CN112680483A

CN112680483A - Application of sanshool dehydrogenase LcADH31 in preparation of citral or product with citral as active substance

Info

Publication number: CN112680483A
Application number: CN202011557706.XA
Authority: CN
Inventors: 赵耘霄; 汪阳东; 陈益存; 高暝; 吴立文; 王民炎
Original assignee: Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Current assignee: Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-04-20
Anticipated expiration: 2040-12-25
Also published as: CN112680483B

Abstract

The invention provides application of sanshoamides dehydrogenase LcADH31 in preparation of citral or products taking the sanshoamides dehydrogenase as an active substance, relates to the technical field of plant molecular biology, and discloses application of sanshoamides dehydrogenase in preparation of citral compounds, wherein the synthesis of citral is catalyzed by the sanshoamides dehydrogenase. The method has the advantages that the substrate is catalyzed by the shanjiao pepper alcohol dehydrogenase LcADH31 to synthesize the citral, the synthesis process is close to the biological natural synthesis process, the synthesis efficiency is high, the impurity content of intermediate products and other non-target products is low, and the pollution of chemical substances in the traditional chemical synthesis is avoided.

Description

Application of sanshool dehydrogenase LcADH31 in preparation of citral or product with citral as active substance

Technical Field

The invention relates to the technical field of plant molecular biology, in particular to application of sanshoamides dehydrogenase LcADH31 in preparation of citral or a product taking the citral as an active substance.

Background

Mountain chicken pepper (Litsea cubeba (Lour.) Pers), also called Litsea cubeba, is perennial shrub or small arbor of Litsea Lam of Lauraceae (Lauraceae), is a light-loving, male and female heterostrain originally produced in Asia and mainly distributed in dozens of provinces in south of the Yangtze river of China. The whole plant of the litsea cubeba contains oil, and the oil content of the fruit at the traditional utilization part can reach 3.0 to 11.0 percent. The litsea cubeba essential oil is one of main products in the flavor and fragrance industry in China, and the production and export quantity are the first in the world for a long time. The essential oil contains abundant terpenoids, so that the essential oil has biological activities of oxidation resistance, disinsection, antibiosis and the like, and has wide development and application prospects.

More than 90% of the litsea cubeba essential oil is terpenoids comprising 41 monoterpenes and 6 sesquiterpenes, wherein citral (including alpha-citral and beta-citral) can reach 60% -90%, and the litsea cubeba essential oil is a plant essential oil with the highest content of natural citral. Citral is C₁₀The monoterpenoid compound has volatility, antibacterial property and aromaticity, participates in the pollination and defense process of plants, can be used in the fields of essences and fragrances, cosmetics, medicines and biopesticides, and creates great economic value.

The biosynthesis pathway of citral in plants is mainly characterized in that isopentenyl pyrophosphate and isomer dimethylallyl pyrophosphate thereof are synthesized through 5-deoxyxylulose phosphate in a plastid, geranyl pyrophosphate is catalyzed by geranyl pyrophosphate synthase to form geranyl pyrophosphate, geraniol is formed under the catalysis of geraniol synthase, and citral is finally synthesized through the catalysis of alcohol dehydrogenase. However, the application of alcohol dehydrogenase participating in citral synthesis in litsea cubeba is only reported, and the research on the sequence characteristics and the catalytic products of the alcohol dehydrogenase has important significance for controlling the biosynthesis of essential oil compounds.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention mainly aims to provide application of the shanjiao zanthoxyl dehydrogenase LcADH31 in preparing citral so as to at least alleviate one of the technical problems in the prior art.

The second purpose of the invention is to provide the application of the shanjiao zanthoxyl dehydrogenase LcADH31 in preparing products taking citral as an active substance.

The invention provides application of sanshool dehydrogenase LcADH31 in preparation of citral.

Furthermore, the amino acid sequence of the behenyl alcohol dehydrogenase LcADH31 is shown in SEQ ID NO. 2.

Further, the nucleotide sequence of the encoding kaempferol dehydrogenase LcADH31 is shown in SEQ ID NO. 1.

Further, the substrate of the shanjiao zanthoxyl dehydrogenase LcADH31 comprises geraniol.

