Encoding gene, application, expression vector, engineering bacteria and preparation method of black meson enzyme

Abstract

The application relates to a coding gene of a black meson enzyme, application, an expression vector, an engineering bacterium and a preparation method. The coding gene of the black meson enzyme is a nucleotide sequence shown in SEQ ID NO: 3. The application also relates to an expression vector containing the coding gene of the black meson enzyme, an engineering bacterium and application thereof, and the application also includes a method for preparing the black meson enzyme and isothiocyanate by using the black meson enzyme. The coding gene of the black meson enzyme can effectively improve the content of the black meson enzyme, is 1.3 times as high as that of the original wild type, has high activity, and can be widely applied in the preparation of the black meson enzyme and isothiocyanate.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and fermentation engineering, specifically relating to a coding gene for mesonema, its application, expression vector, engineered bacteria, and a method for preparing mesonema. This invention also relates to a method for preparing isothiocyanate. Background Technology

[0002] Glucosinolates, sulfur-containing secondary metabolites unique to cruciferous plants, have been identified in more than 120 species. Among them are isothiocyanates [including benzyl isothiocyanate (BITC, also known as benzoyl isothiocyanate, CAS number 532-55-8) and other isothiocyanate compounds], which are chemically stable and have effects such as insect and disease resistance, characteristic flavor formation and anticancer activity.

[0003] Isothiocyanates can be obtained by the specific catalytic hydrolysis of glucosinolates by myrosinase (EC 3.2.1.147). Existing myrosinase mainly relies on extraction from cruciferous plants (such as broccoli and mustard), but this method is limited by problems such as easy enzyme inactivation during cell disruption, complex extraction process, high cost, differences in raw materials, and low expression yield of myrosinase in existing cruciferous plants. These issues limit its large-scale application and thus restrict the production of isothiocyanates.

[0004] Based on the above problems, it is necessary to find a substance and method that can increase the yield of mesonema. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem of low expression levels of black mycosesin enzyme in existing products, and to seek a gene that can increase the yield of black mycosesin enzyme, as well as an expression vector containing the gene, engineered bacteria, and applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a gene encoding mesoneminase, the sequence of which is the nucleotide sequence shown in SEQ ID NO: 3.

[0007] In a second aspect, the present invention provides an expression vector comprising the coding gene as described in the first aspect.

[0008] Thirdly, the present invention provides an engineered bacterium, the engineered bacterium comprising the expression vector as described in the second aspect.

[0009] In this invention, a further preferred embodiment is that the engineered bacteria is a Pichia pastoris engineered bacteria comprising the expression vector described in the second aspect.

[0010] Fourthly, the present invention provides an application of the coding gene as described in the first aspect, wherein the application is at least one of Y1-Y3: The application of the gene encoding Y1 in the production of mesonemazyme; The application of the gene encoding Y2 in the production of isothiocyanates; The application of the gene encoding Y3 in the catalytic hydrolysis of glucosinolates.

[0011] Fifthly, the present invention provides a method for preparing melanocyte enzyme, the method comprising the following steps: obtaining melanocyte enzyme using the encoding gene described in the first aspect.

[0012] In a further preferred embodiment of the present invention, the preparation method includes the following steps: constructing a recombinant plasmid including the coding gene described in the first aspect, then constructing an engineered Pichia pastoris strain including the recombinant plasmid, and then inducing the expression of the coding gene in the engineered Pichia pastoris strain in a culture medium to obtain a fermentation supernatant containing mesosinase, which is the final product.

[0013] Sixthly, the present invention also provides a method for preparing isothiocyanate, wherein the isothiocyanate is obtained through the following steps: S1) Preparation of melanase: Melanase is obtained using the preparation method described in the fifth aspect; S2) Hydrolysis and fermentation: The mesona sesquiterpene enzyme obtained in step S1) is added to the raw material containing glucosinolates and fermented to obtain the final product.

[0014] In this invention, a further preferred embodiment is that the isothiocyanate is benzyl isothiocyanate, the raw material containing glucosinolate is papaya seed, and the glucosinolate is benzyl glucosinolate (BG).

[0015] In this invention, a further preferred embodiment is that step S2) specifically involves: inactivating the mesona chinensis enzyme in papaya seeds, then extracting the papaya seed extract containing benzyl glucosinolates, and then adding the mesona chinensis enzyme obtained in step S1) to the papaya seed extract and fermenting it at a temperature of 10-30°C for 30-60 minutes to obtain the final product.

