Biosynthetic enzyme gene capable of producing borneol and application thereof

Abstract

The present application relates to the technical field of biological enzyme. The present application provides a biological enzyme gene capable of producing borneol and application, including one or more of TPS1, TPS 2, TPS 3, TPS 4, TPS 5 and TPS 6. The gene provided by the present application can be used to prepare a biological enzyme for catalyzing the production of borneol. The preparation of borneol by using the biological enzyme is an efficient method for improving the yield of borneol. Not only can the efficiency be improved, but also a sustainable production mode can be realized. A more efficient and economical preparation method of borneol can be provided, and the method can be widely applied to industrial production and other fields.

Description

Technical Field

[0001] This invention relates to the field of bioenzyme technology, and in particular to a bioenzyme gene capable of producing borneol and its applications. Background Technology

[0002] Borneol, also known as camphor, citrus borneol, artemisia argyi, and camphor fragrans, is actually a product extracted from the stems and leaves of *Artemisia argyi* (a member of the Asteraceae family) or the branches and leaves of *Cinnamomum camphora* (a member of the Lauraceae family) through steam distillation and recrystallization. Its main chemical component is 2-borneol, with the chemical formula C6H2O. 10 H 18 O. It can also be obtained by subjecting turpentine to a series of chemical treatments.

[0003] Borneol has a wide range of applications, including medicine, cosmetics, and fragrances. In traditional Chinese medicine, borneol is used to relieve chest tightness, cough, and nasal congestion, and is also used to treat red and swollen eyes, sore throat, and mouth ulcers. In modern medicine, borneol may be used to treat conditions such as rhinitis and pharyngitis that involve headaches. In the cosmetics and daily chemical industries, borneol is often used to increase the cooling sensation and enhance the aroma of products. In the fragrance industry, borneol is a key ingredient in perfumes and flavor formulations. Additionally, borneol may be used in the production of specialty foods and beverages to add unique flavors, and it is even used as a natural insecticide and preservative, with wide applications in agriculture.

[0004] However, current methods for extracting and preparing borneol suffer from problems such as long processing time, low efficiency, and high cost. Therefore, finding a more efficient and cost-effective method for preparing borneol is of paramount importance. Summary of the Invention

[0005] The purpose of this invention is to provide a biological enzyme gene that can produce borneol and its application, and to find a more efficient and cost-effective method for preparing borneol.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a biological enzyme gene capable of producing borneol, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5 and TPS6;

[0008] The amino acid sequence of TPS1 is shown in SEQ ID NO.1;

[0009] The amino acid sequence of TPS2 is shown in SEQ ID NO.2;

[0010] The amino acid sequence of TPS3 is shown in SEQ ID NO.3;

[0011] The amino acid sequence of TPS4 is shown in SEQ ID NO.4;

[0012] The amino acid sequence of TPS5 is shown in SEQ ID NO.5;

[0013] The amino acid sequence of TPS6 is shown in SEQ ID NO.6.

[0014] The present invention also provides the application of the aforementioned gene in the preparation of bioenzymes for producing borneol.

[0015] The present invention also provides a bioenzyme for producing borneol, said bioenzyme being prepared from the aforementioned gene.

[0016] The present invention also provides the application of the aforementioned bioenzyme in the catalytic preparation of borneol from gerany pyrophosphate.

[0017] This invention provides a bio-enzyme gene capable of producing borneol and its applications, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5, and TPS6. The gene provided by this invention can prepare a bio-enzyme for catalyzing the production of borneol. Using this bio-enzyme to prepare borneol is a highly efficient method to increase borneol yield. It not only improves efficiency but also enables a sustainable production method. It provides a more efficient and economical route for borneol preparation and can be widely applied in industrial production and other fields. Attached Figure Description

[0018] Figure 1 This refers to the biosynthetic pathway of borneol;

[0019] Figure 2 The data are for GC-MS identification of borneol, where a is the GC identification result of borneol with a peak time of 14.05 min; b is the MS identification result of borneol in the sample; c is the MS result of borneol in the database; and d is the information of borneol in the database.

