Gossypol ring-opening enzyme as well as DNA, recombinant plasmid, recombinant strain and application thereof

Abstract

The invention discloses gossypol ring-opening enzyme as well as DNA, recombinant plasmid, recombinant strain and application thereof, and relates to the technical field of microorganisms, the amino acid sequence of the gossypol ring-opening enzyme is shown as SEQ ID No.1, the gossypol ring-opening enzyme recombinant expression strain can secrete the gossypol ring-opening enzyme, and the gossypol ring-opening enzyme can secrete the gossypol ring-opening enzyme. The gossypol ring-opening enzyme is further degraded by opening an aromatic ring of gossypol and / or a gossypol derivative, the toxicity of the gossypol and / or the gossypol derivative subjected to ring opening is reduced, and the product subjected to ring opening is easier to degrade, so that the gossypol ring-opening enzyme and the recombinant expression strain thereof are suitable for detoxification of cottonseed meal.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a cottonpol ring-opening enzyme and its DNA, recombinant plasmid, recombinant strain and application. Background Art

[0002] Cottonpol, chemical name is 1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-(2,2'-binaphthyl)-8,8'-dicarbonaldehyde, its molecular formula is C 30 H 30 O8. Cottonpol can be divided into free cottonpol (FG) and bound cottonpol (BG) according to its existence form. The sum of the two is called total cottonpol. Bound cottonpol is formed by the combination of free cottonpol with proteins and sugars in cotton kernels during high-temperature steaming and frying. It is not absorbed in the digestive system and has very low toxicity. FG has active aldehyde groups and active hydroxyl groups and is highly toxic. If its content exceeds the safety limit, it will lead to growth retardation, poisoning, decreased reproduction rate and death of livestock and poultry.

[0003] Existing methods for removing free cottonpol from cottonseed meal include physical methods such as heat treatment and mechanical pressing, chemical methods such as ferrous sulfate treatment and calcium hydroxide treatment, and organic solvent leaching.

[0004] However, the traditional physical extrusion detoxification method has a low detoxification rate; the chemical method of alkali treatment has high requirements for the corrosion resistance of the equipment and will reduce the palatability of the feed; the organic solvent leaching method will cause solvent residues and other disadvantages. Summary of the invention

[0005] The main purpose of the invention is to provide a cottonpol ring-opening enzyme and its DNA, recombinant plasmid, recombinant strain and application, aiming to solve the problem of high toxicity of free cottonpol in cottonseed meal in the prior art.

[0006] To achieve the above object, the present invention provides a cottonypol ring-opener, the amino acid sequence of the cottonypol ring-opener is shown in SEQ ID No.1.

[0007] The invention provides a DNA molecule, wherein the DNA molecule is used for encoding the aforementioned cottonpol ring-opening enzyme.

[0008] In one embodiment, the nucleotide sequence of the DNA molecule is shown as SEQ ID No.2.

[0009] The present invention provides a recombinant plasmid, which comprises the aforementioned DNA molecule.

[0010] The present invention provides a recombinant strain, which comprises the nucleotide sequence of the aforementioned DNA molecule or the aforementioned recombinant plasmid.

[0011] In one embodiment, the host cell source of the recombinant strain includes Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae.

[0012] The present invention provides an application of the aforementioned recombinant strain in degrading gossypol and / or gossypol derivatives;

[0013] The gossypol derivatives include gossypol acid.

[0014] In one embodiment, the steps include:

[0015] preparing a resting cell suspension, adding a reaction substrate to the resting cell suspension, performing a biotransformation reaction, and obtaining a degradation product;

[0016] The reaction substrate includes gossypol and / or a gossypol derivative.

[0017] In one embodiment, the amount of the reaction substrate added is 0.8-2 mM.

[0018] In one embodiment, the temperature of the bioconversion reaction is 28-37° C., and the pH of the bioconversion reaction is 6.0-8.0.

