CN114350713A - Application of camptothecine synthetase in preparation of terpene compounds and products containing terpene compounds - Google Patents

Abstract

The invention provides application of phellandrene synthetase of sanchi in preparation of terpene compounds and products containing the terpene compounds, and relates to the technical field of biology. The invention discovers that the pheasant phellandrene synthetase can also catalyze and synthesize phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene, and is a synthetase for catalyzing multiple products, so that the invention provides the application of the pheasant phellandrene synthetase in preparation of at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene. The process of synthesizing the terpene compounds by using the substrate catalyzed by the phellandrene synthase of the litsea cubeba is close to the process of biological natural synthesis, 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 camptothecine synthetase in preparation of terpene compounds and products containing terpene compounds

Technical Field

The invention relates to the technical field of biology, in particular to application of a sanshoamides cresene synthetase in preparation of terpene compounds and products containing the terpene compounds.

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, including 41 monoterpenes and 6 sesquiterpenes, and the main terpenoids are monoterpenes such as citral, limonene, phellandrene, alpha-pinene, beta-pinene and the like. The monoterpene compound is C₁₀The terpenoids have volatility, antibacterial property and aromaticity, participate in pollination and defense process of plants, and can be used in the fields of essence, perfume, cosmetics, medicine and biopesticide. The phellandrene is an important component and an active component of the litsea cubeba essential oil, has biological activities such as aromaticity, antibacterial property, oxidation resistance and the like, can be used for preparing a repellent, a bactericide, an insecticide, a pesticide synergist and the like, and has great economic value.

However, the application of terpene synthases in litsea cubeba is rarely reported, and the full utilization of terpene synthases in litsea cubeba has important significance for the development and utilization of synthetic terpene compounds and essential oil products.

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

Disclosure of Invention

The invention aims to provide application of the pheasant phellandrene synthetase in preparation of terpene compounds, so as to solve at least one technical problem in the prior art.

The second purpose of the invention is to provide the application of the pheasant phellandrene synthetase in the preparation of products taking terpene compounds as active substances.

In a first aspect, the invention provides an application of a pheasant phellandrene synthase in preparing a terpene compound, wherein the terpene compound comprises at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene.

Preferably, the terpene compounds include phellandrene, geraniol, α -pinene, β -myrcene, and limonene.

Preferably, the amino acid sequence of the pheasant phellandrene synthetase is shown in SEQ ID No. 2.

Preferably, the nucleotide sequence of the pheasant phellandrene synthetase is shown in SEQ ID NO. 1.

Preferably, the substrate of the pheasant phellandrene synthase comprises geranyl pyrophosphate.

Preferably, the pheasant phellandrene synthase is expressed by a prokaryotic expression system;

preferably, the pheasant phellandrene synthase is expressed by an escherichia coli expression system;

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

Preferably, the pheasant phellandrene synthase is expressed by pET-32 a.

According to another aspect of the invention, the invention also provides application of the bergamotene synthetase in preparation of products taking terpene compounds as active substances, wherein the terpene compounds comprise at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene;

preferably, the terpene compounds include phellandrene, geraniol, α -pinene, β -myrcene, and limonene.

Preferably, the product comprises essential oils and derivatives thereof.

Preferably, the pheasant phellandrene synthase contains an amino acid sequence shown as SEQ ID No. 2;

preferably, the pheasant phellandrene synthase contains a nucleotide sequence shown as SEQ ID No. 1.

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

phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene are important components and active ingredients of the litsea cubeba essential oil, and the synthesis of the geraniol is regulated and controlled by the geraniol synthase at the molecular level. The invention discovers that the pheasant phellandrene synthetase is related to synthesis of phellandrene, and can catalyze and synthesize geraniol, alpha-pinene, beta-myrcene and limonene. The invention discloses a pheasant phellandrene synthase which is a synthase for controlling multiple products, and provides an application of the pheasant phellandrene synthase in preparing terpene compounds, wherein the terpene compounds comprise at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene.

