Biological enzyme gene capable of producing alpha-pinene and application thereof

Abstract

This invention relates to the field of bioenzyme technology. It provides a bioenzyme gene capable of producing α-pinene and its applications, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5, TPS6, and TPS7. The bioenzyme gene of this invention can prepare bioenzymes with high catalytic activity, which can directly catalyze the one-step production of α-pinene from inexpensive substrates, avoiding the extraction process from plants. The operation is simple, the reaction conditions are mild, and the yield is high. This achieves a green and sustainable α-pinene synthesis process, effectively meeting market demand. It not only increases yield and reduces costs but is also environmentally friendly and sustainable, possessing significant application prospects and greatly promoting the development of α-pinene-related industries.

Description

Technical Field

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

[0002] α-Pinene is a colorless liquid compound with the chemical formula C. 10 H 16 α-Pinene belongs to the monoterpene class of compounds. It is found in natural matrices of citrus fruits, herbs, and other plants, and is an important source of aroma. α-Pinene is widely used in the synthesis of fragrances and plasticizers, and has significant industrial applications. Its unique trans structure gives it high processing value; however, traditional extraction and chemical synthesis methods suffer from low yields and high costs.

[0003] Currently, α-pinene is mainly obtained through the extraction of plant essential oils, which suffers from limited sources and low efficiency. Microbial fermentation synthesis of α-pinene could achieve large-scale, low-cost supply. However, existing methods for microbial synthesis of α-pinene have low yields, making industrial application difficult. Summary of the Invention

[0004] The purpose of this invention is to provide a bio-enzyme gene that can produce α-pinene and its application, which increases yield, reduces cost, and is environmentally friendly and sustainable, and has important application prospects.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a bioenzyme gene capable of producing α-pinene, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5, TPS6 and TPS7;

[0007] The amino acid sequence of TPS1 is shown in SEQ ID NO.1;

[0008] The amino acid sequence of TPS2 is shown in SEQ ID NO.2;

[0009] The amino acid sequence of TPS3 is shown in SEQ ID NO.3;

[0010] The amino acid sequence of TPS4 is shown in SEQ ID NO.4;

[0011] The amino acid sequence of TPS5 is shown in SEQ ID NO.5;

[0012] The amino acid sequence of TPS6 is shown in SEQ ID NO.6;

[0013] The amino acid sequence of TPS7 is shown in SEQ ID NO.7.

[0014] This invention also provides the application of the aforementioned gene in the preparation of bioenzymes that produce α-pinene.

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

[0016] This invention also provides the application of the aforementioned bioenzyme in the catalytic preparation of α-pinene from nerolithic pyrophosphate.

[0017] This invention provides a bio-enzyme gene capable of producing α-pinene and its applications, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5, TPS6, and TPS7. The bio-enzyme gene of this invention can prepare bio-enzymes with high catalytic activity, which can directly catalyze the one-step production of α-pinene from inexpensive substrates, avoiding the process of extracting from plants. The operation is simple, the reaction conditions are mild, and the yield is high. It realizes a green and sustainable α-pinene synthesis process, which can effectively meet market demand. It not only increases yield and reduces costs but is also environmentally friendly and sustainable, possessing significant application prospects and greatly promoting the development of α-pinene-related industries. Attached Figure Description

[0018] Figure 1 This represents the biosynthetic pathway of α-pinene;

[0019] Figure 2 The image shows the gas phase detection of pinene, where a is the standard for α-pinene, with a gas phase peak at around 11.3 min, and b is the peak of α-pinene in the sample, with a position at 11.3 min, consistent with the standard.