[0019] In the technical scheme of the present invention, the amino acid sequence of the cottonypol ring-opener is shown in SEQ ID No. 1, and the cottonypol ring-opener recombinant expression strain can secrete the cottonypol ring-opener, and the cottonypol ring-opener further degrades cottonypol and / or cottonypol derivatives by opening their aromatic rings, and the toxicity of the products of cottonypol and / or cottonypol derivatives after ring opening is reduced, and the products after ring opening are more easily degraded, so the cottonypol ring-opener and the recombinant expression strain thereof are suitable for detoxifying cottonseed meal. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0021] Figure 1 This is a graph showing the result of agarose gel electrophoresis detection of PCR amplification of pET28a in Example 1 provided by the present invention;

[0022] Figure 2 The figure is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis) of the purified cottonpol ring-opening enzyme in Example 2 provided by the present invention;

[0023] Figure 3This is a graph showing the results of 48h biotransformation of the recombinant strain, blank control (no strain) and BL21 (DE3) (pET28a) whole cells in Example 3 provided by the present invention;

[0024] Figure 4 The high performance liquid chromatography (HPLC) diagram of the degradation product of the blank control group in Example 3 provided by the present invention;

[0025] Figure 5 This is a HPLC chart of the detected substances of the empty vector control group BL21 (DE3) (pET28a) in Example 3 provided by the present invention;

[0026] Figure 6 This is a HPLC chart of the degradation products of the recombinant strain BL21 (DE3) (pET28a-orf4476) group in Example 3 provided by the present invention;

[0027] Figure 7 The mass spectrum of cottonpol in the blank control group in Example 3 provided by the present invention;

[0028] Figure 8 The mass spectrum of gossypol acid in the blank control group in Example 3 provided by the present invention;

[0029] Fig. 9 This is a graph showing the substance information with a retention time of 5.762 min in the recombinant strain group in Example 3 provided by the present invention;

[0030] Fig.10 This is a graph showing the substance information with a retention time of 5.378 min in the recombinant strain group in Example 3 provided by the present invention;

[0031] Fig.11 This is a diagram for inferring the action principle of the gossypol ring-opening enzyme in Example 3 provided by the present invention.

[0032] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0033] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0034] Cottonpol, chemical name is 1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-(2,2'-binaphthyl)-8,8'-dicarbonaldehyde, its molecular formula is C 30 H 30 O8. Cottonpol can be divided into free cottonpol (FG) and bound cottonpol (BG) according to its existence form. The sum of the two is called total cottonpol. Bound cottonpol is formed by the combination of free cottonpol with proteins and sugars in cotton kernels during high-temperature steaming and frying. It is not absorbed in the digestive system and has very low toxicity. FG has active aldehyde groups and active hydroxyl groups and is highly toxic. If its content exceeds the safety limit, it will lead to growth retardation, poisoning, decreased reproduction rate and death of livestock and poultry.

[0035] Existing methods for removing free cottonpol from cottonseed meal include physical methods such as heat treatment and mechanical pressing, chemical methods such as ferrous sulfate treatment and calcium hydroxide treatment, and organic solvent leaching.

[0036] However, the traditional physical extrusion detoxification method has a low detoxification rate; the chemical method of alkali treatment has high requirements for the corrosion resistance of the equipment and will reduce the palatability of the feed; the organic solvent leaching method will cause solvent residues and other disadvantages.

[0037] In view of this, the present invention provides a cottonypol ring-opener, the amino acid sequence of the cottonypol ring-opener is shown in SEQ ID No.1.

[0038] SEQ ID No.1:

[0039] MLGLKRVHHIAIIATNYAQSKAFYCDILGFTLQSEFYREERDSWKGDLALNGEYVIELFSFPFPPARPSRPEACGLRHLAFSVDDIDAAVAHLEKHGVACEGIRIDPFTGKRFTFFNDPDGLPLEIYQQ.