Further, the sanshool dehydrogenase LcADH31 is expressed by a prokaryotic expression system;

preferably, the prokaryotic expression system comprises an E.coli expression system;

preferably, the e.coli expression system comprises e.coli BL21 strain.

Further, the behenyl alcohol dehydrogenase LcADH31 is expressed by pET-32 a.

In addition, the invention also provides application of the shanjiao zanthoxyl dehydrogenase LcADH31 in preparing products taking citral as an active substance.

Further, the product comprises plant essential oil.

Compared with the prior art, the invention has the following beneficial effects:

the citral is the main component of the litsea cubeba essential oil and the main active component of the physiological activity of the litsea cubeba essential oil, and the synthesis of the citral is regulated and controlled by alcohol dehydrogenase on the molecular level. The invention discovers that the sanshool dehydrogenase catalyzes the synthesis of citral, and provides application of the sanshool dehydrogenase LcADH31 in preparation of citral compounds. The method has the advantages that the substrate is catalyzed by the shanjiao pepper alcohol dehydrogenase LcADH31 to synthesize the citral, the synthesis process is close to the biological natural synthesis process, the synthesis efficiency is high, the impurity content of intermediate products and other non-target products is low, and the pollution of chemical substances in the traditional chemical synthesis is avoided.

Based on the above inventive concept, the application of the shanjiao zanthoxyl dehydrogenase LcADH31 provided by the invention in preparing products taking citral as an active substance also has the same beneficial effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of PCR amplification products in example 1 of the present invention;

FIG. 2 is a SDS-PAGE result of the behenyl alcohol dehydrogenase LcADH31 prepared in example 2 of the present invention;

FIG. 3 is a graph showing the Western Blot analysis result of the behenyl alcohol dehydrogenase LcADH31 prepared in example 2 of the present invention;

FIG. 4 is a graph of the GC-MS identification results of citral provided in example 3 of the present invention;

FIG. 5 is a graph showing the identification result of characteristic peaks of citral ions provided in example 3;

FIG. 6 is a graph showing the comparison of characteristic peaks of citral (2,6-Octadienal,3,7-dimethyl-) ions in the spectral library provided in example 3 of the present invention.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The citral is the main component of the Zanthoxylum bungeanum fruit essential oil, and has various biological activities such as aromaticity, antibacterial property, oxidation resistance, etc. Citral belongs to one of monoterpenes, has a chemical name of 3, 7-dimethyl-2, 6-octadienal, and comprises alpha-citral and beta-citral, and the synthesis of citral is regulated and controlled by alcohol dehydrogenase at molecular level. The invention discovers that the sanshool dehydrogenase LcADH31 can catalyze a proper substrate to synthesize citral.

Based on the discovery that the pheasant alcohol dehydrogenase LcADH31 can catalyze and synthesize citral, on one hand, the invention provides the application of the pheasant alcohol dehydrogenase LcADH31 in the preparation of citral. When the behenyl alcohol dehydrogenase LcADH31 is applied to preparation of citral, citral is separated after the substrate is catalyzed by the behenyl alcohol dehydrogenase LcADH31, the synthesis process is close to a biological natural synthesis process, the synthesis efficiency is high, the impurity content of intermediate products and other non-target products is low, and the pollution of chemical substances in the traditional chemical synthesis is avoided.

Optionally, the amino acid sequence of the behenyl alcohol dehydrogenase LcADH31 is shown in SEQ ID No. 2; the nucleotide sequence of the pheasant alcohol dehydrogenase LcADH31 is shown in SEQ ID NO. 1.

It is understood that the invention is not limited to the addition of sequences such as tag sequences, regulatory elements or selection markers to the amino acid sequence and nucleotide sequence encoding the behenyl alcohol dehydrogenase LcADH31 in the preparation of the recombinant behenyl alcohol dehydrogenase LcADH31 in vitro.

In some preferred embodiments, the substrate of the behenyl alcohol dehydrogenase LcADH31 comprises geraniol. Geraniol is an important product in the isoprene synthesis pathway, and the shanjiao pepper alcohol dehydrogenase LcADH31 can synthesize citral by taking geraniol as a substrate.