The method synthesizes terpene compounds by using the substrate catalyzed by the litsea cubeba phellandrene synthetase, the synthetic process is close to the biological natural synthetic process, the synthetic 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 inventive concept, the application of the pheasant phellandrene synthase provided by the invention in the preparation of products taking terpene compounds as active substances 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 shows PCR amplification products in example 1 of the present invention;

FIG. 2 is a SDS-PAGE identification result of the protein prepared in example 3 of the present invention;

FIG. 3 shows the GC-MS identification results of phellandrene;

FIG. 4 shows the identification result of characteristic peaks of phellandrene ions;

FIG. 5 shows the comparison result of characteristic peaks of phellandrene ion in the spectrum library;

FIG. 6 shows the identification result of geraniol GC-MS;

FIG. 7 shows the identification result of characteristic peaks of geraniol ions;

FIG. 8 shows the comparison of characteristic peaks of geraniol ions in a library;

FIG. 9 shows the GC-MS identification of α -pinene;

FIG. 10 shows the identification result of the alpha-pinene ion characteristic peak;

FIG. 11 shows the comparison result of the characteristic peaks of alpha-pinene ion in the library;

FIG. 12 shows the identification result of characteristic peak of β -pinene ion;

FIG. 13 shows the comparison result of characteristic peaks of β -pinene ion in the library;

FIG. 14 shows the GC-MS identification of β -myrcene;

FIG. 15 shows the identification result of characteristic peaks of beta-myrcene ions;

the comparison result of the characteristic peaks of beta-myrcene ions in the spectrum library of FIG. 16;

FIG. 17 shows the identification of D-limonene by GC-MS;

FIG. 18 shows the identification result of characteristic peaks of D-limonene ions;

FIG. 19 shows the comparison of characteristic peaks of D-limonene ion in a library.

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, the use of "or" means "and/or" unless stated otherwise. Furthermore, 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.

Phellandrene belongs to one of monoterpenes, has a chemical name of (R) -5-isopropyl-2-methyl-1, 3-cyclohexadiene, and the synthesis of phellandrene is regulated and controlled by phellandrene synthetase at a molecular level. The phellandrene is an important component and an active component of the litsea cubeba essential oil, has biological activities of aromaticity, antibacterial property, oxidation resistance and the like, is also a main raw material for synthesizing spices, and can be used for preparing a repellent, a bactericide, an insecticide, a pesticide synergist and the like. The invention discovers that the removal of the pheasant phellandrene synthetase is related to the synthesis of phellandrene, and geraniol, alpha-pinene, beta-myrcene and limonene can be catalytically synthesized, so that the pheasant phellandrene synthetase is a synthetase for controlling multiple products.

The invention provides an application of the bergamotene synthetase in preparation of terpene compounds, wherein the terpene compounds comprise at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene. When the pheasant cresene synthetase is applied to preparation of one of cresene, geraniol, alpha-pinene, beta-myrcene and limonene, the target substance is separated after the substrate is catalyzed by the pheasant cresene synthetase; when the pheasant phellandrene synthase is used for the simultaneous preparation of several of them, for example, beta-pinene and beta-myrcene are prepared; preparing beta-myrcene and D-limonene; or preparing a mixture of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene, and separating out a target substance or removing impurities after catalyzing a substrate by using the campylon-vanillic acid synthetase. Since the jungle pepper phellandrene synthase is a multi-product controlled synthase, terpene compounds that can be synthesized include phellandrene, geraniol, alpha-pinene, beta-myrcene, and limonene, in some alternative embodiments, the jungle pepper phellandrene synthase can be used to synthesize a mixture of phellandrene, geraniol, alpha-pinene, beta-myrcene, and limonene.

The application of the bergamot pepper phellandrene synthetase in preparing terpene compounds can be used for preparing various terpene compounds, including at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene; the method synthesizes terpene compounds by using the substrate catalyzed by the litsea cubeba phellandrene synthetase, the synthetic process is close to the biological natural synthetic process, the synthetic 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.