[0016] The technical effects of this invention are as follows: 1) This invention uses codon optimization technology to obtain a black mycosesin gene suitable for expression in Pichia pastoris. The black mycosesin produced by this gene is 1.3 times higher than that of the wild type (i.e., 2.3 times higher than that of the wild type), and has good activity. The production of black mycosesin is not limited by the season. 2) By using codon optimization technology, the black myrosinase gene is optimized and guided by the α-factor signal peptide. The enzyme solution can be directly secreted into the extracellular space, simplifying downstream purification and yielding black myrosinase with high activity. After the gene is integrated into the Pichia pastoris genome through an expression vector, the specific enzyme activity of the black myrosinase secreted and expressed in Pichia pastoris can reach 0.52±0.06 U / mg. 3) The wild-type black myrosinase gene, codon-optimized gene, expression vector, and expressed black myrosinase of the present invention can be widely used in the production of benzyl isothiocyanate and other fields.

[0017] Other features and aspects of this disclosure will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0018] The accompanying drawings, which are included in and form part of this specification, illustrate exemplary embodiments, features, and aspects of this disclosure together with the specification and serve to explain the principles of this disclosure.

[0019] Figure 1 This is a schematic diagram of the protein expression cassette of the pPICZα-MYR recombinant plasmid in Example 1 of the present invention; Figure 2 Electrophoresis images of the purified black myrosinase from Examples 2 and 3, where: M is the protein molecular weight standard; WT: wild-type gene; OP: codon-optimized gene; Figure 3 This is the BG content standard curve from Example 4; Figure 4 The figures show the HPLC detection results of BG content in different sample extraction methods and enzyme treatments in Example 4. Figure 5 This is the BITC content standard curve from Example 5; Figure 6 This is the HPLC detection and analysis chromatogram of BITC in Example 5; Figure 7 The figure shows the experimental data of the treatment of Fusarium oxysporum in soil with the fungicide prepared from black mustard enzyme and papaya seeds in Example 6. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] In a first aspect, the present invention provides a gene encoding a mesenchyme, the sequence of which is the nucleotide sequence shown in SEQ ID NO: 3 (or simply "Sequence 3"). The gene encoding the mesenchyme shown in Sequence 3 is obtained by codon optimization of the nucleotide sequence shown in SEQ ID NO: 1 (or simply "Sequence 1"). After optimization, the yield of mesenchyme expressed by the mesenchyme is increased by 1.3 times compared to the wild type (Sequence 1) (i.e., the yield is 2.3 times that of the wild type), and the obtained mesenchyme exhibits good activity, thus providing a basis for the application of mesenchyme in the production of isothiocyanates and / or the hydrolysis of glucosinolates.

[0026] The mesonema enzymes expressed by sequences 1 and 3 both have the amino acid sequence shown in SEQ ID NO: 2 (referred to as "Sequence 2"). The specific amino acid sequence of Sequence 2 is: EFVCEENEPFTCNQTKLFNSGNFEKGFIFGVASSAYQVEGGRGRGLNVWDSFTHRFPEKGGADLGNGDTTCDSYTLWQKDIDVMDELNSTGYRFSIAWSRLLPKGKRSRGVNPGAIKYYNGLIDGLVAKNMTPFVTLFHWDLPQTLQDEYNGFLNKTIVDDFKDYADLCFELFGDRVKNWITINQLYTVPTRGYALGTDAPGRCSPKIDVRCPGGNSSTEPYIVAHNQLLAHAAAVDVYRTK YKDDQKGMIGPVMITRWFLPFDHSQESKDATERAKIFFHGWFMGPLTEGKYPDIMREYVGDRLPEFSETEAALVKGSYDFLGLNYYVTQYAQNNQTIVPSDVHTALMDSRTTLTSKNATGHAPGPPFNAASYYYPKGIYY VMDYFKTTYGDPLIYVTENGFSTPGDEDFEKATADYKRIDYLCSHLCFLSKVIKEKNVNVKGYFAWSLGDNYEFCNGFTVRFGLSYVDFANITGDRDLKASGKWFQKFINVTDEDSTNQDLLRSSVSSKNRDRKSLADA.