In some alternative embodiments, the behenyl alcohol dehydrogenase LcADH31 catalyzing the substrate synthesis of citral in vitro is expressed from a prokaryotic expression system, preferably via an E.coli expression system. The shanjiao zanthoxyl dehydrogenase LcADH31 expressed by the prokaryotic expression system has the activity of catalyzing a substrate to synthesize citral in vitro, wherein the prokaryotic expression system preferably uses an escherichia coli expression system, and the escherichia coli expression system has the advantages of clear genetic background, high expression level of target genes, simple operation, short culture period and strong pollution resistance. In some preferred embodiments, escherichia coli BL21 strain (e.coli BL21) with high growth rate and high expression level is preferably used for expressing the shanjiao zanol dehydrogenase lc adh 31.

In some preferred embodiments, the behenyl alcohol dehydrogenase LcADH31 is expressed from an expression vector pET-32a containing the T7 promoter.

According to another aspect of the invention, the invention also provides the application of the shanjiao alcohol dehydrogenase LcADH31 in preparing products taking citral as an active substance.

It should be noted that, in the present invention, when the product has citral as an active ingredient, citral may also include a derivative of citral.

Wherein "derivatives" refer to substances which, when these derivatives are used as one of the active ingredients of a product, are capable of providing the product with citral activity, either directly or indirectly, upon use.

In some preferred embodiments, the product comprises a plant essential oil related product. The citral is an important component of plant essential oil, and has aromatic, antibacterial and antioxidant effects.

The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.

Example 1

Designing a forward primer by taking cDNA obtained by reverse transcription of RNA of the wild chicken prickly ash fruit as a template: 5'-ATGAGTACCCTTGAATCAGA-3' (SEQ ID NO.3), reverse primer: 5'-AAC ATTTGATTTTTCATTG-3' (SEQ ID NO.4), and MCLAB high-fidelity DNA polymerase (Oncodinaceae, Beijing) is used for PCR amplification, and the reaction system is as follows:

mountain chicken pepper cDNA	≤1μg
		2×H Buffer	25μL
F1	2μL
		F2	2μL
ddH₂O	Up to 50μL

The PCR amplification conditions were: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 15 seconds, and 35 cycles; final extension at 72 ℃ for 5 min; the PCR product was detected by 1% agarose gel electrophoresis, and the target fragment was 1,086bp in size, as shown in FIG. 1, M in FIG. 1 is DNA marker DL2000, and lane 1 is the amplification product.

Recovering a target gene fragment by adopting an agarose gel electrophoresis gel recovery kit method, cloning the target fragment in TA, connecting the target fragment to a pClone007 vector, and then transforming the target fragment into an Escherichia coli DH5 alpha clone strain under the transformation conditions of: adding 5 mu L of the ligation product into 50 mu L of competent cells, gently mixing, standing on ice for 25min, performing water bath heat shock at 42 ℃ for 45s, rapidly performing ice bath, standing for 2min, adding 500 mu L of LB culture medium without antibiotics, recovering at 37 ℃ and 200rpm for 1h, taking a proper amount of bacterial liquid, coating the bacterial liquid on a solid LB plate containing antibiotics (Amp), and performing inverted culture at 37 ℃ for 12-16 h. Adopting colony PCR to carry out positive clone screening, wherein the screening method comprises the following steps: randomly picking single colony from the transformation plate, placing the single colony in a liquid culture medium in a 1.5mL centrifuge tube for culture, taking L mu L of each tube as a template for PCR detection, and storing the colony detected as positive in the plate or a glycerol tube for later use. The nucleotide sequence of the cloned pheasant alcohol dehydrogenase LcADH31 gene is shown in SEQ ID NO.1, the nucleotide sequence contains 1,086 basic groups, 362 amino acids are coded in total, and the specific amino acid sequence is shown in SEQ ID NO. 2. Thus, the recombinant plasmid which inserts the gene LcADH31 of the sanshool dehydrogenase into the pClone007 cloning vector is obtained, and the recombinant plasmid is successfully transformed into a positive bacterium of prokaryotic cell Escherichia coli DH5 alpha.