[0020] Figure 3 The relative yields of α-pinene produced by different TPS catalysis. Detailed Implementation

[0021] This invention provides a bioenzyme gene capable of producing α-pinene, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5, TPS6 and TPS7;

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

[0023] MKMSAFTSMALPLQASLPVTAISTRLIFPSSCTKRYVPRRVTALKLSNELTMRRSAYYKPPIWSFEYIQSLKLEYVGGESFKRHINKLKEDVIAMLEGEEMDKDPSHQLELIDTLQRLGLSYHFENEINRILKKVYTKHQGYYGLERHSLYVAALEFRILRQHGYKVPQEIFKSFLNERGNFKPCLKNDCKGMLFLYEASFLSLEGESILDAARTFARNYLSEYVKLNETKDPYLSTLVEHALEFPLHWRMPRMEARWFIEVYKQSPDMNPVLLDLAKLDFNMVQATYQEDLKDASRWWNKSGLGQNLEFIRDRLVENFLWTTGVLFQPQYAYYRRMATQVNALLTTIDDVYDVYGTLDELELFTDVIERWDINAIEQLPDYMKLCFFAVHNSMNQIASDIFQEQGINILPYSKKAWLDLCKSYLIEAKWYHQGYKLSLHEYIDNAVISIAAPLMLIHAYILSSNHITTEVLQYLEEELPNIIRCSSMVLRLADDLGTSPDEMRRGDVPKSIQCYMHETGASEENAREYIQDLIDKTWNKMNKDQFEHSPLPQTLIEAAMNLARMAQFMYKHGDGHSSQDDVMRHSVLSLLINPIPLPAPEESHITA;

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

[0025] MPQHRVTALKLSNELTKRRSAGYKPPIWSFEQIQSLKLEYVEEESFKRHINKLKEDVIAMLEGKEMDKDPSHQLELIDTLQRLGLSYHFENEINIILEKAYTKHQGYYGLEKDSLYVAALEFRILRQHGYKVPQEIFKSFLNERGNFKAHLKQDCKGMLFLYEASFLSLEGETTLVAARTFARNYLNEYVKLNESKDPYLSTLVEHALEFPLHWRMRRMETRWFIEAYQRSPDMNPILLDLAKLDFNMVQATYQEDLKDASRRMATQVNALITTIDDVYDVYGTLDELELFTDAIERWDINAIEQLPDYMKLCFFAVHNSMNQIASDIFQEQGINILPYSKKAWLDLCKTYLIEAKWYHQGYTPSLHEYIDNAVISISAPLILIHGYILSSNNITIEVLKYLEEELPNIIRCSSMVLRLTDDLGTSPDEMRRGDVPKSIQCYMHETRASEEDAREYIQDLIDKTWNKMNKDQFEHSPLPQTLIEAAMNLARMAQFMYKHGDGHSSQDDVMRHRVLSLLINPIPLPGLEESHITA;

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

[0027] MRRSANYKPPIWSFEYIQSLKLEYVEGESFKRHINKLKEDVIAMLEGEEMDKDPSHQLELIDTLQRLGLSYHFENEINRILEKVYTKHQGYYGLERDSLYMAALEFRILRQHGYKVPQEIFKSFLNERGNFKPCLKNDCKGMLFLYEASFLSLEGESTLDAARTFARNYLSEYVKLNETKDPYLSTLVEHALEFPLHWRMPRVEARWFIEVYQQSPNMNPVLLDLAKLDFNMVQATYQEDLKDASRWWKRSGLGQNLEFIRDRLVENFLWTAGVLFQPQYAYFRRMATQVAALVTTIDDVYDVYGTLDELELFTDAIERWDINAIEQLPDYMKLCFFAVHNSMNQIALDIFQEQGINILPYSKKAWLDLCKTYLIEAKWYHRGYKPSLHEYIENAVVSIAAPLVLLHAYILSSNHITAEVLQYLEEELPNIIRCSSMVMRLADDLGTSPDEMRRGDVPKSIQCYMHETGASEEIAREYIQDLIDKTWNKMNKDQFEHSPLPQTLIEAAMNLARMAQFIYKHGDGHSSQDDVMRHRVLSLLINPIPLPGPEES;