[0040] In the technical scheme of the present invention, the amino acid sequence of the cottonyphenol ring-opener is shown in SEQ ID No. 1, and the cottonyphenol ring-opener recombinant expression strain can express the cottonyphenol ring-opener. The cottonyphenol ring-opener can cleave and open the aromatic ring of cottonyphenol and / or cottonyphenol derivatives to degrade them. The toxicity of the products of cottonyphenol and / or cottonyphenol derivatives after ring opening is reduced, and the products after ring opening are more easily degraded. Therefore, the cottonyphenol ring-opener and the recombinant expression strain thereof are suitable for detoxifying cottonseed meal.

[0041] The invention provides a DNA molecule, wherein the DNA molecule is used for encoding the aforementioned cottonpol ring-opening enzyme.

[0042] In some embodiments of the present invention, the nucleotide sequence of the DNA molecule is shown as SEQ ID No.2.

[0043] SEQ ID No.2:

[0044] ATGCTGGGTTTTAAAACGGGTTCACCATATTGCTATCATCGCGACCAATTATGCGCAGAGCAAAGCCTTTTACTGCGATATTCTGGGCTTTACGTTGCAGAGTGAGTTTTATCGTGAAGAGCGCGACTCGTGGAAGGGCGATCTGGCATTGAACGGCGAGTATGTCATTGAACTGTTCTCTTTCCCGTTCCCTCCA GCTCGTCCGAGCCGTCCGGAAGCCTGTGGGCTGCGTCATCTGGCCTTTAGCGTGGATGATTTGACGCGGCGTGGCTCATCTGGAAAAGCATGGCGTGGCCTGTGAGGGAATTCGCATCGATCCTTTTACCGGCAAACGCTTCACCTTTTTTAACGATCCCGACGGCCTGCCGCTGGAGATTTATCAGCAGTAA.

[0045] The present invention provides a recombinant plasmid, which comprises the aforementioned DNA molecule. The recombinant plasmid can express cottonypol ring-opener in microbial cells relatively quickly, and the expressed cottonypol ring-opener has high activity in degrading cottonypol and its derivatives.

[0046] The present invention provides a recombinant strain, the recombinant strain comprises the nucleotide sequence of the aforementioned DNA molecule or the aforementioned recombinant plasmid. The host cell source of the recombinant strain comprises Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae. The host cell source of the recombinant strain is preferably Escherichia coli BL21 (DE3).

[0047] The present invention provides an application of the aforementioned recombinant strain in degrading cottonpol and / or cottonpol derivatives. The cottonpol derivatives include cottonpol acid. In the technical scheme of the present invention, cottonpol and its derivatives can be directly degraded by cottonpol ring-opening enzyme, or a recombinant strain containing a cottonpol ring-opening enzyme gene can be used to degrade cottonpol and its derivatives. Among them, it is preferred to use a recombinant strain containing a cottonpol ring-opening enzyme gene to degrade cottonpol and its derivatives, which has the advantages of removing free cottonpol in cottonseed meal by microbial fermentation. This method is not only low-cost and free of secondary pollution, but also retains many enzymes, vitamins, amino acids and some growth-promoting factors in the microbial fermentation products, thereby increasing the protein content of cottonseed meal, greatly improving the nutritional value and feeding value of cottonseed meal, and increasing the utilization rate of cottonseed meal. In order to reduce the content of free cottonpol in cottonseed feed and reduce the harm to animals, cottonseed cakes detoxified by fermentation with a recombinant strain containing a cottonpol ring-opening enzyme gene can be used as feed, that is, the recombinant strain containing a cottonpol ring-opening enzyme gene can be inoculated into a culture medium containing cottonpol or cottonseed meal, and under certain conditions, the cottonpol is decomposed and utilized or combined with protein amino acids to form combined cottonpol by utilizing the growth, reproduction and metabolism of microorganisms, thereby achieving the purpose of detoxification. The use of a recombinant strain containing a cottonpol ring-opening enzyme gene to degrade cottonpol and its derivatives has low production costs and low investment, and can produce a large amount of enzymes to improve the palatability of feed.