Example 2

The method comprises the following steps of constructing a pET32a expression vector by using the pheasant alcohol dehydrogenase gene LcADH31, transforming prokaryotic cells, performing prokaryotic expression, purifying protein and detecting protein quality, and comprises the following steps:

using the cloned LcADH31 gene as a template, designing a cloning primer carrying a restriction enzyme cutting site, and carrying out PCR amplification on a target fragment:

LcADH31-F:5’-CGGGATCCATGAGTACCCTTGAATCAGAG-3’(SEQ ID NO.5)；

LcADH31-R:5’-CCAAGCTTAACATTTGATTTTTCATTGA-3’(SEQ ID NO.6)。

after the PCR amplification product is recovered, double enzyme digestion reaction is carried out on the PCR product and a pET32a vector, and the system is as follows:

DNA	1μg
		BamHI	2μL
HindIII	2μL
		10X QuickCut Buffer	5μL
ddH₂O	Up to 50μL

after mixing the reaction system, incubation was carried out at 30 ℃ for 10 minutes, and 5. mu.L of the reaction mixture was taken, transformed into E.coli competent cells BL21(DE3), and cultured overnight at 37 ℃. And selecting positive clones, carrying out PCR identification and sequencing on the bacterial liquid, and carrying out induced expression on the recombinant plasmids. Specifically, a single colony containing a recombinant plasmid is inoculated to a plasmid containing ampicillinIn LB medium containing mycin, the cells were cultured at 37 ℃ until OD600 became 0.4, transferred to 18 ℃ and cultured with shaking until OD600 became 0.6, and after induction of expression at 16 ℃ for 20 hours by adding 1mM IPTG, the cells were collected by centrifugation at 5000 rpm. Use of a lysis buffer (50mM NaH)₂PO₄500mM NaCl, 10mM imidazole, pH 8.0), disrupting the cells with ultrasound in an ice-bath beaker until the cells are clear, centrifuging at 13000rpm for 10 minutes at 4 ℃, and collecting the supernatant. 1ml of Ni was aspirated⁺Affinity chromatography beads, to which 10ml of lysis buffer is added, mixed well at 4 ℃, left to stand on ice for 5 minutes, after which the supernatant is aspirated, to which the supernatant of the cell suspension is added, shaken well at 4 ℃ for 2 hours, left to stand for 10 minutes, after which the supernatant is aspirated, and washed with washing buffer (50mM NaH)₂PO₄500mM NaCl, 50mM imidazole, pH 8.0) washing the affinity chromatography column 3 times and discarding the liquid. Add elution buffer (50mM NaH) to the centrifuge tube₂PO₄500mM NaCl, 400mM imidazole, pH 8.0)2ml was shaken for 10 minutes to elute LcADH31 protein, which was stored at-80 ℃. 100 mu L of LcADH31 protein solution is sucked, 25 mu L of LLoading buffer solution is added, denaturation is carried out for 10 minutes at 100 ℃, 20 mu L of LcADH31 protein solution is sucked for SDS-PAGE protein electrophoresis, and Western blot detection is carried out. The results of SDS-PAGE are shown in FIG. 2, in which lane M: marker; lane 1: eluting the protein; lane 2: inducing whole mycoprotein. The Western blot detection results are shown in FIG. 3: wherein lane M is marker and lane 1 is LcADH 31.

Example 3

Biochemical functions of sanshool dehydrogenase LcADH 31:

geraniol is taken as a substrate, and an enzymatic reaction system is as follows: 100mM glycine-NaOH, pH 9.5,1mM NADP +, 1mM geraniol, 30ng of purified protein was added and left at 25 ℃ for 1 h. An Agilent 6890N-5975B gas chromatography-mass spectrometer of Agilent is adopted, volatile substances are detected by adsorbing with an 50/30um DVB/CAR on PDMS extraction head of supelco and adsorbing for 45min at 40 ℃ to detect catalytic products.