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

[0029] MRRSADYKPPIWSFEYIQSLKLEYVEGESFKRHINKLKEDVIAMLEGEEMDKDPSHQLELIDTLQRLGLSYHFENEINRILEKVYTKHQGYYGLERDSLYVAALEFRILRQHGYKVPQEIFKSFLNERGNFKAPLKRDCKEMLFLYEASFLSLEGESTLNVARTFARNYLSEYVKLKESKDPYLSTLVEHALEFPLHWRMPRMETRWFIEFYQQGPNMNPVLLDLAKLDFNMVQATYQEDLKDASRWWKRSGLGQNLNFIRDRLMENFLWTVGILFQPQYAYFRRMITQVAALGTTIDDVYDVYGTLDELELFTDAIERWDINAIEELPDYMKLCFFAVHNSMNQIVSDIFQEQGINILPYSKKAWLNLCKSYLIEAKWYHQGYTPSLHEYIDNAVVSIVGPLALLHAYICSSNHITSEVLQYLVEELPNIIRCSSMIMRLADDLGTSPDEMRRGDVPKSIQCYMHETGASEEIAREYIQDLIDKTWNKMNKDQFEHSPLPQTLIEAAMNIARMAQFMYKHGDGFSSQDDVMRHRVLSLLINPIPSPGPEESHTTA;

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

[0031] MQICHINVYPVKMSPFTSMALPLQASVPVAAISTRLPFPSSCTKRYVPSRVTELKLSNELTMRRSADYKPPIWSFEDIQSLKVDYVEESFGRRINKLKEDVILMLEEKEVDKVPLQQLELIDTLQRLGLSYHFENEIDRILEKVYTNNQGYCYGFDRESLYVAALEFRILRQHGYKVPQEIFKSFLNESGNFKACLNKDCKGMLYLYEASFLSLEGESTLDAARTFARNYLSEYVKLNESKDPYLSTLVEHALEFPLRWRMPRMEARWFVEVYQRCPDMNPLLLDLAKLDFNMVQAMYQEDLKHASMWWKRTGLGQNLGFIRDRLMENFLWTIGELFQPQYGYFRRMAAQVNALVTTIDDVYDVYGTLDELEHFTDAIERWDINAIEQLPDYMKLCFFALHNSMNQIASDIFQKQGINILPYSKKAWLDLCKTYLIEAKWYHQGYTPSLQEYIDVAVISISAPLILLHAYILSSSHIKIEVLQYLEEELPSIIRCSSMVLRLADDLGTSSDEMRRGDVSKSIQCTIYETGVSEDDAREYIQDLIDKTWKKMNKYEFEPSLLPQTLIEAAINLARMAQFMYKHGDGHSSQDDVMRHRILSLLINPIALPRREESYITA;

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

[0033] MSAFTSMALPLQAFIPSQVTTLKSSNELTMRRSADYKPSIWSFEYIQSLKLEYVEEESFRRHVNKLKEDVIAMLEGEEMDKDPSHQLELIDTLRRLGLSYHFENEINKILEKVYTKHQGYYGLERDNLYMAALEFRILRQHGYKVSQEIFKSFVNERGNFKALLKRNCKGMLFLYEASFLSLEGESTLNAARTFMRNYLSEYVKLKESKDPYLSTLVEHALEFPLHWRMPRMETRWFIEVYQQSPDMNPVLLDLAKLDFNMVQATYQEDLKDASRWWKRSGLGQNLNFIRDRLMENFLWSTGILFQPQYAYFRRMITQVGALGTTIDDVYDVYGTLDELELFTDAIERWDINAIEELPDYMKLCFFAVHNSMNQIVSDIFQEQGINILPYSKKAWLNLCKSYLIEAKWYHQGYTPSLHEYIDNAVVSIVGPLILLHAYILSSNHITTEVLQYLEEELPNIIRCSSMVLRLADDLGTSPDEMRRGDVPKSIQCYMHETGASEEIAREYIQDLIDETWNKMNKDQFEHSTLPQTLIEAAMNLARMAQFMYKHGDGHSSQDDVMRHRVLSLLINPIPLPGPEESHITA;

[0034] The amino acid sequence of TPS7 is shown in SEQ ID NO.7:

[0035] .

