Bio-enzyme gene capable of producing beta-pinene and application

Abstract

The invention relates to the technical field of biological enzymes. The invention provides a biological enzyme gene capable of producing beta-pinene and application of the biological enzyme gene. The biological enzyme gene comprises one or more of TPS1, TPS2, TPS3 and TPS4. The gene disclosed by the invention can be used for preparing a biological enzyme for efficiently catalyzing and producing beta-pinene, and the biological enzyme is used as a catalyst, so that the yield of beta-pinene can be increased, and an efficient, environment-friendly and sustainable production mode is realized. The method can become an important production way of beta-pinene in the future, and has a wide application prospect in industrial production and other fields.

Description

Technical Field

[0001] The present invention relates to the technical field of bioenzymes, and particularly to a bioenzyme gene capable of producing β-pinene and its application. Background Art

[0002] β-pinene is a special bicyclic monoterpene compound. Its physical state is oily. It mainly exists in turpentine and is also the double bond position isomer of α-pinene. In the field of chemical engineering, β-pinene is widely used in the formulation of flavors for daily chemical products and the flavor enhancement process of various industrial products. At the same time, it is also the primary raw material for synthesizing fragrances such as citral, citronellol, and linalool. In addition, β-pinene can also be used to produce β-pinene resin and is also an important raw material for preparing vitamin products.

[0003] For a long time, the single or excessive use of pesticides will undoubtedly lead to the development of pesticide resistance in agricultural pests, thus forming cross-resistance and multiple resistance, which has a negative impact on the agricultural production situation. β-pinene can effectively improve the insecticidal efficacy of pesticides. Even though β-pinene itself has relatively low insecticidal performance against insects, its ability is highlighted after being added to a certain pesticide, thereby playing a role in enhancing the efficacy of pesticides.

[0004] However, there are problems such as complexity and high cost in the extraction and synthesis methods of β-pinene. Therefore, it is particularly important to develop a new, efficient, and economical extraction and synthesis method for β-pinene. Summary of the Invention

[0005] The purpose of the present invention is to provide a bioenzyme gene capable of producing β-pinene and its application, and to develop a new, efficient, and economical extraction and synthesis method for β-pinene.

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

[0007] The present invention provides a bioenzyme gene capable of producing β-pinene, including one or more of TPS1, TPS2, TPS3, and TPS4;

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

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

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

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

[0012] The present invention also provides the use of the said gene in the preparation of a bioenzyme for producing β-pinene.

[0013] The present invention also provides a bioenzyme for producing β-pinene, which is prepared from the said gene.

[0014] The present invention also provides the use of the said bioenzyme in catalyzing the preparation of β-pinene from neryl pyrophosphate.

[0015] The present invention provides a bioenzyme gene capable of producing β-pinene and its applications, including one or more of TPS1, TPS2, TPS3 and TPS4. The gene of the present invention can prepare a bioenzyme that efficiently catalyzes the production of β-pinene. Using this bioenzyme as a catalyst can not only increase the yield of β-pinene, but also achieve an efficient, environmentally friendly and sustainable production mode. This method can become an important production route for β-pinene in the future and has broad application prospects in industrial production and other fields. Description of the Drawings

[0016] Figure 1 is the biosynthetic pathway of β-pinene;

[0017] Figure 2 is the analysis of the gas chromatography-mass spectrometry detection results of β-pinene, where a is the gas detection result of β-pinene, and the peak emergence time is about 8.1 min; b is the MS detection result of β-pinene; c is the MS information of β-pinene in the database;

[0018] Figure 3 is the relative content (%) of β-pinene catalyzed by different TPS sequences. Detailed Embodiments

[0019] The present invention provides a bioenzyme gene capable of producing β-pinene, including one or more of TPS1, TPS2, TPS3 and TPS4;

[0020] The amino acid sequence of the said TPS1 is as shown in SEQ ID NO.1:

[0021] MSAFTSVALPLQASVPSRVTALKLSNELTMRRSADYKPPIWSFEYIQSLKLEYVEEEPFRKLINKLKDDVIAMLEGEEMDKDPSRQLELIDTLRRLGLSYHFENEINKILEKVYTKHQGYYGLERDNLYMAALEFRILRQHGYKVPEEIFKSFLNERGNFKAPHKRDCKGMLFLYEASFLSLEGESTLNTARTFARNYLSEYVKLKESKDPYLSTLVEHALEFPLHWRMPRMETRWFIEVYQQSPNMNPVLLDLAKLDFNMVQATYQEDLKDASRWWKRSGLGQNLNFIRDRLMENFLWTTGILFQPQYAYFRRMITQVAALGTTIDDVYDVYGTLDELELFTDAIERWDINAIEELRDYMKLCFFAVHNSINQIVSDVFQEQGINILPYSKKAWLNLCKSYLIEAKWYHQGYTPSLREYIDNAVVSIAGPLALLHAYICSSNHITSEVLQYLVEELPNIIRCSSMIMRLADDLGTSLDEMRRGDVPKSIQCYMHETGASEEIAREYIQDLIDKTWNKMNKDQFEHSPLPQTLIEAAMNIARMAQFMYKHGDGHSSQDDVMRHHVLSLLINPIPSPGPEESHITA;

[0022] The amino acid sequence of TPS2 is shown in SEQ ID NO.2:

[0023] MRRSADYKPSIWTFEHIQSLKVGYAEESLRRRINKLKEDVIVMLEEKEMDKYPLQQLELIDTLQRLGLSYHFENEINRILEKVYTNNQGYYGLERDKLYVAALEFRILRQHGYKVPQEIFKSFLNESGNFKACLSKDCKGMLYLYEASFLSLEGESTLDAARTFTRKHLSEHVKLNESKDPYLSTLVEHSLEFPLRWRTPRMEARWFIEAYQRNPDMKPVLLDLAKLDFNIVQAMYQEDLKHASMWWKRTGLGQNLGFIRDRLMENFLWTTGVLFQPQYEYFRRMATQVTALVTTIDDVYDVYGTLDELELFTDAIERWDINAIEQLPDYMKLCFFALHNSMNQIASDIFQEQGINILPYSKKAWLDLCKTYMIEAKWYHQGYTPSLQEYIDNAVVSISAPLVLVHAYILSSNHITTEVLQYLEEALPNIIRCSSMVLRLADDLGTSPDEMRRGDVPKSIQCYMHETGASEEDAREYIQDLIDKTWNKMNKCEFEPSPLPKTLIEAAMNLARMAQFMYKHGDGHSSQDDVMKNRILSLLINPIPLPVFAWILCWLCSSFSADGYLLARLQYCSMASSYCWMRTARITRSSTLPSTTALLLSHIIDDFKTLTGNRGL;

[0024] The amino acid sequence of TPS3 is shown in SEQ ID NO.3:

[0025] MQICHINVYPVKMSPFTSMALPLQASVPVAAISTRLPFPSSCTKRYVPSRVTELKLSNELTMRRSADYKPPIWSFEDIQSLKVDYVEESFGRRINKLKEDVILMLEEKEVDKVPLQQLELIDTLQRLGLSYHFENEIDRILEKVYTNNQGYCYGFDRESLYVAALEFRILRQHGYKVPQEIFKSFLNESGNFKACLNKDCKGMLYLYEASFLSLEGESTLDAARTFARNYLSEYVKLNESKDPYLSTLVEHALEFPLRWRMPRMEARWFVEVYQRCPDMNPLLLDLAKLDFNMVQAMYQEDLKHASMWWKRTGLGQNLGFIRDRLMENFLWTIGELFQPQYGYFRRMAAQVNALVTTIDDVYDVYGTLDELEHFTDAIERWDINAIEQLPDYMKLCFFALHNSMNQIASDIFQKQGINILPYSKKAWLDLCKTYLIEAKWYHQGYTPSLQEYIDVAVISISAPLILLHAYILSSSHIKIEVLQYLEEELPSIIRCSSMVLRLADDLGTSSDEMRRGDVSKSIQCTIYETGVSEDDAREYIQDLIDKTWKKMNKYEFEPSLLPQTLIEAAINLARMAQFMYKHGDGHSSQDDVMRHRILSLLINPIALPRREESYITA;

[0026] The amino acid sequence of TPS4 is shown in SEQ ID NO.4:

[0027] MKMSAFTSMALPLQASLPVTAISTRLIFPSSCTKRYVPRRVTALKLSNELTMRRSAYYKPPIWSFEYIQSLKLEYVGGESFKRHINKLKEDVIAMLEGEEMDKDPSHQLELIDTLQRLGLSYHFENEINRILKKVYTKHQGYYGLERHSLYVAALEFRILRQHGYKVPQEIFKSFLNERGNFKPCLKNDCKGMLFLYEASFLSLEGESILDAARTFARNYLSEYVKLNETKDPYLSTLVEHALEFPLHWRMPRMEARWFIEVYKQSPDMNPVLLDLAKLDFNMVQATYQEDLKDASRWWNKSGLGQNLEFIRDRLVENFLWTTGVLFQPQYAYYRRMATQVNALLTTIDDVYDVYGTLDELELFTDVIERWDINAIEQLPDYMKLCFFAVHNSMNQIASDIFQEQGINILPYSKKAWLDLCKSYLIEAKWYHQGYKLSLHEYIDNAVISIAAPLMLIHAYILSSNHITTEVLQYLEEELPNIIRCSSMVLRLADDLGTSPDEMRRGDVPKSIQCYMHETGASEENAREYIQDLIDKTWNKMNKDQFEHSPLPQTLIEAAMNLARMAQFMYKHGDGHSSQDDVMRHSVLSLLINPIPLPAPEESHITA。

[0028] The present invention also provides the application of the said gene in the preparation of a bioenzyme for producing β-pinene.

[0029] The present invention also provides a bioenzyme for producing β-pinene, which is prepared from the said gene.

[0030] The present invention also provides the application of the said bioenzyme in catalyzing the preparation of β-pinene from neryl pyrophosphate.

[0031] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1 Obtaining a bioenzyme gene with high activity

[0033] We synthesized the codon-optimized bioenzyme gene sequence from Wuhan Kingcare Bioengineering Co., Ltd. To achieve the high-level expression of the target protein, we selected the pCold vector which is widely used in protein expression. This vector contains an inducible promoter that can initiate the expression of the target protein under low-temperature conditions.

[0034] To construct the recombinant expression vector pCold-TPS, we first linearized the pCold vector using the restriction enzymes Nde1 and EcoR1. After the enzymatic digestion reaction, we performed agarose gel electrophoresis to separate the target band, thereby obtaining the linearized vector.

[0035] Next, we used an agarose recovery kit to purify the linearized vector from the gel. Then, we ligated the purified vector with the enzyme gene fragment to construct the recombinant expression vector pCold-TPS.

[0036] Example 2 Protein Expression and Purification

[0037] 1. The expression of all TPS proteins was successfully achieved using Escherichia coli BL21(DE3) strain.

[0038] a) Starting from the preserved pCold-TPS monoclonal strain or the -80°C frozen storage strain, inoculate it into a small test tube containing 5 mL of LB liquid medium (with 100 μg / mL Amp), and culture it overnight at 37°C and 220 rpm to obtain the seed solution.

[0039] b) Transfer the seed solution to a 50 mL bottle containing LB liquid medium (with 100 μg / mL Amp), and culture it on a shaker at 37°C and 220 rpm to reactivate the bacterial solution.

[0040] c) With an inoculation amount of 1%, transfer the reactivated bacterial solution to an 800 mL bottle containing 2YT liquid medium (with 100 μg / mL Amp), and culture it on a shaker at 37°C and 220 rpm until the OD600 reaches 0.6.

[0041] d) Lower the shaker temperature to 17°C, add isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and induce expression for 15 h.

[0042] e) After the expression is completed, collect the cultured bacterial solution into a bottle, pre-cool the centrifuge to 4°C, and centrifuge at 5500 rpm for 10 min.

[0043] f) Pour off the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex shaker. Centrifuge again at 5500 rpm for 10 min. Pour off the supernatant, add 30 mL of protein purification buffer again, resuspend the bacterial cells using a vortex shaker (make sure there are no solid particles), and pour them into a 50 mL centrifuge tube. Store at -80 °C in the refrigerator.

[0044] 2. Perform high-pressure cell disruption and centrifuge to collect the supernatant.

[0045] a) Take the collected bacterial solution and use a high-pressure low-temperature cell disruptor to disrupt the cells at 4 °C and a pressure of 900 bar for 4 min to fully lyse the cells and release the target protein into the protein buffer.

[0046] b) Centrifugation: Put the disrupted bacterial solution into a pre-cooled 4 °C centrifuge and centrifuge at 8000 rpm for 60 min to obtain the precipitate and supernatant after centrifugation. Prepare the samples and collect the supernatant.

[0047] 3. Use a Ni affinity chromatography column for protein purification.

[0048] a) Purify the supernatant by Ni affinity chromatography. The specific steps are as follows:

[0049] First, wash with double-distilled water for 2 column volumes.

[0050] Then, equilibrate the Ni affinity chromatography column with one column volume of protein buffer.

[0051] b) Take 50 mL of the sample and slowly pass it through the Ni affinity chromatography column. Collect the first few drops of the passed sample.

