CN118109429A - Catalytic protein composition synthesized by natural raspberry ketone, strain and application method thereof - Google Patents
Catalytic protein composition synthesized by natural raspberry ketone, strain and application method thereof Download PDFInfo
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- NJGBTKGETPDVIK-UHFFFAOYSA-N raspberry ketone Chemical compound CC(=O)CCC1=CC=C(O)C=C1 NJGBTKGETPDVIK-UHFFFAOYSA-N 0.000 title claims abstract description 81
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- 239000000203 mixture Substances 0.000 title claims abstract description 22
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- 238000000034 method Methods 0.000 title claims abstract description 20
- VWMVAQHMFFZQGD-UHFFFAOYSA-N p-Hydroxybenzyl acetone Natural products CC(=O)CC1=CC=C(O)C=C1 VWMVAQHMFFZQGD-UHFFFAOYSA-N 0.000 claims abstract description 60
- 238000006243 chemical reaction Methods 0.000 claims abstract description 59
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a catalytic protein composition synthesized by natural raspberry ketone, a strain and a use method thereof, belonging to the technical field of biology. The synthesis method of raspberry ketone takes racemic azalea alcohol and 4-hydroxy benzylidene acetone as substrates, and generates raspberry ketone under the catalysis of panda alcohol dehydrogenase mutant AmADH E-D224A, kefir lactobacillus alcohol dehydrogenase LK-ADH and unexpected raspberry ketone of Alternaria albo-sinensis/Jiang Tongge enzyme AiRZS. According to the invention, the alcohol dehydrogenase is subjected to coenzyme preferential transformation, the 224 th aspartic acid of panda wild type alcohol dehydrogenase (Ailuropoda melanoleuca ADH E) is mutated into alanine, the cofactor regeneration cycle in a reaction system is realized, and the raspberry ketone can be finally obtained at a conversion rate of 92.3%, so that the method has a wide application prospect and a wide market value.
Description
Technical Field
The invention relates to a catalytic protein composition synthesized by natural raspberry ketone, a bacterial strain and a using method thereof, belonging to the technical field of biology
Background
Raspberry ketone (4-hydroxyphenyl) -butan-2-one), a fragrant ingredient found in a variety of fruits and plants. Raspberry ketone has antioxidant and anti-inflammatory effects, is beneficial to lipid metabolism, and is widely used in cosmetics, food industry and pharmaceuticals. The natural content of the raspberry ketone in other fruits such as raspberry, peach or grape is only 1-4 mg/kg, and the extraction cost is too high, so that the raspberry ketone produced by chemical synthesis is dominant in the market at present. However, various toxic byproducts are generated during the chemical synthesis process, which causes environmental pollution, and the product can not be sold as flavor compounds, and the application range is limited. Therefore, the biosynthesis of raspberry ketone is realized, the requirement of industrial production is met, and the raspberry ketone has great economic value.
Ericacumen is an important precursor for producing raspberry ketone, and the enantiomer of the azalea must be oxidized to obtain the final product raspberry ketone. Alcohol Dehydrogenase (ADH), which has a relatively high enantioselectivity and is an enzyme that catalyzes the enantioselective reduction of carbon-based compounds, relies on NAD (P) + and can be used to oxidize chiral azalea alcohols to raspberry ketone. In order to achieve higher conversions, two enantiomers of farinacol are usually contained in the various starting materials separated from natural sources, two alcohol dehydrogenases are required to catalyze the (R) -and (S) -farinacols respectively, while consuming NAD (P) +.
4-Hydroxybenzylideneacetone is the last precursor substrate for de novo synthesis of raspberry ketone. The alpha, beta-unsaturated double bond in the butenyl side chain can be hydrogenated with high specificity by benzalacetone reductase, thereby catalyzing 4-hydroxybenzylidene acetone to generate raspberry ketone. The raspberry ketone/Jiang Tongge enzyme (RZS) is one of benzalacetone reductase, depends on NADPH, can be used in combination with Alcohol Dehydrogenase (ADH), achieves the cyclic action of cofactors NAD (P) + and NAD (P) H in a reaction system, avoids the trouble of continuously supplementing expensive cofactors in the production process, and thus obviously reduces the cost.