[0027] Secondly, the present invention provides an expression vector comprising the coding gene as described in the first aspect, i.e., the expression vector comprising the nucleotide sequence shown in SEQ ID NO: 3. More preferably, the expression vector can be a plasmid; even more preferably, the expression vector is a pPICZαA plasmid.

[0028] Thirdly, the present invention provides an engineered bacterium, which includes the expression vector as described in the second aspect, i.e., the engineered bacterium includes the expression vector, and the expression vector includes the black myrosinase expression gene as described in the first aspect. In a further preferred embodiment of the present invention, the engineered bacterium is a Pichia pastoris engineered bacterium including the expression vector as described in the second aspect. Through testing, the specific enzyme activities of the wild-type (corresponding to sequence 1) and optimized (corresponding to sequence 3) black myrosinase expressed in the Pichia pastoris engineered bacterium of the present invention can reach 0.48±0.05 and 0.52±0.06 U / mg, respectively. Although the enzyme activities are not significantly different, the expression level of the optimized gene is 2.3 times that of the wild-type gene.

[0029] Fourthly, the present invention provides an application of the coding gene as described in the first aspect, wherein the application is at least one of Y1-Y3: The application of the gene encoding Y1 in the production of mesonemazyme; Y2) The application of the encoding gene in the production of isothiocyanates; isothiocyanates are a class of organic compounds with the general formula RN=C=S, which have various biological activities such as anticancer, antioxidant, and antibacterial; examples of isothiocyanates include benzyl isothiocyanate, phenethyl isothiocyanate, methyl isothiocyanate, etc.; furthermore, the isothiocyanate can be exemplified as benzyl isothiocyanate; Y3) The application of the encoding gene in the catalytic hydrolysis of glucosinolates; glucosinolates are nitrogen- and sulfur-containing secondary metabolites in cruciferous vegetables. Based on their side chain structures, they can be divided into three categories: aliphatic, aromatic, and indole. More than 200 species have been discovered. This substance is spatially isolated from myrosinase within plant cells. When the tissue is damaged, enzymatic hydrolysis occurs to produce compounds such as isothiocyanates and nitrile compounds. These compounds can be hydrolyzed under the specific catalysis of myrosinase to produce isothiocyanates and other compounds. Furthermore, the glucosinolates mentioned can be exemplified as benzyl glucosinolates.

[0030] Fifthly, the present invention provides a method for preparing melaninase, the method comprising the following steps: obtaining melaninase using the encoding gene described in the first aspect, i.e., obtaining melaninase by expressing the encoding gene through genetic engineering methods.

[0031] In a further preferred embodiment of the present invention, the preparation method comprises the following steps: constructing a recombinant plasmid comprising the coding gene described in the first aspect; then constructing an engineered Pichia pastoris strain comprising the recombinant plasmid; and then inducing the expression of the coding gene in the engineered Pichia pastoris strain in a culture medium to obtain a fermentation supernatant containing mesosinase, thereby obtaining the product. The recombinant plasmid may be the plasmid of the expression vector described in the second aspect.

[0032] Sixthly, the present invention also provides a method for preparing isothiocyanate, wherein the isothiocyanate is obtained through the following steps: S1) Preparation of melanase: Melanase is obtained using the preparation method described in the fifth aspect; S2) Hydrolysis and fermentation: The mesona sesquiterpene enzyme obtained in step S1) is added to the raw material containing glucosinolates and fermented to obtain the final product.

[0033] In a further preferred embodiment of this invention, the isothiocyanate is benzyl isothiocyanate, the raw material containing glucosinolates is papaya seeds, and the glucosinolates are benzyl glucosinolates. Papaya seeds contain a high concentration of benzyl glucosinolates, which are often a major waste product of the papaya processing industry. By using mesona chinensis enzyme to ferment them to obtain benzyl isothiocyanate, on the one hand, a high yield of mesona chinensis enzyme can be obtained based on the sequence 3 gene of this invention to improve the production efficiency of benzyl isothiocyanate; on the other hand, the amount of waste generated by the papaya processing industry can be reduced, thereby improving the efficiency of the papaya processing industry and saving waste treatment costs.

