Curcumin reductase cfcura, encoding gene and application thereof

Abstract

The application discloses a curcumin reductase CfcurA and a coding gene thereof, and application of the curcumin reductase CfcurA in microbial catalysis preparation of tetrahydrocurcumin. The amino acid sequence of the curcumin reductase CfcurA is shown as SEQ ID NO. 2, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1. The curcumin reductase CfcurA and the coding gene thereof provided by the application can be used for catalytic production of tetrahydrocurcumin by enzyme method, the conversion rate of the method is high, the selectivity is good, 20g / L of curcumin can be catalyzed to generate 16.4g / L of tetrahydrocurcumin within 24h, and the method has a good application prospect.

Description

(I)TECHNICAL FIELD

[0001] The present application relates to a curcumin reductase CfcurA and its encoding gene, and its application in microbial catalytic preparation of tetrahydrocurcumin. (II)TECHNICAL BACKGROUND

[0002] Tetrahydrocurcumin (THC) is one of the main metabolites of curcumin in the human body, which can be obtained by hydrogen reduction of curcumin, and has various biological activities. It is superior to curcumin in stability, water solubility and bioavailability, and is a natural functional whitening raw material, and its structural formula is as shown in Figure 1 THC has strong activity in inhibiting tyrosinase, can inhibit the generation of melanin, and has better whitening effect than arbutin. It has higher antioxidant capacity than curcumin, can effectively inhibit the generation of oxygen free radicals and can remove the formed free radicals, and is widely used in various skin care products for whitening, freckle removal and antioxidant. It also has the effects of anti-tumor, anti-inflammatory, anti-diabetic, treatment of neurological diseases, etc.

[0003] Currently, the synthesis methods of tetrahydrocurcumin mainly include biological synthesis method and chemical synthesis method. The traditional chemical method for preparing THC is mainly through catalytic hydrogenation of curcumin, which has low yield, poor selectivity, complex by-products, and the main by-products are hexahydrocurcumin and octahydrocurcumin, and the product purification is difficult. The biological synthesis method is to synthesize tetrahydrocurcumin by reducing curcumin through endophytic fungi in rhizomes of Curcuma longa (Chem Pharm Bull, 2011, 59, 1042-1044), but the conversion efficiency of this method is not high, and it is not suitable for large-scale production. There are also reports that one-step biological conversion method is used to separate yeasts, filamentous fungi and bacteria from soil or feces, such as Pichia kudriavzevii, Alternaria alternata, Cunningham echinulata, Penicillium brevicompactum, Rhizopus oryzae, and some intestinal microorganisms such as Escherichia coli, Escherichia fergusonii and Bacillus megaterium, etc. (J Agri Food Chem 2017, 65, 3305-3310; Appl Biochem Biotechnol 2013, 170, 1026-1037; Food and Fermentation Industries, 2019, 45, 45-51). Although these microorganisms have the ability to reduce curcumin to synthesize tetrahydrocurcumin by one-step fermentation method, there are problems of complex by-products, which need to be genetically engineered to improve the performance of reducing product specificity. In addition, some enzymes derived from animal cells and microbial cells have been confirmed to have the ability to reduce curcumin and generate tetrahydrocurcumin, and these enzymes include horse liver dehydrogenase, dehydrogenase derived from rat liver cells, some oxidoreductases including curcumin reductase CurA derived from Escherichia coli and Vibrio vulnificus (Proc Natl Acad Sci USA, 2011, 108, 6615-6620; J Agri Food Chem 2006, 54, 756-764;). Enzyme catalytic conversion has the characteristics of relatively specific products, and can realize one-step biological conversion synthesis, so the biological enzyme method for preparing THC has certain advantages. (III) SUMMARY

[0004] The purpose of the present application is to provide a curcumin reductase CfcurA and its encoding gene, and its application in microbial catalytic preparation of tetrahydrocurcumin, which realizes the efficient production of tetrahydrocurcumin by biological enzyme method.

[0005] The technical scheme adopted by the present application is:

[0006] A curcumin reductase CfcurA, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0007] The present application also relates to a gene encoding the curcumin reductase CfcurA. Preferably, the encoding gene nucleotide sequence is shown in SEQ ID NO. 1.