The development of biocatalysis provides a new way for the production of many natural products. Currently, little research is done on the technology of synthesis of raspberry ketone for cofactor recycling. In 2021, yang B et al, in order to recycle cofactors in the reaction system, a combination of raspberry ketone/zingibrone synthase and glucose dehydrogenase was employed, glucose was dehydrogenated to convert glucose to gluconic acid, and also NAD(P)H(Yang B,Zheng P,Wu D,et al.Efficient Biosynthesis of Raspberry Ketone by Engineered Escherichia coli Coexpressing Zingerone Synthase and Glucose Dehydrogenase[J].Journal of Agricultural and Food Chemistry,2021,69(8):2549-56.);2021, becker A et al, in the course of producing raspberry ketone, regenerated oxidase SmNOX with universal cofactor was employed to produce NAD (P) +, and O 2 was catalyzed to H 2 O (Becker A,D,Katzer W,et al.An ADH toolbox for raspberry ketone production from natural resources via a biocatalytic cascade[J].Applied Microbiology and Biotechnology,2021,105(10):4189-97.). However, these methods still have certain limitations, such as the raspberry ketone synthesis method developed by Yang B et al, in which glucose is introduced to realize cofactor circulation in the system, and the produced gluconic acid affects the pH value of the reaction system; the synthesis method of raspberry ketone developed by Becker A et al does not affect the reaction system, but the nature of cofactor regenerated oxidase SmNOX limits the reaction conditions for raspberry ketone synthesis and affects the substrate conversion rate. These systems require additional addition of enzymes for cofactor recycling and additional addition of substrates. Therefore, there is a need to develop efficient synthesis of raspberry ketone with cofactor self-circulation.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for synthesizing raspberry ketone by using biological enzyme as a catalyst, which uses racemic farinacol and 4-hydroxybenzylidene acetone as substrates and uses panda alcohol dehydrogenase mutant (Ailuropoda melanoleuca ADH E-D224A), kefir lactobacillus alcohol dehydrogenase (Lactobacillus kefir ADH) and unexpected raspberry ketone of Alternaria sinica/Jiang Tongge enzyme (Albidovulum inexpectatum RZS) as the substrates to catalyze the substrates to generate raspberry ketone.
The beneficial effects of the invention are as follows: compared with the prior disclosed raspberry ketone synthesis technical route, the method can realize the cofactor self-circulation in the reaction system, simultaneously avoid the generation of byproducts, has high substrate conversion rate and has important application prospect.
The technical scheme adopted by the invention is as follows:
A catalytic protein composition synthesized from natural raspberry ketone comprises panda alcohol dehydrogenase mutant, kefir lactobacillus alcohol dehydrogenase and unexpected raspberry ketone of Alternaria parvula/Jiang Tongge enzyme.
Further improvement, the amino acid sequence of the panda alcohol dehydrogenase is shown as SEQ ID NO.2, the amino acid sequence of the kefir lactobacillus alcohol dehydrogenase is shown as SEQ ID NO.3, and the amino acid sequence of the unexpected raspberry ketone/gingerol synthase of the Leuconostoc is shown as SEQ ID NO. 4.
Further improved, the molar ratio of the panda alcohol dehydrogenase, the kefir lactobacillus alcohol dehydrogenase and the unexpected raspberry ketone/gingerol synthase is 5-15:1-4:1.
Further improved, the molar ratio of the panda alcohol dehydrogenase, the kefir lactobacillus alcohol dehydrogenase and the unexpected raspberry ketone/gingerol synthase is 12:2:1.