[0034] In a further preferred embodiment of this invention, step S2) specifically involves: inactivating the mesona chinensis enzyme in papaya seeds, then extracting the papaya seed extract containing benzyl glucosinolates, and then adding the mesona chinensis enzyme obtained in step S1) to the papaya seed extract and fermenting at 10-30°C for 30-60 minutes. Since the enzymes in papaya seeds react with the substrate after crushing, they cannot be stored for long periods, and this reaction interferes with enzyme activity assays when detecting exogenous mesona chinensis enzymes. To avoid the mesona chinensis enzymes in papaya seeds affecting the storage time and the accuracy of exogenous mesona chinensis enzyme activity testing, this invention employs a method of first inactivating the mesona chinensis enzymes in papaya seeds (i.e., inactivating the mesona chinensis enzymes naturally present in papaya seeds) before extraction and fermentation with the addition of exogenous mesona chinensis enzymes. Furthermore, before soaking the papaya seeds, they are pulverized to a fineness of ≥100 mesh, which improves extraction efficiency. For the extraction solution, deionized water is preferred, with a material-to-liquid ratio of 1:10. The extraction temperature is preferably a constant 70℃, and the extraction time is preferably 4 hours. For enzymatic fermentation, the temperature is further preferably 20-30℃, and the fermentation time is further preferably 40 minutes. Under these conditions, the molar conversion rate of glucosinolates is ≥90%. For inactivation treatment, the papaya seeds can be heated to 85-100℃ to destroy the activity of mesosinase. More preferably, the papaya seeds can be mixed with water and heated for inactivation.

[0035] Isothiocyanates (such as benzyl isothiocyanate) can be used as antibacterial agents, anticancer agents, and insecticidal agents; furthermore, they can be used as fungicides to treat Fusarium oxysporum in soil.

[0036] The present invention will be further illustrated by the following embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0037] Example 1: Construction of Pichia pastoris expression vector for wild-type Arabidopsis thaliana black mysinase gene 1. The Arabidopsis thaliana black myrosinase gene sequence TGG1 (GenBank: AY090382.1) was retrieved from the NCBI website, and sequence 1 (nucleotide sequence shown in SEQ ID NO: 1) was obtained through gene synthesis. 2. Based on sequence 1, design a pair of primers upstream and downstream of its ORF frame, introducing a 6 × His tag coding sequence downstream to facilitate subsequent protein purification. The amplification primers for the sequence 1 gene are designed as follows: Upstream primer P1 (SEQ ID NO.4) 5'-AGAAAAGAGAGGCTGAAGCTGAGTTTGTTTGTGAAGAGAAC-3'; Downstream primer P2 (SEQ ID NO.5): 5'-TGTCTAAGGCGAATTAATTCCTAGTGATGGTGATGGTGATGTGCATCTGCAAGACTCTTCCG-3'; The reaction system and conditions for its amplification are shown in Table 1 below and related content below: Table 1: Composition of the amplification reaction system

[0038] The amplification conditions were as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 55℃ annealing for 45 s, and 72℃ extension for 45 s for 30 cycles; 72℃ extension for 5 min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, yielding a band of approximately 1600 bp. The PCR product was recovered using a small-volume DNA recovery kit, yielding the mature peptide coding sequence of the black myrosinase gene (i.e., the nucleotide sequence obtained by amplifying the primer sequence based on the nucleotide sequence shown in SEQ ID NO: 1).

[0039] 3. The mature peptide coding sequence of the black myrosinase gene was ligated into the linearized pPICZαA plasmid containing XhoI and XbaI to obtain the recombinant plasmid pPICZα-MYR. WT Protein expression cassettes, such as Figure 1 As shown.