[0008] The present application also relates to a vector containing the coding gene and a recombinant genetically engineered bacterium.

[0009] The present application also relates to the application of the genetically engineered bacterium in the microbial catalytic preparation of tetrahydrocurcumin.

[0010] Specifically, the application is as follows: a recombinant genetically engineered bacterium containing the coding curcumin reductase gene is constructed, the bacterium obtained by fermentation culture of the genetically engineered bacterium or the enzyme-containing preparation obtained by crushing the bacterium is used as a catalyst, and curcumin is used as a substrate to prepare tetrahydrocurcumin by catalytic reaction.

[0011] The catalytic reaction is carried out under the synergistic action of NAD(P) + / NAD(P)H coenzyme cycle system. Figure 2

[0012] In the present application, the alcohol dehydrogenase LbADH (J. Mol. Biol. (2005) 349, 801-813) derived from Lactobacillus brevis and isopropanol are added as the coenzyme NAD(P) + / NAD(P)H cycle system, and 0.05-2.0 mmol / L of NAD(P) + (0.2 mmol / L of NADP + is preferably added as a coenzyme factor. Curcumin is used as a reaction substrate, Tween-80 is added as a cosolvent, the substrate is fully dissolved to obtain a catalytic reaction system, and the reaction is carried out under stirring at 20-40°C (preferably 30°C).

[0013] In the catalytic reaction system, the cell density is 5-50 (preferably 20), the addition amount of curcumin is 1-50 g / L (preferably 10 g / L), the addition amount of the cosolvent Tween-80 is 0.05%-5% (preferably 0.15%), and the addition amount of isopropanol is 0.5%-5% (v / v) (preferably 3%, v / v).

[0014] ​The recombinant E. coli engineering bacteria of the curcumin reductase can be prepared by the following method: (1) firstly, gene amplification and expression strain construction, the genomic DNA of Citrobacter freundii IEF113 isolated from the biological fertilizer sample is used as the template, the gene of curcumin reductase is obtained by PCR amplification, and then the gene is cloned into the E. coli expression plasmid pET28a to construct a recombinant strain containing the curcumin reductase gene. The strain is subjected to shake flask fermentation to obtain a fermentation broth, which is inoculated in a seed culture medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm to the mid-logarithmic growth phase to obtain a seed liquid; the final concentration of the seed culture medium is composed of: yeast powder 5 g / L, peptone 10 g / L, NaHPO4·12H2O 8.9 g / L, KH2PO4 3.4 g / L, NH4Cl 2.67 g / L, Na2SO4 0.71 g / L, MgSO4·7H2O 0.49 g / L, and the solvent is deionized water, pH 7.0. The fresh seed liquid is inoculated into a fermentation medium containing 50 μg / mL kanamycin at a volume concentration of 1%, and cultured at 37°C for 4 h, and then 0.5 mmol / L IPTG is added, and the culture is continued at 22-24°C for 12 h to obtain a fermentation broth containing curcumin reductase cells. The fermentation broth is centrifuged, the wet bacterial cells are collected, the bacterial cells are resuspended with 100 mmol / L phosphate buffer, the cells are broken by a high-pressure cell homogenizer, and a crude enzyme solution is obtained, which is the cell broken liquid containing curcumin reductase. The final concentration of the fermentation medium is composed of: 10 g / L of peptone, 5 g / L of yeast extract powder, 15 g / L of glycerol, 9 g / L of Na2HPO4, 3.4 g / L of KH2PO4, 3 g / L of NH4Cl, 0.71 g / L of Na2SO4, and 5 g / L of MgSO4, and the solvent is deionized water, pH 6.5-7.5.