A strain containing a gene expressing the amino acid sequence of panda alcohol dehydrogenase shown as SEQ ID NO. 2, the amino acid sequence of kefir lactobacillus alcohol dehydrogenase shown as SEQ ID NO. 3 or the amino acid sequence of Raspberry ketone/gingone synthase of Excellent white oomycete shown as SEQ ID NO. 4.
Further improvement, the nucleotide sequence of the gene for expressing the panda alcohol dehydrogenase is shown as SEQ ID NO. 5, the nucleotide sequence of the gene for expressing the kefir lactobacillus alcohol dehydrogenase is shown as SEQ ID NO. 6, and the nucleotide sequence of the gene for expressing the unexpected raspberry ketone/gingerol synthase of the oomycete is shown as SEQ ID NO. 7.
Further improvements include E.coli, pichia pastoris and Bacillus subtilis.
A method of using a catalytic protein composition for synthesis of natural raspberry ketone, the catalytic protein composition for synthesis of natural raspberry ketone being as shown above;
The using method comprises the following steps:
the racemic azalea alcohol and 4-hydroxy benzylidene acetone are used as substrates, and the catalytic protein composition is used as a catalyst to react to obtain the natural raspberry ketone.
Further improvement, the using method is as follows:
10nmol of the catalytic protein composition, 0.1-10 mM of racemic azalea alcohol and 0.1-10 mM of 4-hydroxybenzylidene acetone are contained in each 200 mu L of reaction system, 10 mu M-10 mM of NADP + and 10 mu M-10 mM of NADPH are taken as cofactors, the pH value of the reaction system is controlled to be 6.0-9.0, and then the reaction is stopped by adding hydrochloric acid for example after 15 minutes to 6 hours at the temperature of 30-50 ℃ to obtain a reaction solution containing natural raspberry ketone.
Further, the reaction system was controlled to contain 10nmol of the catalytic protein composition, 0.5mM of racemic azalea alcohol and 0.5mM of 4-hydroxybenzylidene acetone, 0.1mM of NADP +, 0.1mM of NADPH as cofactor per 200. Mu.L of the reaction system, pH=8.0, and then, after 2 hours of reaction at 40℃the reaction was terminated by adding hydrochloric acid for example and a reaction solution containing natural raspberry ketone was obtained.
The beneficial effects of the invention are as follows:
The invention provides a high-efficiency synthesis method of raspberry ketone, which utilizes alcohol dehydrogenase and raspberry ketone/gingerol synthase to realize cofactor circulation in a reaction system, simultaneously avoids the generation of byproducts, efficiently converts racemic azalea alcohol and 4-hydroxybenzylidene acetone into raspberry ketone, and has important application prospect.
The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
Drawings
FIG. 1 is a schematic diagram showing the synthetic route of preparing raspberry ketone by converting substrate (R/S) -rhododendron alcohol and substrate 4-hydroxybenzylideneacetone with alcohol dehydrogenase and raspberry ketone/Jiang Tongge enzyme in example 7 of the present invention.
FIG. 2 shows the retention times of (R/S) -azalea alcohol samples and raspberry ketone samples in example 3 of the present invention.
FIG. 3 shows substrate conversion of 2 hours of reaction of substrate (R/S) -azalea alcohol under the co-catalysis of LK-ADH and AmADH E-D224A in example 3 of the present invention at 40℃and pH range of 6.5-9.0.
FIG. 4 shows the retention times of the raspberry ketone sample and the 4-hydroxybenzylidene acetone sample in example 4 according to the invention.
FIG. 5 shows substrate conversion of the substrate 4-hydroxybenzylideneacetone of example 4 of the present invention by AiRZS reaction for 15 minutes at 40℃and pH ranging from 6.0 to 9.0.
FIG. 6 shows substrate conversions of the dual substrate racemic azalea and 4-hydroxybenzylidene acetone of example 5 of the present invention, at 40℃and pH=8.0, under the catalysis of LK-ADH, amADH1E-D224A and AiRZS at different enzyme ratios (total enzyme amount 10 nmol).