In some alternative embodiments, the amino acid sequence of the pheasant phellandrene synthase is shown in SEQ ID No. 2. In some alternative embodiments, the nucleotide sequence of the phellandrene synthase from cubeb pepper 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 screening markers to the amino acid sequence and nucleotide sequence encoding the pheasant phellandrene synthase when preparing in vitro recombinant pheasant phellandrene synthase.

In some alternative embodiments, the substrate of the pheasant phellandrene synthase comprises geranyl pyrophosphate; geranyl pyrophosphate (GPP) is an important product in the isoprene synthesis pathway, and is a synthetic precursor of various terpenoids, and pheasant phellandrene synthase can synthesize terpene compounds using geranyl pyrophosphate as a substrate.

In some alternative embodiments, the phellandrene synthase catalyzing the substrate synthesis of the terpene compounds in vitro is expressed by a prokaryotic expression system, preferably via an e. The pheasant phellandrene synthetase expressed by a prokaryotic expression system has the activity of catalyzing a substrate to synthesize terpene compounds including phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene 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, simplicity in operation, short culture period and strong anti-pollution capability. In some preferred embodiments, escherichia coli BL21 strain (e.coli BL21) which has a high growth rate and high expression level is preferably used for expressing the phellandrene synthase from the shanjiao pepper.

In some preferred embodiments, the pheasant phellandrene synthase is expressed by an expression vector pET-32a containing a T7 promoter, the T7 promoter is regulated by T7 RNA polymerase, and the gene transcription of a host containing the expression vector with T7 as the promoter does not compete with a T7 expression system, so that the expression vector pET-32a in the host can utilize most of resources in the host for synthesizing foreign proteins, and the expression amount of the foreign proteins regulated by the T7 expression system can account for more than 50% of the total protein of the host cell, so that the pET-32a can efficiently express the pheasant phellandrene synthase in the host.

According to another aspect of the invention, the invention also provides application of the bergamotene synthetase in preparation of products taking terpene compounds as active substances, wherein the terpene compounds comprise at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene. The product using terpene compounds as active substances can be selected from one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene as an active ingredient, or can be selected from a plurality of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene as active ingredients, for example, beta-pinene and beta-myrcene are used as active ingredients; beta-myrcene and D-limonene are taken as active ingredients; since the pheasant cresene synthase is a synthase for controlling multiple products, terpene compounds which can be synthesized include α -pinene, geraniol, phellandrene, α -pinene, β -myrcene, and limonene, in some alternative embodiments, the product has a mixture of α -pinene, geraniol, phellandrene, α -pinene, β -myrcene, and limonene as an active ingredient.

In the present invention, when the product uses at least one of phellandrene, geraniol, α -pinene, β -myrcene and limonene, or uses a mixture of phellandrene, geraniol, α -pinene, β -myrcene and limonene as an active ingredient, phellandrene may further include derivatives of phellandrene, geraniol may further include derivatives of geraniol, α -pinene may further include derivatives of α -pinene, β -pinene may further include derivatives of β -pinene, β -myrcene may further include derivatives of β -myrcene, and limonene may further include derivatives of limonene. Wherein "derivatives" refer to substances which are capable of providing, when such derivatives are used as one of the active ingredients of a product, the product, in use, with either direct or indirect phellandrene activity, geraniol activity, α -pinene activity, β -myrcene activity or D-limonene activity.

In some preferred embodiments, the product comprises essential oils and derivatives thereof. Alpha-pinene, geraniol, phellandrene, alpha-pinene, beta-myrcene and limonene are important components of natural plant essential oil. In some preferred embodiments, the pheasant phellandrene synthase for preparing essential oil and products derived therefrom has an amino acid sequence as shown in SEQ ID No. 2; the pheasant phellandrene synthetase preferably contains a nucleotide sequence shown as SEQ ID NO. 1.

The invention is further illustrated by the following examples. The materials in the examples are prepared according to known methods or are directly commercially available, unless otherwise specified.

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'-ATGGCATTGCAAATGACTGT-3', reverse primer: 5'-CCCAGAAGATCAGGGAACTAC-3' 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 30 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 1,815bp in size, as shown in FIG. 1, wherein M in FIG. 1 is DNA marker DL2000, and lanes 1 and 2 are amplification products.