[0015] Further, the NAD(P) +The NAD(P)H coenzyme cycle system is a commonly used coenzyme cycle system, and the alcohol dehydrogenase LbADH and isopropyl alcohol from Lactobacillus brevis are preferably used. The alcohol dehydrogenase LbADH crude enzyme solution is prepared as follows: the recombinant E. coli engineering bacteria E. coli IEF-LbADH (CN112143764A) containing LbADH enzyme frozen and stored in a glycerol tube is activated by streaking on a LB plate containing 50 μg / mL kanamycin. A single colony is inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm until the logarithmic growth phase, to obtain a seed solution. Fresh seed solution is inoculated into fermentation medium containing kanamycin 50 μg / ml at a volume concentration of 1%, and cultured at 30-37°C for 4h, and then IPTG is added at a final concentration of 0.5mmol / L, and the fermentation is continued at 22-25°C for 12-18h. The fermentation broth is centrifuged, and the wet bacterial cells are collected and resuspended in 100mmol / L phosphate buffer. The cells are broken by a high-pressure cell homogenizer to obtain a crude enzyme solution. The final concentration of the seed culture medium and the fermentation medium is the same as above.

[0016] The beneficial effects of the present application mainly include: the present application provides a curcumin reductase CfcurA and its encoding gene, which can be used for enzymatic catalytic production of tetrahydrocurcumin. The conversion rate of the method is high, the selectivity is good, 20g / L of curcumin can be catalyzed to generate 16.4g / L of tetrahydrocurcumin within 24h, and the method has good application prospect. (V)DETAILED DESCRIPTION

[0017] Figure 1 Molecular structural formula of tetrahydrocurcumin.

[0018] Figure 2 Process of curcumin reductase catalyzing curcumin to generate tetrahydrocurcumin.

[0019] Figure 3 Liquid chromatogram of tetrahydrocurcumin standard sample.

[0020] Figure 4 Liquid chromatogram of curcumin and tetrahydrocurcumin mixed standard.

[0021] Figure 5 Liquid chromatogram of CfcurA reaction end point sample. (V)DETAILED DESCRIPTION

[0022] The present application will be further described below in conjunction with specific examples, but the protection scope of the present application is not limited to this:

[0023] In the examples, the methods used are conventional methods unless otherwise specified, and the reagents used can be obtained from commercial channels.

[0024] LB medium: yeast powder 5.0 g / L, protein peptone 10.0 g / L, NaCl 10.0 g / L, solvent is deionized water, pH = 6.8-7.0.

[0025] Fermentation medium: yeast powder 12.0 g / L, protein peptone 15.0 g / L, Na2HPO4·12H2O 8.9 g / L, KH2PO4 3.4 g / L, NH4Cl 2.67 g / L, Na2SO4 0.71 g / L, MgSO4·7H2O 0.49 g / L, solvent is deionized water, pH = 6.8-7.0.

[0026] 100 mmol / L phosphate buffer solution (pH 6.5): Na2HPO4·12H2O 33.9 g / L, NaH2PO4·2H2O 0.41 g / L.

[0027] Example 1: Construction of recombinant strains of curcumin reductase from different sources

[0028] (1) Amplification of curcumin reductase gene curA.

[0029] The genomic DNA from Escherichia coli K-12 in the laboratory, the genomic DNA of Citrobacter freundii IEF113 isolated from biological fertilizer samples, the genomic DNA of Kosakonia cowanii IEF58 isolated from enzyme fermentation, the genomic DNA of Salmonella enterica ATCC13076 preserved in the laboratory, the genomic DNA of Xanthomonas campestris pv. campestris 8004 (Beijing), and the genomic DNA of Acinetobacter junii isolated from soil samples were used as templates, respectively, and the primers shown in Table 1 were used. PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novozyme Bio-tech Co., Ltd. to obtain the corresponding PCR amplification products of curcumin reductase gene curA. The PCR amplification program was as follows: 95℃, 3min; 95℃, 15s; 53℃, 15s; 72℃, 3min; 30 cycles; 72℃, 5min; 4℃ storage. The PCR products were purified using a PCR product recovery kit.