[0020] Figure 3 The relative content of borneol synthesized using different TPS catalysts. Detailed Implementation

[0021] This invention provides a biological enzyme gene capable of producing borneol, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5 and TPS6;

[0022] The amino acid sequence of TPS1 is shown in SEQ ID NO.1:

[0023] MRSLSIALPLPLPLHTTVPRVILSGRMLPPSSRSCYASSQVRSMSAINMSEQAIARRSADYRPPIWSFEYIQSLKTEYAGEAYSRRRDKLKEDVRRMLQKEVDEDQLELIDNLQRLGLSYHFEDEIKNTLDRIYIKNHGNYGLMKDSLYATALGFRILRQHGYNVPQEIFNGFRDESGNFKACLGEDYKGMLYLYEASFHSVEGESILDVAKAFATKHLKEYIKLNQAKDMYLSTLVSHALELPLHWRMLKMEARWFIEVYQQSPHVNPILLDFAKLDFNLVQATYQEDLKHASSWWKRTGLGEKLGFIRDRLVENFLWPIGELYEPQYGYYRRMATQVNTLITTIDDIYDIYGTLDELELFTEAIERWDINRIDKLPDYMKLCFFEVHNSINQMAADIFREQGLNILPYSKKVWTDLCKAYLLEAKWYHSGYKPTLQEYLENAVVTIAAPVILHYAYVLVSNQITRESLECFEQEYPNVIHCSAMLLRLADDLGTSTDEMKRGDVPKSIQCYMNETGASEEDARKYIWHLIDITWKKMNKDQHKTSSLPPALIGAAMNLGRTAQFMYLYGDGHSSQDHVTRNRILSLLVNPIT;

[0024] The amino acid sequence of TPS2 is shown in SEQ ID NO.2:

[0025] MRSLSIALSLPLPLRTTDPRVILSGRMLPPSSRSCYASSQVRSMSAINMSDQAIARRSADYQPPIWSFEYIQSLKTEYAGEAYSRRRDKLKEDVRRMLQKEVDEDQLELIDNLQRLGLSYHFEDEIKNTLDRIYSKNHGNYGLMKDSLYATALEFRILRQHGYNVPQEIFNGFRDESGNFKACLGEDYKGMLYLYEASFHSVEGESILDVAKAYATKHLKEYIKLNQGKDMYLSTLVSHALELPLHWRMLRMEARWFTEVYQQSPHVNPILLDFAKLDFNLVQATYQEDLKHASSWWKRTGLGEKLGFIRDRLVENFLWPIGELYEPQYGYYRRMATQVNTLITTIDDIYDVYGTLDELELFTEAIERWDINRIEKLPDYMKLCFFELHNSINQMAADIFREQGLNILPYSKKVWTDLCKAYLLEAKWYHSGYKPTLEEYLDNAVVTIAAPVILHYAYVLVSNQITRKGLECFEQEYPNAIHCSAMLLRLADDLGTSTDEMKRGDVPKSVQCYMNETGASEEDAREYIWHLIDITWKKMNKDRHKTSSLPPALIGAAMNLARTAQFMYLYGDGHSSQDHVTRNRILSLLVNPVA;

[0026] The amino acid sequence of TPS3 is shown in SEQ ID NO. 3:

[0027] MRSVSMALSLPLPLHTSVPRVILSGRMLPPSSRSCYASSSSQVRSMSAINMSDARRSADFQPSIWSFEYIQSLKTEYAGEAYNRRRDKLKEDVRVMLQDEVDKDQLELMDNLQRLGLSYHFEDEIKKILDRIYNKNNGNYGLKKDSLYAAALEFRILRQHGYNVSQEIFNGFRDESGNFKACLGEDYKGMLYLYEASFHSVEGESILDAAKAFATKHLKEYIKLNQGKDMYLSTLVSHALELPLHWRMLRMEARWFMEVYQQSRHVNPILLDLAKLDYNLVQATYQEDLKHASSKNVCREKRYPFLYHFSKSCQTGKVCKSYSFFSITFTCIKKILWWKRTGLGQNLGFIRDRLMENFLWATGILFQPQYEYFRRMTTQVGALVTTIDDVYDVYGTLDELELFTNAIERWDINAIEQLPDYMKLCFFALHNSMNQIASDIFQEQRINILPYSKKAWLDLCKTYLIEVKWYHQGYTPSLQEYIDNAVVSISAPLILLHAYILSSNHITTEVLQYLEEELPNIIRCSSMVVRLADDLGTSHDEMRRGDVPKSIQCYMHETGASEEDAREYIHDLIDKTWNKMNKDEFEPSPLPKTLIEAAMNLARIAQFMYKHGDGHSSQDDVMRNRILSLLINPIPLPGLDESHITTA;

[0028] The amino acid sequence of TPS4 is shown in SEQ ID NO.4:

[0029] MRSLSIALPLPLPLHTTVPRVILSGRMLPPSSRSCYASSQVRSMSAINMSEQAIARRSADYRPPIWSFEYIQSLKTEYAGEAYSRRRDKLKEDVRRMLQKEVDEDQLELIDNLQRLGLSYHFEDEIKNTLDRIYIKNHGNYGLMKDSLYATALEFRILRQHGYNVPQEIFNGFRDESGNFKACLGEDYKGMLYLYEASFHSVEGESILDVAKAFATKHLKEYIKLNQAKDMYLSTLVSHALELPLHWRMLRMEARWFIEVYQQSPHVNPILLDFAKLDFNLVQATYQEDLKHASSWWKRTGLGEKLGFIRDRLVENFLWPIGELYEPQYGYYRRMATQVNTLITTIDDIYDIYGTLDELELFTEAIERWDINRIDKLPDYMKLCFFEVHNSINQMAADIFREQGLNILPYSKKVWTDLCKAYLLEAKWYHSGYKPTLQEYLENAVVTIAAPVILHYAYVLVSNQITRESLECFEQEYPTVIHCSAMLLRLADDLGTSTDEMKRGDVPKSIQCYMNETGASEEDAREYIWHLIDITWKKMNKDRRKTSSLPPALIGAAMNLGRTAQFMYLFASFNGYKKCNVQTCLFACVNNVSFFGGFGSRDKLELKAVNDGSASYAALEKKAELYEKLVRGELSDEEDREVLCRFLQKGPGRTDGVVDRDEHKRFVREVHEEANQARKKVSELKLRRQKQAAARREKLRQAYLHKQLQKLKAASNTQQT;

[0030] The amino acid sequence of TPS5 is shown in SEQ ID NO.5:

[0031] MFVQVTESLPAPPTNQSKPEINRRSAGFHPSIWGDHFLSYGSNNIMQSEEEKREHLKLKEKLKEKLMAVVDEPLKKLELIDVIQRLGVSYHFQKEIDEILGQILQVMNQREFKAGSCKDDDLCTISLTFRLLRQHGYRISSEMFKKFKDRGGNFKESLIDDVKGMLSLYEAAHLRVPGEHILDQALSFTTTHLLSMATSLGSPSLAAQVTNALHQPFHTGIPRLEARRFIPVYQADPSHDKTLLSFARLDFNMLQRLHQREISDIAKWWKDLDFSTKLPFARDRLIECYFWMLGLYFEPEYLLARRFSTKVIVLSSVIDDIYDVYGTLDELELLTEAIQRWDINAIDQMPEYMQIHFRAFLDIYREIEEQMAAQGKLYRVHYAKESMKRLVRAYFQEAKWFHEKYAPTLDEYMPIALITSGYEMLAVTCIVGLGDVATEDSFIWLMSRPKIVTASEVIARLMDDIATHKFEQKRGHVASSIECYMTQYGSTEEEAVEELGKLITKAWMDINEECLRPTAIPMPLLTCVVNLARVIEFLYKSEDGYTHVGKRMKESVASLLIDRVPEKCILPGSGFAGSASPRHPPVSYEELELPNQQADLVEDNAQKHGPKIPSVRLSPAITLRLREN;

[0032] The amino acid sequence of TPS6 is shown in SEQ ID NO.6:

[0033] .