Detailed Description
The technical contents of the present invention are further described below with reference to examples: the following examples are illustrative, not limiting, and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The strains and growth conditions used in the present invention are as follows:
Cloning hosts TOP10 and BL21 (DE 3) were purchased from Invitrogen, all E.coli were cultivated in LB medium containing 100mg/l ampicillin at 37 ℃.
Wherein, the formula of the LB liquid medium is as follows: peptone 10g/L, yeast extract 5g/L, naCl 10g/L, pH 7.0; LB solid culture medium 20g/L agar is added into LB liquid culture medium; sterilizing with steam at 121deg.C under high temperature and high pressure for 20min.
All plasmids were pETDuet-1 (purchased from Novagen corporation) derived plasmids for expression of the gene of interest.
Example 1 construction of E.coli genetically engineered strains expressing alcohol dehydrogenase and raspberry ketone/zingibnone synthase:
(1) The encoding gene (amadh E) of alcohol dehydrogenase (AmADH E) from panda (Ailuropoda melanoleuca), the encoding gene (amadh E-D224A) of alcohol dehydrogenase mutant (AmADH E-D224A) from panda, the encoding gene lkadh of alcohol dehydrogenase (LK-ADH) from Lactobacillus kefir (Lactobacillus kefir) and the encoding gene airzs of raspberry ketone/Jiang Tongge enzyme (AiRZS) in Achillea-forming bacteria (Albidovuluminexpectatum) are subjected to codon optimization according to the codon preference of escherichia coli, the nucleotide sequence of amadh E after optimization is shown as SEQ ID NO:5, the nucleotide sequence of amadh E-D224A is shown as SEQ ID NO:6, the nucleotide sequence of lkadh is shown as SEQ ID NO:7, and the nucleotide sequence of airzs is shown as SEQ ID NO: 8.
(2) Sending the optimized gene sequence in the step (1) to Jin Weizhi company (GENEWIZ) for gene synthesis, and synthesizing amadh e to the middle of BamHI and HindIII cleavage sites of pETDuet-1 to obtain pETDuet-amadh e; lkadh is synthesized to the middle of BamHI and HindIII cleavage sites of pETDuet-1 to obtain recombinant expression plasmid pETDuet-lkadh; airzs was synthesized intermediate the BamHI and HindIII cleavage sites of pETDuet-1 to obtain pETDuet-airzs.
(3) The recombinant plasmid is transformed into an expression host E.coli BL21 (DE 3) to obtain recombinant expression strains E.coli BL21 (DE 3)/pETDuet (1) -amadh e, E.coli BL21 (DE 3)/pETDuet (1) -lkadh and E.coli BL21 (DE 3)/pETDuet (1) -airzs. Inoculating the recombinant strain into LB culture medium containing 25-50 mL, culturing at 37 ℃ at 160-180 rpm until the bacterial liquid OD 600 is 0.6-0.8, then placing the shake flask on ice for 10 minutes, adding IPTG with the final concentration of 1mM for induction, and inducing expression at 16 ℃ at 160-180 rpm for 16-18 h. Total protein from whole cells was analyzed by SDS-PAGE, and the results showed that the recombinant expression strain had distinct expression bands of alcohol dehydrogenase AmADH1E, LK-ADH and raspberry ketone/Jiang Tongge enzyme AiRZS, respectively, after induction with IPTG (isopropyless-D-1-thiogalactopyranoside), and the molecular weight of the bands was consistent with the expected molecular weight. Recombinant strains were stored at-20℃with 15% glycerol.
Example 2 construction of E.coli genetically engineered strain expressing panda alcohol dehydrogenase mutant AmADH E-D224A:
(1) And (3) sending the optimized panda alcohol dehydrogenase mutant nucleotide sequence SEQ ID NO. 6 to Jin Weizhi company (GENEWIZ) for gene synthesis, and synthesizing amadh e-D224A to the middle of BamHI and HindIII enzyme cutting sites of pETDuet-1 to obtain pETDuet-amadh e-D224A.