[0030] (2) The target gene was cloned into the pET28a plasmid to obtain a recombinant plasmid, and the recombinant plasmid was transformed into an expression host E. coli BL21(DE3). The specific operation is as follows:

[0031] The plasmid pET28a was extracted, and after EcoRI / NdeI enzyme digestion, the linearized plasmid after enzyme digestion was purified by a DNA gel recovery kit. The purified PCR product and the linearized plasmid were subjected to recombination connection reaction, and then transformed into E. coli BL21(DE3) cells. 10 μL of the recombinant plasmid was added to 100 μL of E. coli BL21(DE3) competent cells, and the tube wall was shaken several times and mixed. It was placed in an ice water bath for 30 min. 42°C heat shock for 45 s, ice water incubation for 3 min. Add 900 μL of LB medium without antibiotics, incubate at 37°C for 60 min. Take 200 μL of bacterial liquid and evenly coat on the LB plate containing 50 mg / mL kanamycin. Invert the plate and incubate at 37°C overnight. After colony PCR verification as a positive clone, it was purified by streaking on an LB plate containing 50 μg / mL kanamycin and shaking culture in LB liquid medium. The plasmid was extracted and verified by enzyme digestion and sequencing. Finally, recombinant E. coli E.coil IFE-EccurA, E.coil IFE-CfcurA, E.coil IFE-KccurA, E.coil IFE-SecurA, E.coil IFE-XcccurA, E.coil IFE-AjcurA were obtained for catalytic activity analysis.

[0032] Table 1: Amplification primers of curA genes from different sources

[0033]

[0034]

[0035] Example 2: Preparation of alcohol dehydrogenase LbADH for coenzyme cycle

[0036] Curcumin reductase belongs to oxidoreductase, which needs NADPH as coenzyme. The application of coenzyme NAD(P) + / NAD(P)H cycle system avoids the use of expensive NADH raw materials, and uses relatively low-priced NAD(P) +The raw material is reduced significantly, thereby reducing the production cost. Lactobacillus brevis-derived alcohol dehydrogenase LbADH and isopropyl alcohol are selected for coenzyme circulation. The laboratory-preserved recombinant Escherichia coli IEF-LbADH (CN112143764A) containing LbADH is activated by plate streaking, and a single colony is inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm to the mid-logarithmic growth phase to obtain a seed solution.

[0037] The fresh seed solution is inoculated into fermentation medium containing 50 μg / mL kanamycin at a volume concentration of 1%, and cultured at 30-37°C for 4 h. IPTG is added at a final concentration of 0.5 mmol / L, and the fermentation is continued at 22-25°C for 12-18 h. The fermentation broth is centrifuged, and the wet bacterial cells are collected. The bacterial cells are resuspended in 100 mmol / L phosphate buffer, and the cells are broken by a high-pressure cell homogenizer to obtain a crude enzyme solution containing LbADH.

[0038] Example 3: Fermentation expression of different recombinant Escherichia coli for curcumin reductase

[0039] The recombinant expression strains of different curcumin reductases prepared in Example 1 are activated by plate streaking in LB containing 50 μg / mL kanamycin, and a single colony is inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm to the mid-logarithmic growth phase to obtain a seed solution.

[0040] The seed solution is inoculated into Escherichia coli fermentation medium containing 50 μg / mL kanamycin at a volume concentration of 5%, and cultured at 37°C for 3 h. IPTG is added at a final concentration of 1.0 mmol / L, and the fermentation temperature is controlled at 24°C. The fermentation is continued for 10 h to obtain a fermentation broth.

[0041] The fermentation broth is centrifuged, and the bacterial cells are resuspended in 100 mmol / L phosphate buffer. The cells are broken by a high-pressure cell homogenizer to obtain a crude enzyme solution of curcumin reductase CurA from different sources.

[0042] Example 4: Comparison of the activities of six different sources of curcuminase in catalyzing the preparation of THC from curcumin

[0043] The crude enzyme solutions of curcumin reductase CurA and alcohol dehydrogenase LbADH are used for enzymatic catalytic preparation of THC. The catalytic reaction system is as follows: 76% (v / v) of CurA crude enzyme solution, 19% (v / v) of LbADH crude enzyme solution, 10 g / L of curcumin, 0.2 mmol / L of NADP +, 5% (v / v) isopropanol, 0.15% Tween 80. The above 10 mL reaction solution was placed in a 50 mL round-bottom flask and magnetically stirred in a 30°C constant-temperature water bath for 12 h.