[0034] The present invention also provides the application of the aforementioned gene in the preparation of bioenzymes for producing borneol.

[0035] The present invention also provides a bioenzyme for producing borneol, said bioenzyme being prepared from the aforementioned gene.

[0036] The present invention also provides the application of the aforementioned bioenzyme in the catalytic preparation of borneol from gerany pyrophosphate.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1: Obtaining a Highly Active Bioenzyme Gene

[0039] A codon-optimized bioenzyme gene sequence was synthesized from Wuhan Jinkairui Biotechnology Co., Ltd. To achieve efficient expression of the target protein, the pCold vector, widely used in protein expression, was selected. This vector contains an inducible promoter, enabling the expression of the target protein under low-temperature conditions.

[0040] To construct the recombinant expression vector pCold-TPS, the pCold vector was first linearized using restriction endonucleases Nde1 and EcoR1. Following enzyme digestion, agarose gel electrophoresis was performed to separate the target band, thus obtaining the linearized vector.

[0041] Next, the linearized vector was purified from the gel using an agarose gel extraction kit. The purified vector was then ligated with the enzyme gene fragment to construct the recombinant expression vector pCold-TPS.

[0042] Example 2 Protein Expression and Purification

[0043] 1. Using Escherichia coli BL21(DE3) strain, the expression of all TPS proteins was successfully achieved.

[0044] a) Starting with the preserved pCold-TPS monoclonal strain, inoculate it into a small test tube containing 5 mL of LB liquid medium (Amp content 100 μg / mL) and culture it overnight at 37°C and 220 rpm to obtain the seed culture.

[0045] b) Transfer the seed culture to a 50 mL bottle containing LB liquid medium (Amp content 100 μg / mL) and incubate on a shaker at 37 °C and 220 rpm to reactivate the bacterial culture.

[0046] c) Using a 1% inoculum, transfer the reactivated bacterial culture to an 800 mL bottle containing 2YT liquid medium (Amp content 100 μg / mL) and incubate on a shaker at 37°C and 220 rpm until the OD600 is 0.6.

[0047] d) Reduce the temperature of the shaker to 17°C, add isopropyl thio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM, and induce expression for 15 h.

[0048] e) After expression is complete, collect the culture medium into a bottle, pre-cool the centrifuge to 4°C, and centrifuge at 5500 rpm for 10 min.

[0049] f) Discard the supernatant, add 30 mL of protein purification buffer, and resuspend the bacteria using a vortex mixer. Centrifuge again at 5500 rpm for 10 min. Discard the supernatant, add another 30 mL of protein purification buffer, resuspend the bacteria using a vortex mixer (ensuring no solid particles remain), and transfer to a 50 mL centrifuge tube. Store at -80°C.

[0050] 2. Perform high-pressure crushing and centrifuge to collect the supernatant.

[0051] a) Take the collected bacterial culture and use a high-pressure cryogenic disruptor to disrupt the bacteria at 4°C and 900 bar for 4 minutes to fully lyse the cells and release the target protein into the protein buffer.

[0052] b) Centrifugation: Place the broken bacterial culture into a pre-cooled 4℃ centrifuge and centrifuge at 8000rpm for 60min to obtain the precipitate and supernatant after centrifugation. Prepare samples and collect the supernatant.

[0053] 3. Protein purification was performed using a Ni affinity chromatography column.

[0054] a) The supernatant was purified by nickel affinity chromatography, and the specific steps are as follows:

[0055] First, wash twice the column volume with double-distilled water.

[0056] Then, equilibrate the Ni affinity chromatography column with protein buffer at a ratio of one.

[0057] b) Take 50 mL of sample and slowly pass it through the Ni affinity chromatography column, collecting the first few drops of sample.

[0058] c) Elute with protein buffers containing 20 mM, 50 mM, 100 mM, 200 mM and 300 mM imidazole to remove bound contaminating proteins. Use the first few drops of the flow-through sample for sample preparation and perform detection using 12% SDS-PAGE.