[0040] Example 2: Construction of a recombinant Pichia pastoris expression vector with optimized codons for the Arabidopsis thaliana black myrosinase gene. 1. The wild-type nucleotide sequence of Pichia pastoris was optimized according to its codon preference to obtain sequence 3 (nucleotide sequence shown in SEQ ID NO: 3). 2. Based on sequence 3, design a primer pair upstream and downstream of its ORF frame, introducing a 6 × His tag coding sequence downstream to facilitate subsequent protein purification. The amplification primers for the sequence 3 gene are designed as follows: Upstream primer P1 (SEQ ID NO.6, or sequence 6) 5'-AGAAAAGAGAGGCTGAAGCTGAATTTGTTTGTGAGGAAAAC-3'; Downstream primer P2 (SEQ ID NO.7, or sequence 7): 5'-TGTCTAAGGCGAATTAATTCCTAGTGATGGTGATGGTGATGAGCATCAGCCAAAGATTTC-3'; The gene sequence 3 was amplified using the same amplification reaction system as in Example 1 (but with the upstream and downstream primers replaced by sequences 6 and 7, respectively, while keeping the rest unchanged) and the same amplification conditions as in Example 1. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, yielding a band of approximately 1600 bp. The PCR product was recovered using a small-volume DNA recovery kit, yielding the amplified sequence of the black myrosinase gene (i.e., the nucleotide sequence obtained by amplifying the primer sequence based on the nucleotide sequence shown in SEQ ID NO: 3).

[0041] 3. The amplified black myrosinase gene sequence was ligated into the linearized XhoI and XbaI pPICZαA plasmid to obtain the recombinant plasmid pPICZα-MYR. OP Protein expression cassettes, such as Figure 1 As shown.

[0042] Example 3: Construction of recombinant strain of black myrosinase and protein purification Recombinant plasmid pPICZα-MYR WT or pPICZα-MYR op After linearization with SacI, GS115 was chemically transformed, and Zeocin™-resistant single colonies were screened. Positive strains GS115 / pPICZα-MYRwt or GS115 / pPICZα-MYR were verified by PCR. op Positive strains were cultured in BMGY medium at 28°C for 24 h, then transferred to BMMY medium and induced with 0.5% methanol every 24 h. After 96 h, the supernatant was collected by centrifugation at 20,000 × g for 30 minutes.

[0043] The supernatant was mixed with three volumes of 20 mM phosphate buffer, and the protein in the fermentation supernatant was purified using the affinity of a nickel ion affinity column and the 6×His tag at the protein terminus. Protein concentration was determined using a BCA protein assay kit, and protein purity was assessed using SDS-PAGE. Figure 2As shown, a band of approximately 60 kDa was visible on the SDS-PAGE gel. Calculations showed that the protein expression level of the codon-optimized gene in GS115 was approximately 2.3 times that of the wild-type gene, demonstrating that codon optimization helps improve the gene's expression level in the host bacterium. After 96 h of methanol induction, the target protein expression level reached 11.3 mg / L.

[0044] Example 4: Extraction of benzyl glucosinolates from papaya seeds 50g of fresh papaya seeds were placed in 50mL of water and boiled for 10min to inactivate myrosinase. After drying the seeds at 50℃, they were crushed and sieved, then added to 500mL of boiling water and extracted with magnetic stirring for 10min. Subsequently, they were incubated in a 70℃ constant temperature water bath for 4h. After cooling, the supernatant of the upper aqueous phase was collected, which was the crude BG extract of the heat-treated group.

[0045] As a control, 50g of fresh papaya seeds were dried at 50℃, then crushed and sieved, and added to 400mL of boiling water. Extraction was carried out with magnetic stirring for 10min, followed by incubation in a 70℃ constant temperature water bath for 4h. After cooling, the supernatant of the upper aqueous phase was taken as the BG crude extract control group.

[0046] Take 20 mL of the crude BG extract from the heat-treated group, add 1 U of recombinant black mycosesin, and react at room temperature for 3 h to obtain the recombinant enzyme treatment group.

[0047] Preparation of standard curve: Prepare glucosinolate standards of different concentrations (0.05-1 mg / mL) and dissolve them in water.

[0048] A high-performance liquid chromatography (HPLC) system equipped with a Luna C18 column (Phenomenex, 250 mm × 4.6 mm, 5 µm) was used, with the column temperature set at 30°C. The mobile phase composition was: Phase A, 0.1% (v / v) trifluoroacetic acid (TFA) aqueous solution; Phase B, 0.1% (v / v) trifluoroacetic acid methanol solution. The elution gradient was as follows: 0–10 min, 100% A; 10–15 min, 80% A, 20% B; 15–25 min, 50% A, 50% B; 25–35 min, 100% B; 35–45 min, maintain 100% B; then switch back to 100% A at 45–55 min and equilibrate to the end. The flow rate was 1.0 mL / min. -1 The absorbance was measured at a wavelength of 227 nm using a Shimadzu UV-Vis detector, and the standard curve is shown below. Figure 3 As shown.