(2) Competent cells BL21 (DE 3) were transformed, and the extracted plasmids were subjected to sequencing and identification to obtain recombinant expression strains E.coli BL21 (DE 3)/pETDuet (1) -amadh e-D224A.
Example 3 Synthesis of raspberry ketone from (R/S) -azalea alcohol Using the pure enzyme Lactobacillus kefir (Lactobacillus kefir) alcohol dehydrogenase LK-ADH and the pure enzyme panda (Ailuropoda melanoleuca) alcohol dehydrogenase mutant AmADH E-D224A together:
(1) Recombinant strains E.coli BL21 (DE 3)/pETDuet (1) -lkadh and E.coli BL21 (DE 3)/pETDuet (1) -amadh e-D224A obtained in examples 1 and 2 are respectively inoculated into LB culture medium containing 200-1000 mL, cultured at 37 ℃ and 160-180 rpm until bacterial liquid OD 600 is 0.6-0.8, cooled on ice for 10 minutes, added with IPTG with the final concentration of 1mM for induction, and induced and expressed at 16 ℃ and 160-180 rpm for 16-18 hours to obtain culture liquid.
(2) The bacterial liquid was transferred to a 200mL centrifuge bottle, bacterial cells were collected at 6000rpm for 10min, the bacterial cells were washed 2 times with PBS buffer, and then 50mL of cell lysate was added to prepare cell disruption. The cell lysate formulation is as follows: 50mL of 25mM imidazole buffer, 30. Mu. LTriton X100, 15. Mu.L of beta-mercaptoethanol, 200. Mu. LPMSF and 50. Mu.L of nuclease were added in this order and prepared. After re-suspending, the bacteria are crushed for 2 minutes at 4 ℃ under high pressure, the pressure is 800-900 bar, cell lysate is collected, and the cells are centrifuged for 50 minutes at 3500rpm at 4 ℃ in a refrigerated centrifuge. The supernatant was immediately passed through a filter membrane having a pore size of 0.22. Mu.m, and the filtered sample was purified by Ni + column affinity chromatography. Washing 10mL deionized water by column, washing a nickel column by 25mM imidazole buffer solution, washing a cell supernatant by column, and then washing small molecule hybrid protein by 5mL25mM imidazole buffer solution in sequence; 5mL of 50mM imidazole buffer solution competes for target protein, and effluent samples are collected; 10mL 200mM imidazole buffer solution competes for target protein, and effluent samples are collected; the nickel column was washed with 5mL of 1M imidazole buffer. And finally, flushing the nickel column with deionized water. The collected effluent was mixed with a 3×protein loading buffer, boiled for 10 minutes for denaturation, and then subjected to SDS-PAGE to examine the purification result. The obtained protein sample is mixed with high-concentration imidazole, ultrafiltered and changed at 4 ℃ and 3000-4000 rpm, and diluted 10 times by Tris-HCl with pH of 8.0 each time, and repeated 3 times, so as to obtain the protein sample after changing and concentrating. Finally, mixing the protein solution with the protein storage solution according to the volume ratio of 1:1 to finish the preparation of the enzyme. Packaging, quick freezing with liquid nitrogen, and storing at-80deg.C. Wherein the formula of the protein storage solution is 5% glycerol and Tris-HCl mixed solution with the pH of 8.0.
(3) Constructing a reaction system by taking the enzyme purified in the step (2) as a catalyst, wherein the addition amount of the enzyme is 0.2nmol, the total reaction system is 200 mu L, 1mM of substrate (R/S) -azalea alcohol (namely racemic azalea alcohol) is added, 1mM of NADP + is used as a cofactor, the pH range is 6.5-9.0, and the reaction condition is 40 ℃ for 2 hours. After completion of the reaction, the reaction was terminated by adding 15. Mu.L of 8M hydrochloric acid to the reaction system to obtain a reaction solution.