[0059] d) Concentration and buffer replacement: The eluent containing the target protein was concentrated by centrifugation (4°C, 3400 rpm) using a 10 kDa Amicon ultrafiltration tube (Millipore) to a final volume of 1 mL. Then, 10 mL of protein buffer was added to concentrate the protein to a final volume of 1 mL. This step was repeated once to reduce the imidazole concentration in the protein and obtain a purified protein sample.

[0060] 4. Protein concentration determination

[0061] We used the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to determine protein concentration.

[0062] First, we initially determined the protein concentration by measuring the absorbance at 280 nm, and then diluted the protein to a concentration of 0.5-1 mg / mL based on the preliminary results. Next, we prepared the reaction solution by mixing reagent A and reagent B in a 50:1 ratio.

[0063] We took 200 μL of the reaction solution into the ELISA plate and added 25 μL of diluted protein sample. After mixing by pipetting, we incubated the reaction solution at 37°C for 30 minutes.

[0064] We then placed the ELISA plate into an ELISA reader to measure the absorbance at 562 nm and processed the data according to the protein standard curve to determine the protein concentration.

[0065] Example 3 Enzyme Activity Assay

[0066] Each enzyme activity assay used a 200 mL reaction mixture containing 50 mM Tris-HCl (pH 7.2), 10 mM MgCl2, 5 mM DTT, 1 mM PMSF, and 3 mM GPP substrate. Different concentrations of purified protein (1 mg / mL) were added at the start of the catalytic reaction, and the reaction mixture was incubated at 30 °C for 15 min. The reaction was stopped 5 min after incubation and immediately quenched on ice. To promote dephosphorylation of the product, 3 mL of calf intestinal alkaline phosphatase (TaKaRa) was added, and the mixture was incubated at 37 °C for 1 h. Subsequently, 300 mL of n-hexane was added, and the mixture was stirred for 15 min to extract monoterpenoids for GC-MS analysis. GC-MS analysis was performed using an Agilent 7890A gas chromatograph system equipped with a DB-5MS capillary gas chromatograph column coated with 5% diphenyl and 95% dimethylpolysiloxane (30 m long, 250 μm inner diameter, 0.25 mm film thickness, J&W Scientific, USA). The oven temperature program was set as follows: initial temperature 50 °C, hold for 2 min, then ramp to 180 °C at 5 °C / min (hold for 5 min), followed by ramp to 230 °C at 10 °C / min. The temperatures of the injection inlet, transfer line, and ion source were controlled at 250 °C, 290 °C, and 230 °C, respectively. Only 1 mL of sample was added in split mode, with an injection ratio adjusted to 10:1. Finally, mass spectrometry data were acquired in full scan mode, covering the m / z range of 35–650, with a solvent delay of 7.5 min.

[0067] GC-MS results for TPS-catalyzed borneol production are shown in [reference needed]. Figure 2 The elution time of borneol was approximately 14.05 min. The mass spectrometry information of borneol in the sample matched the borneol mass spectrometry information in the database, confirming that borneol was detected. The relative contents of borneol synthesized by different sequences are shown in [reference needed]. Figure 3 Among them, TPS6 has the highest yield, reaching 31.0 mg / L.

[0068] As can be seen from the above embodiments, the present invention provides a bio-enzyme gene capable of producing borneol and its applications, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5, and TPS6. The gene provided by the present invention can prepare a bio-enzyme for catalyzing the production of borneol. Using this bio-enzyme to prepare borneol is a highly efficient method to increase borneol yield. It not only improves efficiency but also enables a sustainable production method. It provides a more efficient and economical route for borneol preparation and can be widely applied in industrial production and other fields.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A biological enzyme for producing borneol, characterized in that, The bioenzyme is TPS 6, and the amino acid sequence of TPS 6 is shown in SEQ ID NO.

6.

2. The application of the bioenzyme described in claim 1 in the catalytic preparation of borneol from geraniol pyrophosphate.

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