[0048] In some embodiments of the present invention, the following steps are included: preparing a resting cell suspension, adding a reaction substrate to the resting cell suspension, performing a biotransformation reaction, and obtaining a degradation product; the reaction substrate includes gossypol and / or a gossypol derivative.

[0049] In the technical solution of the present invention, since the resting cells are already in a metabolically active state and do not need to go through a growth cycle, the biotransformation reaction can be completed in a shorter time. The use of resting cell biotransformation can quickly obtain cottonypol degradation products.

[0050] In some embodiments of the present invention, the amount of the reaction substrate added is 0.8-2 mM. The amount of the reaction substrate added within the above range can ensure that the ring-opening reaction of cottonpol is faster.

[0051] In some embodiments of the present invention, the temperature of the bioconversion reaction is 28-37° C., and the pH of the bioconversion reaction is 6.0-8.0.

[0052] In the technical solution of the present invention, the temperature and pH of the bioconversion reaction are within the above ranges to ensure that the ring-opening reaction of cottonpol is faster and more cottonpol degradation products can be obtained faster.

[0053] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0054] Experimental Materials

[0055] Luria-Bertani (LB) liquid medium: Peptone 10.0 g, Yeast extract 5.0 g, NaCl 10.0 g, distilled water 1000 mL, sterilized at 121°C for 25 min.

[0056] LB medium containing the antibiotic kanamycin (Km): 50 μg / L kanamycin was added to the above LB liquid medium.

[0057] LB solid medium: Add 1.6% agar powder to the above LB liquid medium.

[0058] Example 1 Construction of recombinant plasmid

[0059] 1. Linearization of pET28a vector

[0060] Preparation of competent cells of Escherichia coli BL21 (DE3): Streak the Escherichia coli BL21 (DE3) strain on an LB solid plate, culture it at 37°C overnight, then pick a single colony in 5 mL LB liquid medium, culture it at 37°C, 200 r / min overnight to obtain a cultured bacterial solution. Inoculate the cultured bacterial solution into 100 mL LB liquid medium at a ratio of 1:100, and culture it at 37°C, 200 r / min for 3 hours until the strain reaches the logarithmic growth phase, that is, OD 600 Between 0.4 and 0.6, and the culture solution was obtained. The culture solution was placed on ice for precooling for 15 minutes, and then centrifuged at 6000r / min and 4°C for 10 minutes in a sterilized 50mL centrifuge tube to collect the bacteria and discard the supernatant. Then 15mL of 0.1mol / L CaCl2 solution precooled on ice was added to the bacteria, and the bacteria were suspended by blowing with a pipette, and continued to be placed on ice for 30 minutes, and then centrifuged at 6000r / min and 4°C for 10 minutes, and the supernatant was discarded to collect the cell pellet. The cell pellet was suspended in 0.1mol / L CaCl2 solution containing 20% ​​glycerol to obtain BL21(DE3) competent cells.

[0061] pET28a was transformed into competent cells of BL21(DE3) to obtain recombinant bacteria BL21(DE3)(pET28a), which were then spread on LB solid plates containing 50μg / mL Km and cultured overnight at 37°C to obtain cultured BL21(DE3)(pET28a) colonies, from which single colonies were picked and inoculated into 5mL LB liquid medium containing 50μg / mL Km, and cultured overnight at 37°C and 200r / min. The plasmid of the cultured strain was extracted using a plasmid extraction kit, and the extracted plasmid DNA was used as a template to perform PCR amplification using the designed upstream primer pET28a-F and downstream primer pET28a-R. The PCR amplification program was as follows: 95°C pre-denaturation for 5min, 95°C denaturation for 30s, 62°C annealing for 30s, 72°C extension for 1min, 30 cycles, and 72°C extension for 8min.