[0049] Testing revealed that the heat-treated group had the highest BG content in its crude BG extract, while the control group showed a decrease. This is likely because the enzyme in the papaya seeds reacted with the substrate during the crushing process, leading to a reduced yield. The recombinant enzyme reacted with the heat-treated group's crude BG extract, degrading the substrate; only a small peak, similar in size to the standard, was observed on the HPLC chromatogram. Figure 4 As shown in the figure, the content of benzyl glucosinolates in papaya seeds was approximately 36 μmole / g dry weight.

[0050] Example 5: Determination of benzyl isothiocyanate (BITC) 1. Determination of black mustard enzyme activity Principle Overview: Black myrosinase catalyzes the formation of benzyl glucosinolate (BITC) from papaya seeds; the reagent benzo-1,2-dithiol (BDT) undergoes a cyclization reaction with benzyl isothicyanate to generate 1,3-benzodithiole-2-thione, a product detectable by absorbance and reversed-phase high-performance liquid chromatography (RP-HPLC).

[0051] 2. Preparation and analysis of standards and reagents (1) Potassium phosphate buffer (10 mM, pH 8.5): Weigh 0.174 g K2HPO4, dissolve it in ultrapure water and bring the volume up to 100 mL.

[0052] (2) BITC standard stock solution: Accurately transfer 1 µL of pure BITC, dilute to 10 mL with methanol, vortex to mix, and obtain a stock solution with a concentration of 0.754 mM.

[0053] (3) BDT reagent solution: Accurately transfer 4.59 µL of BDT and dilute to 10 mL with methanol to prepare BDT solution.

[0054] (4) Sample pretreatment and derivatization reaction Take the sample containing BITC and add it to a glass sample vial with a PTFE-lined screw cap. Add 10 mM potassium phosphate buffer (pH 8.5) to bring the volume to 1 mL, and then add 1 mL of 4 mM benzene-1,2-dithiol (BDT) reagent solution.

[0055] (5) Sample preparation and reaction Place the sample to be tested (containing BITC) into a glass bottle with a Teflon-lined screw cap. Add 10 mM potassium phosphate buffer (pH 8.5) and bring the volume to 1 mL. Add 1 mL of 4 mM BDT solution to prepare for the reaction.

[0056] (6) Sample derivatization and HPLC analysis The sample pretreatment procedure follows the standard curve preparation method: accurately transfer an appropriate amount of sample, add 10 mM potassium phosphate buffer (pH 8.5) to a final volume of 1 mL, and then add 1 mL of 4 mM BDT reagent solution.

[0057] Example: 50 µL of oil sample (may contain BITC) + 950 µL of phosphate buffer + 1 mL of BDT solution, for a final volume of 2 mL. While preparing the sample, preheat or microwave the water bath to 60°C. After adding the sample and standards, tighten the cap, vortex to mix, and place in the 60°C water bath for reaction. Ensure the sample vial is completely submerged and does not float. The reaction should continue for 3 hours, checking the water level and manually shaking gently once per hour. After the reaction is complete, remove the sample vial and allow it to cool to room temperature.

[0058] Precipitation may occur in most samples (including blank controls), requiring filtration of all standards and samples using a syringe filter fitted with a 0.20 µm PES membrane. After filtering each sample, the syringe and needle must be rinsed with ultrapure water at least three times to prevent cross-contamination.

[0059] Chromatographic separation was performed on a Shimadzu Premier C18 column (5 µm, 120 Å, 25 cm × 4.6 mm) with a mobile phase of 80% methanol (HPLC grade) and 20% water (HPLC grade) at an elution flow rate of 1 mL / min. -1 The absorbance was measured at 365 nm using a Shimadzu UV-Vis detector. The BITC content standard curve is shown below. Figure 5 As shown, the HPLC peak chromatogram of 1,3-benzodithiole-2-thione is as follows. Figure 6 As shown in the figure. Testing revealed that the hydrolysis conversion rate of glucosinolates in papaya seeds can reach over 90%.