(4) And (3) detecting a product: centrifuging the reaction solution obtained in the step (3) at 12000rpm for 10min, filtering the supernatant by a filter membrane with the aperture of 0.22 mu m, detecting the yield of raspberry ketone by an HPLC method, using an Agilent 1290 information II high performance liquid chromatograph, setting the column temperature to be 30 ℃ by using an Eclipse Plus-C18 column with the column temperature of 2.1 multiplied by 50mm, setting the detection wavelength of (R/S) -rhododendron alcohol and raspberry ketone to be 195nm, setting the mobile phase to be 80 percent deionized water containing 0.1 percent phosphoric acid and 20 percent acetonitrile, and setting the flow rate to be 0.5mL/min. As shown in FIG. 2, the retention time of the (R/S) -azalea alcohol sample was about 4.2min and the retention time of the raspberry ketone sample was about 5.3min. The results show (see FIG. 3) that the conversion of racemic azalea alcohol was 74.2% in 2 hours at 40℃and pH 8.0.
Example 4 synthesis of raspberry ketone using Raspberry ketone/Jiang Tongge enzyme AiRZS pure enzyme from Accident Alternaria (Albidovuluminexpectatum):
(1) The method for obtaining the pure enzyme is the same as in the steps (1) and (2) of example 3. The reaction system is constructed by taking pure enzyme as a catalyst, the addition amount of the enzyme is 0.2nmol, the total reaction system is 200 mu L, 1mM 4-hydroxybenzylidene acetone is added as a substrate, 1mM NADPH is taken as a cofactor, the pH range is 6.0-9.0, and the reaction condition is 40 ℃ for 15 minutes. After completion of the reaction, the reaction was terminated by adding 15. Mu.L of 8M hydrochloric acid to the reaction system to obtain a reaction solution.
(2) The detection method of the product is the same as that of the step (4) of the example 3, and the detection wavelength of the 4-hydroxybenzylidene acetone is 195 or 310nm. As shown in FIG. 4, the retention time of the raspberry ketone sample was about 5.3 minutes and the retention time of the 4-hydroxybenzylideneacetone sample was about 5.8 minutes. The results show (see FIG. 5) that the conversion of racemic azalea alcohol was 79.2% in 15 minutes at 40℃and pH 7.5.
Example 5 Synthesis of raspberry ketone from (R/S) -rhododendron alcohol and 4-hydroxybenzylideneacetone using three pure enzymes, giant panda alcohol dehydrogenase mutant AmADH E-D224A, kefir lactobacillus alcohol dehydrogenase LK-ADH, unexpected raspberry ketone Alternaria parvula/Jiang Tongge enzyme AiRZS:
(1) The method comprises the steps of constructing a reaction system by taking pure enzyme as a catalyst, controlling the total amount of AmADH E-D224A, LK-ADH and AiRZS pure enzyme to be 10nmol according to different enzyme proportions, adding 0.5mM (R/S) -azalea alcohol (racemic azalea alcohol) and 0.5mM 4-hydroxybenzylideneacetone as substrates into the total reaction system to be 200 mu L, taking 0.1mM NADP + and 0.1mM NADPH as cofactor, wherein the pH=8.0, and the reaction condition is 40 ℃ for 2 hours. After completion of the reaction, the reaction was terminated by adding 15. Mu.L of 8M hydrochloric acid to the reaction system to obtain a reaction solution.