[0062] pET28a-F: 5'-aagcttgcggccgcactc-3';

[0063] pET28a-R: 5'-catggtatatctccttcttaaagttaaacaaaattat-3'.

[0064] The amplified PCR products were separated by 0.8% agarose gel electrophoresis and analyzed by gel imaging. Figure 1 shown.

[0065] Figure 1 M in the figure indicates DNA Marker DL 15000, and lane 1 indicates the PCR product. Figure 1 It can be seen that there is a band of about 5000 bp in lane 1, which is consistent with the theoretical size of the linearized vector pET28a, 5251 bp, indicating that the PCR amplification of the vector is successful and the linearized pET28a vector is obtained.

[0066] 2. Preparation of E. coli DH5α competent cells

[0067] Streak the E. coli DH5α strain on an LB solid plate and culture it overnight at 37°C. Pick a single colony and place it in 5 mL of LB liquid medium without resistance and culture it overnight at 37°C and 200 r / min. Inoculate the cultured bacterial solution into 100 mL of LB liquid medium at a ratio of 1:100 and culture it at 37°C and 200 r / min for 3 hours until the strain reaches the logarithmic growth phase. The OD 600Between 0.4 and 0.6, and the culture solution was obtained. The culture solution was placed on ice for precooling for 15 minutes, and then centrifuged at 6000r / min and 4°C for 10 minutes in a sterilized 50mL centrifuge tube to collect the bacteria and discard the supernatant. Then 15mL of 0.1mol / L CaCl2 solution precooled on ice was added to the bacteria, and the bacteria were suspended by blowing with a pipette, and the cells were placed on ice for another 30 minutes, and then centrifuged at 6000r / min and 4°C for 10 minutes, and the supernatant was discarded to collect the cell precipitate. The cell precipitate was suspended in 0.1mol / L CaCl2 solution containing 20% ​​glycerol to obtain E. coli DH5α competent cells.

[0068] 3. Construction of recombinant plasmid

[0069] (1) The nucleotide sequence of the DNA molecule (SEQ ID NO. 2) was connected to the linearized pET28a vector using ClonExpress Ultra One Step Cloning Kit (purchased from Nanjing Novogene Biotechnology Co., Ltd., catalog number C116-01) to obtain a connection product. The specific reaction system is shown in Table 1.

[0070] Table 1 Infusion connection system

[0071] Components Volume (μL) Linearized pET28a vector 1.0 The nucleotide sequence of a DNA molecule 2.0 Exnase 1.0 5×CE II buffer 2.0 <![CDATA[ddH2O]]> 4.0

[0072] In Table 1, Exnase and 5×CE II buffer were purchased from Nanjing Novozymes Biotech Co., Ltd.

[0073] (2) Incubate the ligation product in a 37°C water bath for 30 minutes. After the incubation, place the ligation product on ice to cool. Take out the DH5α competent cells from the -80°C refrigerator, place them on ice to completely thaw, take 10μL of the ligation product and introduce it into 100μL of DH5α competent cells, mix with a pipette tip and rotate three times each, and place it on ice for 30 minutes. Then place it in a 42°C water bath for 45 seconds, and then place it on ice for 2 minutes. Then add 700μL of liquid LB culture medium, mix well, and culture and recover for 1 hour at 37°C and 200r / min shaking to obtain a culture.

[0074] 50 μL of the culture was spread on a solid LB plate containing Km. The remaining culture was centrifuged at 8000 r / min for 2 min, the supernatant was removed, 100 μL of the supernatant was retained and used to resuspend the precipitated cells to obtain a suspension, and the suspension was spread on a solid LB plate containing Km for culture.