[0052] c) Elute with protein buffer containing 20 mM, 50 mM, 100 mM, 200 mM, and 300 mM imidazole to remove the bound impurity proteins. Take the first few drops of the flow-through sample for sample preparation and detect using 12% SDS-PAGE.

[0053] d) Concentration and buffer exchange: Centrifuge and concentrate the eluate containing the target protein using a 10 kDa Amicon ultrafiltration tube (Millipore) at 4 °C and 3400 r / min to 1 mL. Then, add 10 mL of protein buffer and concentrate again to 1 mL. Repeat this step once to reduce the imidazole in the protein and obtain a purified protein sample.

[0054] 4. Protein concentration determination

[0055] We used the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to determine the protein concentration.

[0056] First, we preliminarily determined the protein concentration by measuring the absorbance of the protein at 280 nm, and diluted the protein to the range of 0.5 - 1 mg / mL according to the preliminary measurement results. Next, we prepared the reaction solution by mixing reagent A and reagent B in a ratio of 50:1.

[0057] We took 200 μL of the reaction solution in an enzyme-linked immunosorbent assay (ELISA) plate and added 25 μL of the diluted protein sample to it. By pipetting up and down to mix evenly, we incubated the reaction solution at 37 °C for 30 min.

[0058] Subsequently, we put the ELISA plate into an enzyme-labeling instrument to measure the absorbance at 562 nm, and processed the data according to the protein standard curve to determine the protein concentration.

[0059] Example 3 Enzyme Activity Assay

[0060] Each enzyme activity assay experiment 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 NPP as the substrate. We added different concentrations of purified protein (1 mg / mL) at the start of the catalytic reaction and incubated the reaction mixture at 30 °C for 15 min. The reaction was stopped 5 min after incubation and immediately quenched on ice. To promote the dephosphorylation of the product, we added 3 mL of calf intestinal alkaline phosphatase manufactured by TaKaRa and incubated it at 37 °C for 1 h. Subsequently, we added 300 mL of n-hexane and stirred the mixture for 15 min to extract monoterpenoids and performed GC-MS analysis. The GC-MS analysis was carried out using an Agilent 7890A gas chromatography system equipped with a DB-5MS capillary gas chromatography column coated with 5% diphenyl and 95% dimethyl polysiloxane (length 30 m, inner diameter 250 μm, film thickness 0.25 mm, J&W Scientific, USA). The oven temperature program was set as follows: the initial temperature was 50 °C, held for 2 min, then heated at a rate of 5 °C per minute to 180 °C (held for 5 min), and subsequently heated at a rate of 10 °C per minute to 230 °C. The temperatures of the injection port, transfer line, and ion source were controlled at 250 °C, 290 °C, and 230 °C, respectively. Only 1 mL of the sample was added in split mode, and the injection ratio was adjusted to 10:1. Finally, mass spectrometry data were obtained in full scan mode, covering the m / z range of 35 - 650, and a solvent delay of 7.5 min was set.

[0061] The GC-MS results of β-pinene produced by TPS catalysis are shown in Figure 2, where the peak time of β-pinene is around 8.2 min, and the mass spectrometry detection information is consistent with that in the database. The relative contents of β-pinene catalytically synthesized by different sequences are shown in Figure 3 , where the yield of TPS2 is the highest, reaching 14 mg / L.

[0062] As can be seen from the above embodiments, the present invention provides a bioenzyme gene capable of producing β-pinene and its application, including one or more of TPS1, TPS2, TPS3 and TPS4. The gene of the present invention can prepare a bioenzyme for highly efficient catalytic production of β-pinene. Using this bioenzyme as a catalyst can not only increase the yield of β-pinene, but also achieve an efficient, environmentally friendly and sustainable production mode. This method can become an important production route for β-pinene in the future and has broad application prospects in industrial production and other fields.

[0063] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A bioenzyme gene capable of producing β-pinene, characterized in that, including one or more of TPS1, TPS2, TPS3, and TPS4; the amino acid sequence of the said TPS1 is as shown in SEQ ID NO.1; the amino acid sequence of TPS2 is as shown in SEQ ID NO.2; the amino acid sequence of TPS3 is as shown in SEQ ID NO.3; the amino acid sequence of TPS4 is as shown in SEQ ID NO.

4.

2. Use of the gene according to claim 1 in the preparation of a bioenzyme for producing β-pinene.

3. A bio-enzyme for producing β-pinene, characterized in that, The said bioenzyme is prepared from the gene according to claim 1.

4. Use of the bioenzyme according to claim 3 in the catalysis of neryl pyrophosphate to prepare β-pinene.

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