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 the ligation product uniformly, standing the mixture on ice for 25min, performing water bath heat shock at 42 ℃ for 45s, rapidly performing ice bath, standing the mixture for 2min, adding 500 mu L of LB culture medium without antibiotics, recovering the mixture at 37 ℃ and 200rpm for 1h after the mixture is uniformly mixed, centrifuging the bacterial liquid at 3000rpm for 1min, removing 400 mu L of supernatant, suspending the bacterial liquid, coating the suspension 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: single colonies were randomly picked from the transformation plates and cultured in liquid medium in 1.5mL centrifuge tubes. Each tube is numbered, one microliter of each tube is used as a template for PCR detection, the rest culture is stored at 4 ℃, and colonies which are detected to be positive are stored on a plate or a glycerol tube for later use. The nucleotide sequence of the cloned pheasant phellandrene synthetase gene is shown in SEQ ID NO.1, the phellandrene synthetase gene contains 1815 basic groups, 605 amino acids are coded in total, and the specific amino acid sequence is shown in SEQ ID NO. 2. And then the positive bacterium of prokaryotic cell Escherichia coli DH5 alpha is successfully transformed by the recombinant plasmid which inserts the pheasant phellandrene synthetase gene into pClone007 cloning vector.

Example 2

The construction of an expression vector and the transformation of a prokaryotic cell by the phellandrene synthetase gene comprises the following steps:

the cloned target fragment is transformed into a pET32a linearized vector by means of homologous recombination. The ligation reaction conditions were: mixing the reaction systems, placing the mixed reaction systems at 37 ℃ for incubation for 30min, then at 20 ℃ for 1h, adding 5 mu L of reaction liquid into 50 mu L of escherichia coli DH5 alpha competent cells, uniformly mixing, standing in an ice bath for 30min, gently taking out, heating at 42 ℃ for 60s, immediately performing ice bath for 2min, adding 500 mu L of LB culture medium, and culturing at 37 ℃ for 1 h; 100. mu.L of the bacterial suspension was applied to an Amp-resistant LB plate and cultured overnight. And selecting positive colonies obtained by screening antibiotics (Amp) and extracting plasmids. Transferring the prokaryotic expression vector into an escherichia coli BL21 strain, culturing at 37 ℃ and 200rpm until OD600 is 0.6, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.l mM into a test tube culture solution, and then respectively performing induced expression at 15 ℃ and 37 ℃ to obtain a large amount of expression pheasant phellandrene synthetase gene bacterial solution.

Example 3

Prokaryotic expression, protein purification and protein quality detection of phellandrene synthetase comprise the following steps:

selecting a single colony containing the recombinant plasmid to a 3mL LB liquid culture medium (Amp resistance), culturing overnight at 37 ℃, and preserving the strain at-20 ℃; respectively selecting single colonies containing the recombinant plasmids into 3mL LB liquid culture medium (Amp resistance), and performing shake culture at 37 ℃ until the OD600 is about 0.6; taking part of the bacterial liquid as a control group, adding IPTG inducer (final concentration is 1mM) into the rest bacterial liquid, and performing shake culture at 37 ℃ for 3 h; 0.15mL of the two groups of bacterial liquid are respectively taken, the two groups of bacterial liquid are centrifuged for 2min at 12000 Xg, and the bacterial precipitation is resuspended and cracked by 40 mu L of 1 Xloading buffer and is detected by SDS-PAGE. Inoculating 100 μ L of strain stored at-20 deg.C into 100mL LB liquid medium (Amp resistant), and shake culturing overnight; inoculating 100mL of bacterial liquid into 2000mL of LB liquid medium, carrying out amplification culture at 37 ℃ until the OD600 is about 0.6, and reducing the culture temperature to 30 ℃; adding IPTG inducer to the final concentration of 0.5mM, and continuing shaking culture at 30 ℃ for 3 h; centrifuging at 8000rpm for 3min, collecting thallus, suspending in 50mL precooling NTA-0 buffer solution, and ice-cooling for 30 min; ultrasonically crushing thalli, setting parameters to be 200W of power, 3s of working, 4s of pause and 99 cycles; centrifuging at 16000rpm at 4 deg.C for 50min, and collecting supernatant and precipitate; a small amount of the supernatant and the precipitate were subjected to SDS-PAGE, and the remaining supernatant and the precipitate were placed at 4 ℃ for further use. Filtering the supernatant protein solution by using a 0.22 mu m filter for later use; preparing a Ni-NTA column; loading the supernatant protein solution at a flow rate of 1 mL/min; washing the column with NTA-0 buffer (pH 8.0) until the effluent is free of protein (G250 detection solution is not discolored); eluting with 20mM, 60mM, 200mM and 500mM imidazole respectively, and collecting the eluate in stages until the G250 detection solution is not discolored; washing the column material with deionized water of 3 times of the column volume, and sealing the column with 20% ethanol; and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection on the collected eluate, and carrying out Western blot detection on the gene of the phellandrene synthetase of the litsea cubeba. The results of SDS-PAGE are shown in FIG. 2, in which lane M: marker; lane 1 before induction; lane 2 post induction; lane 3 elution of protein. The Western blot detection results are shown in FIG. 3: wherein Lane M is marker, Lanes 1-4 are sequentially marker, column-loading effluent liquid, protein flowing out and protein eluting.

Example 4

Biochemical function of pheasant cresene synthetase

Geranyl pyrophosphate (GPP) is taken as a substrate, and an enzymatic reaction system is as follows: HEPES, 25 mM; KCl, 100 mM; MgCl ₂10 mM; 10% (v/v) glycerol; DTT, 5 mM; GPP, 30 mM; 30ng of purified protein was added and left at 30 ℃ for 1 h. An Agilent 6890N-5975B gas chromatography-mass spectrometer of Agilent is adopted, volatile substances are detected by adsorbing with an 50/30 mu m 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. 3-19:

the result of GC-MS detection shows that the pheasant phellandrene synthetase can catalyze GPP to synthesize geraniol, phellandrene, alpha-pinene, beta-myrcene and limonene, and the peak-off time and the ion characteristic peak can be matched with NIST08 standard spectrum library retrieval, so that the catalytic product of the enzyme can be qualitative; because the enzyme content is limited, the enzyme cannot be quantified in an experiment, and only can the enzyme be proved to have enzyme catalytic activity and be a multi-product enzyme.

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.

Claims (10)

1. The application of the bergamotene synthetase in preparing terpene compounds comprises at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene.

2. The use according to claim 1, wherein the terpene-based compounds include phellandrene, geraniol, α -pinene, β -myrcene and limonene.

3. The use according to claim 1, wherein the amino acid sequence of the pheasant phellandrene synthase is shown as SEQ ID No. 2.

4. The use according to claim 1, wherein the nucleotide sequence of the pheasant phellandrene synthase is shown as SEQ ID No. 1.

5. The use according to claim 1, wherein the substrate of the jatropha phellandrene synthase comprises geranyl pyrophosphate.

6. The use according to any one of claims 1 to 5, wherein said phellandrene synthase is expressed from a prokaryotic expression system;

optionally, the prokaryotic expression system comprises an escherichia coli expression system;

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

7. The method of claim 6, wherein the pheasant phellandrene synthase is expressed by pET-32 a.

8. The application of the bergamotene synthetase in preparing products taking terpene compounds as active substances is disclosed, wherein the terpene compounds comprise at least one of phellandrene, geraniol, alpha-pinene, beta-myrcene and limonene;

preferably, the terpene compounds include phellandrene, geraniol, α -pinene, β -myrcene, and limonene.

9. Use according to claim 8, wherein said products comprise essential oils and derivatives thereof.

10. The use according to claim 8 or 9, wherein the pheasant phellandrene synthase comprises an amino acid sequence as shown in SEQ ID No. 2;

preferably, the pheasant phellandrene synthase contains a nucleotide sequence shown as SEQ ID No. 1.

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