[0075] (3) Extraction and verification of positive transformant plasmids

[0076] Pick 3-5 single colonies from the LB solid plate containing Km and inoculate them into 5 mL of LB liquid medium containing Km, culture them overnight at 37°C and 200 r / min, extract the plasmid, and detect the plasmid by 0.8% agarose gel electrophoresis. Three bands can be observed, which are supercoiled, linear and open-circular plasmid DNA of different configurations, indicating that the plasmid extraction was successful.

[0077] The plasmid was identified by double digestion with restriction endonucleases to confirm whether the target gene was correctly inserted into the plasmid. The double digestion system was as follows: 8 μL of plasmid, 1 μL of 10× buffer M, 0.5 μL each of Xba I and Hind III. Digestion was performed at 37°C for 1 hour, and then agarose electrophoresis was performed. Two bands were detected by electrophoresis, one at about 5000 bp and the other at about 400 bp, which were consistent with the vector pET28a and gene orf4476 in size, respectively. This indicated that the recombinant plasmid pET28a-orf4476 was successfully constructed.

[0078] Example 2 Expression and identification of cottonpol ring-opener

[0079] The recombinant plasmid pET28a-orf4476 in Example 1 was transformed into E. coli BL21 (DE3) to obtain a recombinant strain. A single colony was picked and placed in 5 mL of LB liquid medium containing Km, and cultured at 37°C with shaking overnight. A 1% inoculum was added to 100 mL of LB liquid medium and cultured at 37°C with shaking until the OD 600 The concentration of isopropyl-β-D-thiogalactoside (IPTG) was 0.4, and the final concentration was 0.1mM. The cells were cultured at 16℃ for 16h. After the culture was completed, the cells were centrifuged at 8000r / min for 8min to collect the bacteria. The precipitate was resuspended and washed 2-3 times with phosphate buffer solution (PBS). Then, the solution was clarified by ultrasonic disruption (interval 5s; disruption 3s; power 500W). After disruption, the supernatant was collected by low-temperature centrifugation at 1200r / min for 30min, which was the crude enzyme solution.

[0080] First, the nickel column was pretreated. After 20% ethanol was drained, the nickel column was activated by eluting with 20mM imidazole for 20min (flow rate 1mL / min) after washing. The crude enzyme solution was loaded with 0.5mL / min flow rate. After the crude enzyme solution was loaded, the protein was eluted with 20mM, 40mM, 80mM, 120mM, and 200mM imidazoles in sequence. Each gradient was washed for 30-40min, and about 15mL of sample was connected. After the elution was completed, the protein was thoroughly eluted with 500mM imidazole for 30min, and finally washed with ddH2O for 30min, 20% ethanol for 3 times the column volume, and finally the nickel column was soaked with 20% ethanol to obtain a purified protein sample.

[0081] 10 μL of the purified protein sample was added to 2 μL of 6× protein loading buffer (purchased from Takara) to obtain a mixed solution. The mixed solution was boiled at 100°C for 10 min, cooled naturally and centrifuged at 10000 r / min for 2 min. 10 μL of the supernatant was taken for SDS-PAGE electrophoresis. The results are as follows: Figure 2 shown.

[0082] Depend on Figure 2 It can be seen that the purified target band in lane 1 is consistent with the theoretical value, and the protein purification is successful. The molecular weight of cottonpol ring-opening enzyme Orf4476 is calculated to be 14.6 KDa.

[0083] Example 3 Degradation of cottonpol by cottonpol ring-opening enzyme and identification of degradation products

[0084] The resting cell biotransformation of cottonpol was carried out as follows: a single colony of BL21(DE3)(pET28a-orf4476) was inoculated into 5 mL of LB solid medium containing 50 μg / L kanamycin, cultured overnight, and then the cultured strain was added into 100 mL of LB liquid medium at a 1% inoculum and cultured at 37°C with shaking until the OD 600 The concentration of isopropyl-β-D-thiogalactoside (IPTG) was 0.4, and then 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added and cultured at 16°C for 16 h. After the culture was completed, the bacteria were collected by centrifugation at 8000 r / min for 8 min, and the precipitated bacteria were washed twice with phosphate buffered saline (PBS). The resting cell transformation substrate was 0.8 mM cotton pol.