[0036] This invention also provides the application of the aforementioned gene in the preparation of bioenzymes that produce α-pinene.

[0037] The present invention also provides a bioenzyme for producing α-pinene, said bioenzyme being prepared from the aforementioned gene.

[0038] This invention also provides the application of the aforementioned bioenzyme in the catalytic preparation of α-pinene from nerolithic pyrophosphate.

[0039] 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.

[0040] Example 1: Obtaining a Highly Active Bioenzyme Gene

[0041] 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.

[0042] 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.

[0043] The linearized vector was purified from the gel using an agarose extraction kit. The purified vector was then ligated with an enzyme gene fragment to construct the recombinant expression vector pCold-TPS.

[0044] Example 2 Protein Expression and Purification

[0045] 1. Using Escherichia coli BL21(DE3) strain, the expression of all TPS proteins was successfully achieved.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] d) Reduce the temperature of the shaker to 16°C, add isopropyl thio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM, and induce expression for 15 h.

[0050] 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.

[0051] 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.

[0052] 2. Perform high-pressure crushing and centrifuge to collect the supernatant.

[0053] a) Take the collected bacterial culture and use a high-pressure cryogenic disruptor to disrupt the bacteria at 4°C and 900 bar for 5 minutes to fully lyse the cells and release the target protein into the protein buffer.

[0054] 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.

[0055] 3. Protein purification was performed using a Ni affinity chromatography column.

[0056] a) The supernatant was purified by nickel affinity chromatography, and the specific steps are as follows:

[0057] First, wash twice the column volume with double-distilled water.

[0058] Then, equilibrate the Ni affinity chromatography column with protein buffer at a ratio of one.

[0059] b) Take 50 mL of sample and slowly pass it through the Ni affinity chromatography column, collecting the first few drops of sample that flow through.

[0060] 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.

[0061] 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.

[0062] 4. Protein concentration determination

[0063] We used the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to determine protein concentration.

[0064] 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.

[0065] 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 min.

[0066] 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.

[0067] Example 3 Enzyme Activity Assay

[0068] 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 NPP 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 analysis. GC 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% dimethyl polysiloxane (30 m long, 250 μm 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 the injection ratio adjusted to 10:1.

[0069] GC-MS results for TPS-catalyzed production of α-pinene are shown in [Figure Number]. Figure 2 The peak elution time of α-pinene was approximately 11.3 min, consistent with that of the standard. The relative contents of α-pinene synthesized by different sequences are shown in [reference needed]. Figure 3 Among them, TPS1 has the highest yield, reaching 35.0 mg / L.

[0070] As shown in the above embodiments, this invention provides a bio-enzyme gene capable of producing α-pinene and its applications, including one or more of TPS1, TPS2, TPS3, TPS4, TPS5, TPS6, and TPS7. The bio-enzyme gene of this invention can prepare bio-enzymes with high catalytic activity. These enzymes can directly catalyze the one-step production of α-pinene from inexpensive substrates, avoiding the process of extracting from plants. The operation is simple, the reaction conditions are mild, and the yield is high. It realizes a green and sustainable α-pinene synthesis process, which can effectively meet market demand. It not only increases yield and reduces costs but is also environmentally friendly and sustainable, possessing significant application prospects and greatly promoting the development of α-pinene-related industries.

[0071] 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. Use of a biological enzyme capable of producing α-pinene in the catalytic production of α-pinene from neryl pyrophosphate, characterized in that, The biological enzyme is TPS1; the amino acid sequence of the TPS1 is shown as SEQ ID NO.

1. The biological enzyme is TPS1; the amino acid sequence of the TPS1 is shown as SEQ ID NO. 1.

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