The detection method comprises the following steps: GC-MS conditions: a chromatographic column: DB-5MS (60m 0.25mm ID 0.25 μm film thickness). Temperature programming parameters: the initial temperature is 50 ℃, the temperature is kept for 2min, the temperature is increased to 80 ℃ at the speed of 3 ℃/min and kept for 2min, the temperature is increased to 180 ℃ at the speed of 5 ℃/min and kept for 1min, the temperature is increased to 230 ℃ at the speed of 10 ℃/min and kept for 5min, and finally the temperature is increased to 250 ℃ at the speed of 20 ℃/min and kept for 3 min. Sample inlet temperature: at 220 ℃, pulse without shunt, sample injection of 1 μ L, carrier gas of high-purity helium (99.999%), column flow rate: 1.5 mL/min. Interface temperature of chromatography-mass spectrometry: at 250 ℃ to obtain a mixture. Ion source temperature: 230 ℃ to 230 ℃. An ionization mode: EI. Electron energy: 70 eV. The scanning mass range is 50-500 m/z. Qualitative and quantitative: the components are respectively searched and matched by a NIST08 standard spectrum library, the fragments are compared, and the qualitative determination is carried out by combining related literature reports, relative retention time of each component and the like. And quantitatively calculating the relative content of each peak area according to a peak area normalization method. The experimental results are shown in fig. 4-6:

through GC-MS detection, the result shows that the shanji pepper alcohol dehydrogenase LcADH31 can catalyze geraniol to synthesize citral, and the peak-off time and the ion characteristic peak can be matched with the NIST08 standard library search, so that the enzyme catalysis product can be qualitative; because the enzyme content is limited, the enzyme cannot be quantified in the experiment, and only the enzyme has enzyme catalytic activity.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> subtropical forestry research institute of China forestry science research institute

Application of sanchi alcohol dehydrogenase LcADH31 in preparation of citral or products taking citral as active substance