(2) The method for detecting the product is the same as in the step (4) of the example 3. As a result, as shown in FIG. 6, when the ratio of AmADH E-D224A, LK-ADH to AiRZS was 12:2:1 at 40℃and pH 8.0, the conversion rate of the dual-substrate racemic azalea and 4-hydroxybenzylidene acetone was 92.3% in 2 hours, and the addition amount of cofactor was reduced by 10%.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (10)
1. A catalytic protein composition synthesized by natural raspberry ketone is characterized by comprising a panda alcohol dehydrogenase mutant, kefir lactobacillus alcohol dehydrogenase and unexpected raspberry ketone of Alternaria albopictus/Jiang Tongge enzyme.
2. The catalytic protein composition for synthesizing natural raspberry ketone according to claim 1, wherein the amino acid sequence of the panda alcohol dehydrogenase mutant is shown in SEQ ID NO.2, the amino acid sequence of the kefir lactobacillus alcohol dehydrogenase is shown in SEQ ID NO.3, and the amino acid sequence of the unexpected raspberry ketone/gingerol synthase is shown in SEQ ID NO. 4.
3. The catalytic protein composition for the synthesis of natural raspberry ketone according to claim 2, wherein the molar ratio of giant panda alcohol dehydrogenase, kefir alcohol dehydrogenase and unexpected raspberry ketone/gingerol synthase is 5-15:1-4:1.
4. The catalytic protein composition for the synthesis of natural raspberry ketone according to claim 2, wherein the molar ratio of giant panda alcohol dehydrogenase, kefir lactobacillus alcohol dehydrogenase and unexpected raspberry ketone/gingerol synthase is 12:2:1.
5. A strain is characterized in that the strain contains genes for expressing the amino acid sequence of panda alcohol dehydrogenase shown as SEQ ID NO. 2, the amino acid sequence of kefir lactobacillus alcohol dehydrogenase shown as SEQ ID NO. 3 or the amino acid sequence of unexpected raspberry ketone/gingone synthase of the oomycete shown as SEQ ID NO. 4.
6. The strain of claim 5, wherein the nucleotide sequence of the gene expressing the panda alcohol dehydrogenase is shown in SEQ ID NO. 5, the nucleotide sequence of the gene expressing the kefir lactobacillus alcohol dehydrogenase is shown in SEQ ID NO. 6, and the nucleotide sequence of the gene expressing the surprising small oomycete raspberry ketone/zingibnone synthase is shown in SEQ ID NO. 7.
7. The strain of claim 5, wherein the strain comprises escherichia coli, pichia pastoris, and bacillus subtilis.
8. A method of using a catalytic protein composition for the synthesis of natural raspberry ketone, wherein the catalytic protein composition for the synthesis of natural raspberry ketone is as shown in any of claims 1-4;
The using method comprises the following steps:
the racemic azalea alcohol and 4-hydroxy benzylidene acetone are used as substrates, and the catalytic protein composition is used as a catalyst to react to obtain the natural raspberry ketone.
9. A method of using the catalytic protein composition for the synthesis of natural raspberry ketone as claimed in claim 8, wherein the method of using is as follows:
10nmol of the catalytic protein composition, 0.1-10 mM of racemic azalea alcohol and 0.1-10 mM of 4-hydroxybenzylidene acetone are contained in each 200 mu L of reaction system, 10 mu M-10 mM of NADP+, and 10 mu M-10 mM of NADPH are taken as cofactors, the pH value of the reaction system is controlled to be 6.0-9.0, and then hydrochloric acid is added for stopping the reaction after 15 minutes to 6 hours at 30-50 ℃ to obtain a reaction solution containing natural raspberry ketone.
10. The method of using the catalytic protein composition for synthesis of natural raspberry ketone according to claim 9, wherein 10nmol of the catalytic protein composition, 0.5mM of racemic rhododendron alcohol and 0.5mM of 4-hydroxybenzylidene acetone are contained in each 200 μl of the reaction system, 0.1mM of NADP + and 0.1mM of NADPH are used as cofactors, the ph=8.0 of the reaction system is controlled, and then the reaction is terminated by adding hydrochloric acid after 2 hours at 40 ℃ to obtain a reaction solution containing natural raspberry ketone.
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