[0044] The catalytic reaction solution was used for HPLC analysis to compare the yield of THC, and the results are shown in Table 2. The curcumin reductase CfcurA from Citrobacter freundii had the highest reductive activity on curcumin.

[0045] Table 2: Comparison of catalytic activities of curcumin reductases from different sources on curcumin

[0046]

[0047] Liquid chromatography detection conditions. Sample pretreatment: 500 μL of the reaction solution was extracted in 1.0 mL of ethyl acetate under magnetic stirring, centrifuged at 12000 rpm for 5 min, and the upper ethyl acetate solution was taken and the ethyl acetate was removed by nitrogen blowing, and then 500 μL of chromatographic methanol was used for dissolution; the filtrate was filtered through a 0.22 μm microporous filter and added to a liquid phase sample bottle for liquid phase detection and analysis. Detector: Agilent 1260 high performance liquid chromatograph, chromatographic column: Agilent Eclipse XDB-C18 column; column temperature: 30°C; mobile phase: C2H3N (acetonitrile): H2O: CH3COOH (glacial acetic acid) = 45:55:0.01 (volume ratio); flow rate: 1 mL·min -1 ; detector: ultraviolet detector; detection wavelength: 280 nm; injection volume: 10 μL. The peak time of the substrate curcumin is generally 13-14 min, and the peak time of the product THC is generally 5.3-5.4 min and 10-11 min (two peaks with ketone and enol structures). Figure 2 HPLC chromatogram of tetrahydrocurcumin standard. Figure 3 HPLC chromatogram of curcumin and tetrahydrocurcumin mixed standard.

[0048] Example 5: Application of CfcurA in preparation of tetrahydrocurcumin

[0049] (1) Fermentation preparation of CfcurA and coenzyme LbADH

[0050] The recombinant E. coli E.coil IFE-CfcurA and the recombinant E. coli E.coil IFE-LbADH were activated in seed culture medium (same as in Example 3) containing 50 μg / ml kanamycin, respectively, and cultured at 37°C, 200 rpm to the mid-logarithmic growth phase to obtain seed solution.

[0051] E. coli IFE-CfcurA fermentation broth and E. coli IEF-LbADH fermentation broth were prepared respectively using a 2L fermenter. The fermentation conditions were controlled as follows: fresh seed liquid was inoculated into E. coli fermentation medium containing 50 mg / L kanamycin at a volume concentration of 5%; 30°C was used for cultivation for 4h, and then the final concentration of 40 g / L glycerol was added, and the fermentation was continued to 4.5h, and then the final concentration of 20 g / L lactose was added, the fermentation temperature was controlled at 23°C, the dissolved oxygen DO was controlled to be greater than 20%, the fermentation pH was controlled at 6.8 using 25% ammonia water, and the fermentation was continued for 12h to obtain the fermentation broth, which was recorded as CfcurA fermentation broth and LbADH fermentation broth respectively.

[0052] Preparation of CfcurA crude enzyme liquid and LbADH crude enzyme liquid: the fermentation broth of CfcurA and the fermentation broth of LbADH were centrifuged at 4000xg and 4°C to collect cells; the collected cells were resuspended in 100 mmol / L PBS buffer (pH 6.5) to control the OD 600 of the cell suspension liquid to be 40; the cells were broken by a high-pressure cell homogenizer to obtain the crude enzyme liquid of CfcurA and the crude enzyme liquid of LbADH, which were used for catalytic reaction as soon as possible to avoid long-term storage.