[0060] Table 2: BITC Standard Curve Development

[0061] Example 6: Preparation of fungicide from black mustard enzyme and papaya seeds for treating Fusarium oxysporum in soil (… Fusarium oxysporum ) The core principle of using black myrosinase and papaya seeds to prepare a bio-fungicide for treating Fusarium oxysporum in soil is based on the "thioglucosinolate-black myrosinase" bio-fumigation system to produce active isothiocyanates, which destroy the cell structure and metabolism of the pathogen, thereby achieving antibacterial effect.

[0062] Take 200g of fresh papaya seeds, mix with an equal volume of water, heat at 90°C to inactivate the myrosinase, remove the water, dry, and pulverize to prepare papaya seed powder. Mix the papaya seed powder and myrosinase powder in a certain proportion to prepare a fungicide. Mix the fungicide with soil in different proportions. Inoculate the soil with Fusarium oxysporum spores at a rate of 1×10⁷ CFU / g and mix thoroughly. Add half the volume of water to the soil to completely moisten it. Treat at room temperature for 3 days. Take an equal volume of soil sample (5g) and resuspend it in 5 times the volume of sterile water. Take an equal volume of soil suspension to prepare fungal suspensions of different gradients and spread them evenly on PDA medium. After culturing for 2 days, count the colonies and calculate the fungicide effect. Figure 7 As shown, the number of spores in the soil gradually decreases with the increase of fungicide content, while an addition of 1% can kill almost all Fusarium oxysporum spores in the soil.

[0063] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0064] Unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning understood by one of ordinary skill in the art to which this application pertains. The terms "a," "an," "the," and similar words used in this application do not indicate quantity limitation and may indicate singular or plural. The terms "comprising," "including," "having," and any variations thereof used in this application are intended to cover non-exclusive inclusion. The terms "connected," "linked," "coupled," and similar words used in this application are not limited to physical or mechanical connections but include electrical connections, whether direct or indirect. The term "multiple" used in this application refers to two or more, and "and / or" describes the relationship between related objects, indicating that three relationships may exist. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. The terms "first," "second," "third," etc. used in this application are merely to distinguish similar objects and do not represent a specific ordering of objects.

[0065] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications or improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A gene encoding a mesoneminase, characterized in that, The sequence of the gene is the nucleotide sequence shown in SEQ ID NO:

3.

2. An expression vector for the gene as described in claim 1, characterized in that, The expression vector includes the coding gene as described in claim 1.

3. An engineered bacterium, characterized in that, The engineered bacteria include the expression vector as described in claim 2.

4. The engineered bacteria according to claim 3, characterized in that, The engineered strain is a Pichia pastoris engineered strain including the expression vector as described in claim 2.

5. An application of the gene encoding according to claim 1, characterized in that, The application is at least one of Y1-Y3: The application of the gene encoding Y1 in the production of mesonemazyme; The application of the gene encoding Y2 in the production of isothiocyanates; The application of the gene encoding Y3 in the catalytic hydrolysis of glucosinolates.

6. A method for preparing mesoneminase, characterized in that... The preparation method includes the following steps: obtaining mesoneminase using the encoding gene described in claim 1.

7. The method for preparing mesonetinase according to claim 6, characterized in that... The preparation method includes the following steps: constructing a recombinant plasmid containing the encoding gene of claim 1, then constructing an engineered Pichia pastoris strain containing the recombinant plasmid, and then inducing the expression of the encoding gene in the engineered Pichia pastoris strain in a culture medium to obtain a fermentation supernatant containing mesosinase, which is the final product.

8. A method for preparing isothiocyanate, characterized in that, The isothiocyanate is prepared by the following steps: S1) Preparation of melanase: Melanase is obtained using the preparation method described in claim 6 or 7; S2) Hydrolysis and fermentation: The mesona sesquiterpene enzyme obtained in step S1) is added to the raw material containing glucosinolates and fermented to obtain the final product.

9. The method for preparing isothiocyanate according to claim 8, characterized in that, The isothiocyanate is benzyl isothiocyanate, the raw material containing glucosinolate is papaya seed, and the glucosinolate is benzyl glucosinolate.

10. The method for preparing isothiocyanate according to claim 9, characterized in that... Step S2) specifically involves: inactivating the mesona chinensis enzyme in papaya seeds, then extracting the papaya seed extract containing benzyl glucosinolates, and then adding the mesona chinensis enzyme obtained in step S1) to the papaya seed extract and fermenting it at 10-30℃ for 30-60 minutes to obtain the final product.

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