fig. 4, the CsGT2 and CsGT3 are used as examples for explanation. The enzyme has high catalytic activity within the pH range of 6.5-8.5, and the optimal pH is 7.0-8.5;
reaction solution: the enzyme has higher biological activity in Tris-HCl and phosphate buffer solution, and the main PH can be in a proper range.
Donor selectivity: the enzyme has the ability to synthesize galactoside and glucuronide in addition to the formation of glucoside, but glucose is the best donor, as shown in the figure.
(5) Examples of the applications
Example 1
Benzyl alcohol is used as a substrate, glucose is used as a donor, the reaction is carried out for 1 hour at 30 ℃ in Tris-HCl as a reaction solution (pH is 7.5), a product is extracted by ethyl acetate and then directly used for LC-MS detection (a mobile phase is methanol and water), the production of benzyl alcohol glucoside can be seen, and the conversion rate is more than 75%.
Example 2
Linalool oxide is used as a substrate, glucose is used as a donor, the linalool oxide reacts in a phosphoric acid reaction solution (pH is 7.0) at 35 ℃ for 0.5 hour, a product is extracted by ethyl acetate and then directly used for LC-MS (mobile phase is methanol and water), the production of linalool oxide glucoside can be seen, the reaction rate is 19.5nmol/min/ug, and the efficiency is very high, as shown in figure 6.
Example 3
The cis-3 hexenol is used as a substrate, glucose is used as a donor, the reaction is carried out under the condition of pH 7.0, the reaction rate is 27.8nmol/min/ug, and the efficiency is very high, as shown in figure 7.
Example 4
Geraniol is used as a substrate, glucose is used as a donor, the reaction is carried out under the condition that the pH value is 8.5, the product is extracted by ethyl acetate and then directly used for LC-MS detection (a mobile phase is methanol and water), the synthesis of geraniol glucoside can be seen, and mass spectrum detection is carried out.
Example 5
Taking n-octanol as a substrate and glucose as a donor, reacting under the condition of pH 8.5, extracting a product with ethyl acetate, directly using the extracted product for LC-MS detection (a mobile phase is methanol and water), and synthesizing the visible n-octanol glucoside, and performing mass spectrum detection.
The invention is not limited to the above embodiments, and any simple changes or equivalent substitutions which can be obviously made by those skilled in the art within the technical scope of the invention are within the technical scope of the invention.
SEQUENCE LISTING
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Gly Pro Leu His Ile Leu Met Asn Gln Ile Asn Asp Asp Ser Leu Lys
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Leu Asp Thr Lys Gly Pro Asn Ser Val Val Tyr Val Asn Phe Gly Ser
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Ala Asn Ser Asn Gln Thr Phe Leu Trp Val Ile Arg Pro Asp Leu Val
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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. A synthetic alcohol-series aroma glycoside biological enzyme is characterized by consisting of CsGT1, CsGT2 and CsGT3, wherein the nucleotide of CsGT1 is shown as SEQ ID NO. 1, and the amino acid is shown as SEQ ID NO. 2; the nucleotide of the CsGT2 is shown as SEQ ID NO. 3, and the amino acid is shown as SEQ ID NO. 4; the nucleotide of the CsGT3 is shown in SEQ ID NO. 5, and the amino acid is shown in SEQ ID NO. 6.
2. The synthetic alcohol-based aroma glycoside bioenzyme according to claim 1, wherein the reaction system of the bioenzyme CsGT2 and CsGT3 is: buffer solution: Tris-HCl and phosphate buffer, pH6.5-8.5, temperature 20-45 deg.
3. The synthetic alcohol-based aroma glycoside bio-enzyme according to claim 2, wherein said PH is in the range of 7.0 to 8.5 and the temperature is 30 to 35 degrees.
4. The synthetic alcohol-based aromaculoside bioenzyme according to claim 1, wherein the CsGT1 is active on benzyl alcohol, phenethyl alcohol, geraniol, linalool oxide, 4-hydroxycoumarin, and cis/trans-3-hexenol.
5. The method for producing a synthetic alcohol-based aroma glycoside bio-enzyme according to claim 1, comprising the steps of: the CsGT1, CsGT2, CsGT3 nucleotide sequences of claim 1 were ligated into pGEX-4T1 vector, transformed into BL21 or Rosetta E.coli, cultured at 37 ℃ to OD600After addition of 1mM IPTG (0.6-0.8), the mixture was induced at 16-18 ℃ or 37 ℃ for 20-24 hours, purified with GST-resin, and the purified protein was detected with SDS-Page.
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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
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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
CN109628421B (en) * 2019-01-11 2022-11-01 安徽农业大学 Glycosyl transferase for specifically synthesizing furanone glucoside and application thereof

Citations (4)

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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
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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

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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
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