[0053] (2) Preparation of tetrahydrocurcumin when the substrate addition amount is 10 g / L

[0054] Take 16 mL of CfcurA crude enzyme liquid into a 50 mL round-bottom flask, then add 4 mL of LbADH crude enzyme liquid, 1 mL of isopropyl alcohol, 0.15% of Tween 80 and 0.2 mmol / L of NADP + with a final concentration, and finally add 0.2 g of curcumin, i.e. the substrate addition amount is 10 g / L. Catalytic reaction was carried out at 30°C under magnetic stirring at 500 rpm, and the reaction was ended after 24h of catalysis. Take 0.2 mL of the reaction liquid into 1 mL of ethyl acetate, centrifuge at 12000 rpm for 5 min, take the upper ethyl acetate solution, remove the ethyl acetate by nitrogen blowing, dissolve in 1 mL of chromatographic methanol, filter through a 0.22 μm microporous filter membrane, and then add the filtrate into a liquid phase sample bottle for liquid phase detection and analysis. Detector: Agilent 1260 high performance liquid chromatograph, chromatographic column: Agilent Eclipse XDB-C18 column; column temperature: 30°C; mobile phase: C2H3N (acetonitrile): H2O: CH3COOH (glacial acetic acid) = 45:55:0.01 (volume ratio); flow rate: 1 mL·min -1Detector: UV detector; Detection wavelength: 280 nm; Injection volume: 10 μL. The elution time of the substrate curcumin is generally 13-14 min, while the elution time of the product THC is generally 5.2-5.3 min and 10-11 min (possessing keto and enol structures). After 16 h of reaction, the conversion rate of the substrate curcumin is greater than 98%. Based on the peak area calculated by HPLC, the yield of tetrahydrocurcumin is approximately 8.9 g / L, containing a small amount of dihydrocurcumin.

[0055] (3) Preparation of tetrahydrocurcumin with a substrate addition of 20 g / L

[0056] Take 16 mL of CfcurA crude enzyme solution into a 50 mL round-bottom flask, then add 4 mL of LbADH crude enzyme solution, 1 mL of isopropanol, 0.15% Tween 80, and NADP to a final concentration of 0.2 mmol / L. + Finally, 0.4 g of curcumin was added, resulting in a substrate feed rate of 20 g / L. The catalytic reaction was carried out at 30°C with magnetic stirring at 500 rpm for 24 hours. The reaction was then terminated, and HPLC analysis was performed. The results are as follows: Figure 5 As shown, the conversion rate of the substrate curcumin was 90.3%, the yield of tetrahydrocurcumin was 16.4 g / L, and it contained a certain amount of dihydrocurcumin.

[0057] (4) Preparation of tetrahydrocurcumin with a substrate addition of 30 g / L

[0058] Take 16 mL of CfcurA crude enzyme solution into a 50 mL round-bottom flask, then add 4 mL of LbADH crude enzyme solution, 1 mL of isopropanol, 0.15% Tween 80, and NADP to a final concentration of 0.2 mM. + Finally, 0.6 g of curcumin was added, resulting in a substrate feed rate of 30 g / L. The catalytic reaction was carried out at 30°C and 500 rpm with magnetic stirring, and the reaction was terminated after 48 hours. Liquid phase analysis showed that the conversion rate of the substrate curcumin was 76.3%, the yield of tetrahydrocurcumin was 20.4 g / L, and it also contained a certain amount of dihydrocurcumin.

[0059] Therefore, the substrate concentration should be 10-20 g / L, preferably 10 g / L.

Claims

1. A method for preparing tetrahydrocurcumin using curcumin reductase CfcurA microbial catalysis, the method comprising: A recombinant genetically engineered bacterium containing a gene encoding curcumin reductase with an amino acid sequence as shown in SEQ ID NO.2 was constructed. The tetrahydrocurcumin was prepared by using the enzyme-containing preparation obtained by fermenting and culturing the recombinant genetically engineered bacterium and then crushing the bacterial cells as a catalyst and curcumin as a substrate.

2. The method as described in claim 1, characterized in that... The nucleotide sequence of the gene encoding the curcumin reductase is shown in SEQ ID NO.

1.

3. The method as described in claim 1, characterized in that... The catalytic reaction is enhanced with alcohol dehydrogenase LbADH and isopropanol as coenzymes NADP. + / NADPH circulation system, and add 0.05~2.0 mmol / L NADP. + As a coenzyme factor.

4. The method as described in claim 1, characterized in that... The catalytic reaction uses curcumin as the substrate and Tween-80 as a co-solvent, and is carried out under stirring conditions at 20~40℃.

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