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1152

<212> DNA

<213> Artificial sequence

<400> 1

atgagtaccc ttgaatcaga gaaacaagtt actggatggg ctgctagaga cgcttcaggt 60

gttttatcac cttactccta cacactaagg agcacaggtc ctgaagatgt ggttctaaag 120

gtatggtact gtggcatttg ccacaccgac atccaccagc tgaagaacca ccttggcatg 180

tccaactacc ccatggttcc cgggcatgaa gttgtagggg aagtgacaga ggtgggatca 240

gatgtgaaga aattcaaggt gggggacact ataggggtgg gcctgattgt gggctgttgc 300

aggacctgtg gtccctgcaa gtccaacatt gagcaatact gcaataagag gatctggtct 360

tacaatgatg tttatgttga tggaaaacct acacaaggag gatttgcttc gtctctggtt 420

gtagaccaaa agtttgcagt gaaaatccca gcaggactgg ccccagagca agcagcccca 480

ctattatgtg ccggggtgac agtttacagc ccgttaaggc attttgggct gaaaagaagt 540

ggcctgaggg gagcaattct gggtctaggt ggggttgggc atatgggtgt caagatagcc 600

aaggcaatgg gccaccatgt aacagtaatc agctcctctg acaagaaaag agaggaggcg 660

cttgatcact tgggtgctga tgcctatctg gtcagctctg atgtgtccaa gatgcaagaa 720

gctgccgata gcctggacta catccttgac accgcccctg cccatcatcc acttgagcca 780

tacctttcgc ttttgaaagt cgacggaaaa atagtgctga tgggggtcat ctcagagcca 840

ttgcaattcg tttcacccat gatcatgcta gggagaaaga ccattactgg gactttcatt 900

ggaagcatgg aggagaccca agagatgcta gagttctgtg aggaaaaggg tcttacatca 960

atgatagaga ttgtaaagat ggattatgtg aatgaggccc tcgagagatt ggagaaaaat 1020

gacgtaagat acaggtttgt ggtggacgtt gctggcagca atctcaatga aaaatcaaat 1080

gtttgaactg aggaaaagaa aatcagttgc catgatttac gtggtgcgtg catacggtta 1140

gagcatgctt ag 1152

<210> 2

<211> 361

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<213> Artificial sequence

<400> 2

Met Ser Thr Leu Glu Ser Glu Lys Gln Val Thr Gly Trp Ala Ala Arg

1 5 10 15

Asp Ala Ser Gly Val Leu Ser Pro Tyr Ser Tyr Thr Leu Arg Ser Thr

20 25 30

Gly Pro Glu Asp Val Val Leu Lys Val Trp Tyr Cys Gly Ile Cys His

35 40 45

Thr Asp Ile His Gln Leu Lys Asn His Leu Gly Met Ser Asn Tyr Pro

50 55 60

Met Val Pro Gly His Glu Val Val Gly Glu Val Thr Glu Val Gly Ser

65 70 75 80

Asp Val Lys Lys Phe Lys Val Gly Asp Thr Ile Gly Val Gly Leu Ile

85 90 95

Val Gly Cys Cys Arg Thr Cys Gly Pro Cys Lys Ser Asn Ile Glu Gln

100 105 110

Tyr Cys Asn Lys Arg Ile Trp Ser Tyr Asn Asp Val Tyr Val Asp Gly

115 120 125

Lys Pro Thr Gln Gly Gly Phe Ala Ser Ser Leu Val Val Asp Gln Lys

130 135 140

Phe Ala Val Lys Ile Pro Ala Gly Leu Ala Pro Glu Gln Ala Ala Pro

145 150 155 160

Leu Leu Cys Ala Gly Val Thr Val Tyr Ser Pro Leu Arg His Phe Gly

165 170 175

Leu Lys Arg Ser Gly Leu Arg Gly Ala Ile Leu Gly Leu Gly Gly Val

180 185 190

Gly His Met Gly Val Lys Ile Ala Lys Ala Met Gly His His Val Thr

195 200 205

Val Ile Ser Ser Ser Asp Lys Lys Arg Glu Glu Ala Leu Asp His Leu

210 215 220

Gly Ala Asp Ala Tyr Leu Val Ser Ser Asp Val Ser Lys Met Gln Glu

225 230 235 240

Ala Ala Asp Ser Leu Asp Tyr Ile Leu Asp Thr Ala Pro Ala His His

245 250 255

Pro Leu Glu Pro Tyr Leu Ser Leu Leu Lys Val Asp Gly Lys Ile Val

260 265 270

Leu Met Gly Val Ile Ser Glu Pro Leu Gln Phe Val Ser Pro Met Ile

275 280 285

Met Leu Gly Arg Lys Thr Ile Thr Gly Thr Phe Ile Gly Ser Met Glu

290 295 300

Glu Thr Gln Glu Met Leu Glu Phe Cys Glu Glu Lys Gly Leu Thr Ser

305 310 315 320

Met Ile Glu Ile Val Lys Met Asp Tyr Val Asn Glu Ala Leu Glu Arg

325 330 335

Leu Glu Lys Asn Asp Val Arg Tyr Arg Phe Val Val Asp Val Ala Gly

340 345 350

Ser Asn Leu Asn Glu Lys Ser Asn Val

355 360

<210> 3

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<213> Artificial sequence

<400> 3

atgagtaccc ttgaatcaga 20

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence

<400> 4

aacatttgat ttttcattg 19

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence

<400> 5

cgggatccat gagtaccctt gaatcagag 29

<210> 6

<211> 28

<212> DNA

<213> Artificial sequence

<400> 6

ccaagcttaa catttgattt ttcattga 28

Claims

1. Application of sanshool dehydrogenase LcADH31 in preparation of citral.

2. The use of claim 1, wherein the amino acid sequence of the behenyl alcohol dehydrogenase LcADH31 is shown in SEQ ID No. 2.

3. The use according to claim 1, wherein the nucleotide sequence encoding the behenyl alcohol dehydrogenase LcADH31 is shown in SEQ ID No. 1.

4. Use according to any one of claims 1 to 3, wherein the substrate of the behenyl alcohol dehydrogenase LcADH31 comprises geraniol.

5. The use according to any one of claims 1 to 3, wherein the behenyl alcohol dehydrogenase LcADH31 is expressed by a prokaryotic expression system;

preferably, the e.coli expression system comprises e.coli BL21 strain.

6. The use according to claim 5, wherein the behenyl alcohol dehydrogenase LcADH31 is expressed from pET-32 a.

7. Application of sanshool dehydrogenase LcADH31 in preparing products with citral as active substance is provided.

8. Use according to claim 7, wherein the product comprises a plant essential oil.

9. The use according to claim 7, wherein the amino acid sequence of the behenyl alcohol dehydrogenase LcADH31 is shown in SEQ ID No. 2.

10. The use according to claim 7, wherein the nucleotide sequence encoding the behenyl alcohol dehydrogenase LcADH31 is shown in SEQ ID No. 1.