[0085] The recombinant strain E. coli BL21 (DE3) (pET28a-orf4476), blank control (no inoculation strain) and empty vector BL21 (DE3) (pET28a) were used for whole cell biotransformation for 48 h. Figure 3 shown.

[0086] Figure 3 CK represents the blank control, 1 represents BL21(DE3)(pET28a), and 2 represents the recombinant strain BL21(DE3)(pET28a-orf4476). Figure 3 It can be seen that after 48 hours, the colors of the recombinant strain, blank control and BL21 (DE3) (pET28a) groups were different. The blank control group was yellow, the negative control BL21 (DE3) (pET28a) group was khaki, and the recombinant strain group was brown. The pH was adjusted to 5.0 with dilute hydrochloric acid, and the extraction was performed and high-performance liquid chromatography (HPLC) was performed. The HPLC results of the blank control, negative control and recombinant strain groups were as follows: Figure 4 , Figure 5 and Figure 6 shown.

[0087] Depend on Figure 4 , Figure 5 and Figure 6 It can be seen that compared with the negative control and the blank control group, the recombinant strain BL21 (DE3) (pET28a-orf4476) has product peaks with peak areas of 66 mAU.s and 156 mAU.s at retention times of 1.37 min and 1.48 min, indicating that the cottonpol ring-opening enzyme expressed in the recombinant strain degraded the cottonpol substrate.

[0088] The products of the biotransformation of the blank control group and the recombinant strain group BL21 (DE3) (pET28a-orf4476) resting cells were identified by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), and four substances with mass-to-charge ratios of m / z 517.1863, 549.1760, 581.1666 and 617.1879 were detected. The quality and molecular structure of the degradation products were analyzed using the "element composition" tool of the liquid chromatography-mass spectrometry (LC-MS) analysis software: MestReNova 14.0 software, and the parameters were set as follows: error range: 10ppm, equivalent double bonds 0.5-20.0, and element restrictions of 0-40 atoms of the four elements C, H, N, and O. The analysis results are shown in Table 2.

[0089] Table 2 Accurate mass and elemental composition analysis of biotransformation products

[0090]

[0091] Figure 7 is the mass spectrum of cottonpol, Figure 7 A is the TIC diagram of the blank control group, B is the primary mass spectrum at the retention time of 7.77min, and C is the molecular structure diagram of m / z=517.1860. Figure 7 It can be seen that the mass-to-charge ratio of cottonpol is 517.18630 ​​(MH), and the actual value differs from the theoretical value by 0.49 mDa. Therefore, it is inferred that the molecular structure is C 30 H 30 O8. The calculation error of the "element composition" tool is less than 1mDa, which has a high credibility. There is also a substance with a mass-to-charge ratio of 549.1760 (MH) in the blank control group, and the calculated molecular structure is C 30 H 30 O 10 , also known as gossypol acid, is the product of the oxidation of two aldehyde groups of gossypol to form carboxyl groups. It may be a substance generated by the self-oxidation of gossypol. Its mass spectrum is shown in Figure 8 shown. Figure 8In the figure, A is the TIC diagram of the blank control group, B is the primary mass spectrum at the retention time of 7.27 min, and C is the molecular structure diagram of m / z = 549.1760.

[0092] The mass spectrometry results of the recombinant strain group and the blank control group were analyzed, and the product structure was inferred based on the molecular formula, matching the aromatic ring cleavage product. In the recombinant strain BL21 (DE3) (pET28a-orf4476) group, two new substances were found in the UHPLC-MC results: substance 1: retention time 5.762min, m / z = 581.1666; substance 2: retention time 5.378min, m / z = 617.1879. The elemental composition of these two substances was analyzed (Table 2). The molecular structure of substance 1 (m / z = 581.1666) is C 30 H 30 O 12 , structure see Fig. 9 ; Substance 2 (m / z = 617.1879) has a molecular structure of C 30 H 34 O 14 , structure see Fig.10 It is speculated that substance 1 may be the meta-ring opening product of the two benzene rings of the gossypol molecule or the meta-ring opening product of one benzene ring in gossypol acid. The ring opening product with gossypol as the substrate is (2E,2'E)-3,3'-(2,2'-dihydroxy-6,6'-dimethyl-3,3'-bis(2-oxoacetyl)-[1,1'-biphenyl]-4,4'-diyl)bis(2-hydroxy-4-methylpent-2enoic acid), and the meta-ring opening product with gossypol acid as the substrate is It is 2-(3-(8-carboxy-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene-2-yl)-2-hydroxy-6-isobutyl-4-methylphenyl)-3-hydroxymaleic acid; it is speculated that substance 2 may be the ortho-ring opening product of gossypol acid: 2,2'-(4,4'-bis(1-carboxy-2-methylpropyl)-2,2'-dihydroxy-6,6'-dimethyl-[1,1'-biphenyl]-3,3'-diyl) dimalonic acid. It should be noted that: Fig. 9 In the figure, A is the TIC diagram of BL21(DE3)(pET28a-orf4476), B is the primary mass spectrum at the retention time of 5.76 min, and C is the molecular structure diagram of m / z=581.1666; Fig.10 In the figure, A is the TIC diagram of BL21(DE3)(pET28a-orf4476), B is the primary mass spectrum at the retention time of 5.378 min, and C is the molecular structure diagram of m / z=617.1879.

[0093] Therefore, it is speculated that cottonypol is degraded under the action of the recombinant strain BL21(DE3)(pET28a-orf4476), specifically, the aromatic ring of cottonypol is opened to generate a ring-opened product, and the cottonypol ring-opener expressed in the recombinant strain also acts on cottony acid to break the aromatic ring of cottony acid, so the substrate of cottonypol ring-opener can be cottonypol or cottony acid. Fig.11 shown.

[0094] In summary, since the aromatic ring in the cottonpol structure is more easily degraded after being opened, the toxicity of the opened product will also be reduced. Using this enzyme to detoxify cottonseed meal can realize the resource utilization of cottonseed meal and reduce its harm to animals and humans, which is of great significance.

[0095] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A gossypol ring-opening enzyme, characterized in that The amino acid sequence of the cottonpol ring-opener is shown in SEQ ID No.

1.

2. A DNA molecule, characterized in that The DNA molecule is used to encode the cottonpol ring-opener as claimed in claim 1.

3. The DNA molecule according to claim 2, characterized in that The nucleotide sequence of the DNA molecule is shown as SEQ ID No.

2.

4. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the DNA molecule as claimed in claim 3.

5. A recombinant strain, characterized in that: The recombinant strain comprises the nucleotide sequence of the DNA molecule according to claim 3 or the recombinant plasmid according to claim 4.

6. The recombinant strain according to claim 5, characterized in that The host cell sources of the recombinant strain include Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae.

7. Use of the recombinant strain according to claim 5 or 6 in degrading gossypol and / or gossypol derivatives; The gossypol derivatives include gossypol acid.

8. The use according to claim 7, characterized in that The following steps are involved: preparing a resting cell suspension, adding a reaction substrate to the resting cell suspension, performing a biotransformation reaction, and obtaining a degradation product; The reaction substrate includes gossypol and / or a gossypol derivative.

9. The use according to claim 8, characterized in that The amount of the reaction substrate added is 0.8-2 mM.

10. The use according to claim 8, characterized in that The temperature of the bioconversion reaction is 28-37° C., and the pH of the bioconversion reaction is 6.0-8.0.

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