Bienzyme immobilized resin, preparation method and application thereof

Abstract

The invention discloses double-enzyme immobilized resin as well as a preparation method and application thereof. The double-enzyme immobilized resin comprises a resin carrier, curcumin reductase and alcohol dehydrogenase, wherein the curcumin reductase and the alcohol dehydrogenase are connected to the resin carrier; the preparation method comprises the following steps: step 1, preparing a double-enzyme crude enzyme mixed solution containing curcumin reductase and alcohol dehydrogenase; step 2, adding a resin carrier into the double-enzyme crude enzyme mixed solution, and reacting to obtain a double-enzyme immobilized resin suspension covalently bound with curcumin reductase and alcohol dehydrogenase; 3, carrying out suction filtration and washing, and adding the resin into a cross-linking terminator; and 4, carrying out suction filtration, washing and drying to obtain the double-enzyme immobilized resin. The preparation method has the advantages that the preparation method is simple, the prepared double-enzyme immobilized resin is high in enzyme activity and cycling stability, the enzyme activity recovery rate reaches 92% or above, the protein recovery efficiency reaches 93%, cyclic utilization of coenzyme is effectively improved, and the excellent characteristics of being high in mechanical strength, good in biocompatibility, high in environmental tolerance, not prone to inactivation and high in stability are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized enzymes, and relates to a dual-enzyme immobilized resin, a preparation method and an application thereof, and more specifically to a dual-enzyme immobilized resin loaded with curcumin reductase and alcohol dehydrogenase, a preparation method and an application thereof. Background Art

[0002] Tetrahydrocurcumin is a natural metabolite of curcumin, which exists in the plant turmeric. Studies have shown that tetrahydrocurcumin has similar biological activities to curcumin and can be used to treat cancer, central nervous system diseases, protect cardiovascular and whiten the skin. However, tetrahydrocurcumin is more effective than curcumin in treating some cancer diseases. In addition, tetrahydrocurcumin has better water solubility, better stability under physiological pH environment, stronger antioxidant properties, and its product is colorless and has no dyeing effect. Therefore, while tetrahydrocurcumin has good biological activity, it avoids some shortcomings of curcumin in application and has good applications in food, cosmetics, and medicine.

[0003] Existing tetrahydrocurcumin products mainly include biosynthesis and chemical synthesis. The chemical synthesis method is prepared by chemical hydrogenation. Its advantages are high catalytic reaction yield, relatively thorough hydrogenation reaction, and good product appearance, but its disadvantage is that it is easy to produce over-reduced hydrogenation byproducts, including hexahydrocurcumin and octahydrocurcumin, and it is difficult to separate and remove. In the biosynthesis method, there are microbial cell transformation, plant cell transformation and enzyme catalysis transformation, among which enzyme catalysis has a single reduction product, simple catalytic reaction conditions, green and environmentally friendly, and has good industrial application value. Curcumin reductase has been reported to be used to prepare tetrahydrocurcumin. For example, Chinese patent 202310255061.1 discloses a method for efficiently synthesizing tetrahydrocurcumin by cascade enzyme catalysis based on NADPH cofactor cycle, which uses Escherichia coli BL21 (DE3) as a host cell to express NADPH-dependent curcumin convertase (CurA) and formate dehydrogenase (Fdh). Fermentation is carried out using LB medium, and ultrasonic cell disruption is used to obtain a crude enzyme solution of CurA and Fdh. NADPH-dependent CurA is responsible for the conversion of curcumin into tetrahydrocurcumin. Fdh is responsible for converting the generated NADP + Reduced to NADPH to achieve efficient recycling of NADPH cofactor. In addition, the solubility of curcumin in the reaction system was improved by ionic liquid. Through the optimization of substrate and ionic liquid concentration, the final curcumin conversion rate was only 40.5%, the tetrahydrocurcumin yield was only 48.66%, and the yield was 11.01 mg / L.

[0004] In order to solve the above problems, enzyme immobilization is adopted to realize the recycling of enzymes, and at the same time, the tolerance of enzymes to the environment is enhanced. Existing immobilized enzyme methods can be divided into physical methods and chemical methods. The immobilization methods of physical methods mainly include embedding, adsorption, ionic bonding, etc. These methods can obtain a higher enzyme activity recovery rate because they do not destroy the three-dimensional structure of the protein. However, due to its relatively weak interaction force and instability, the fixed enzyme is easy to fall off, affecting the cyclic stability of the immobilized enzyme. The chemical method mainly includes covalent cross-linking between the carrier and the enzyme. Unlike the physical method, the covalent connection directly combines the enzyme with the carrier by a method of covalent bonding of chemical bonds. Due to the strong interaction of the covalent bond, the cyclic stability of the immobilized enzyme obtained is good. However, due to changes in the flexibility and kinetic characteristics of the covalently linked amino acid residues, it is easy to affect the catalytic activity pocket of the enzyme, damage the structure and catalytic function of the enzyme, and cause a low enzyme activity recovery rate. Therefore, when the enzyme is covalently immobilized, it is necessary to screen and optimize the immobilized material and the immobilized material to obtain an immobilized enzyme with high enzyme activity recovery rate and good stability. However, the immobilization process of curcumin reductase is often accompanied by a loss of high enzyme activity. In addition to the usual causes of enzyme tertiary structure distortion and chemical inactivation, the dissociation of single subunits of polymeric enzymes and the dissociation of coenzyme factors are also important influencing factors, which will lead to the loss of high enzyme activity during the immobilization process of curcumin reductase. Summary of the invention

[0005] In order to overcome the problems of difficulty in enzyme recovery, long reaction time, poor environmental tolerance, etc. when curcumin reductase is used to prepare tetrahydrocurcumin in the prior art, the present invention provides a dual-enzyme immobilized resin, a preparation method and application thereof, which are used for hydrogenation reduction of curcumin to prepare tetrahydrocurcumin.

[0006] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions:

[0007] A dual-enzyme immobilized resin, characterized in that the dual-enzyme immobilized resin comprises a resin carrier and curcumin reductase and alcohol dehydrogenase connected to the resin carrier.

[0008] As a preference of the present application, the resin carrier is any one of epoxy resin and primary amino resin or a combination of two thereof.

[0009] As a preference of the present application, the epoxy resin is any one or more combinations of LX-1000EPA epoxy resin, LX-103B epoxy resin, EP-109F, EP-115F, ES-103B, and ES-109 epoxy resin.

[0010] As a preference of the present application, the epoxy resin is any one or more combinations of LX-1000EPA epoxy resin, LX-103B epoxy resin, and ES-109 epoxy resin.

[0011] As a preferred embodiment of the present application, the epoxy resin is any one of ES-109 and LX-103B or a combination of two thereof, and ES-109 is the most preferred.

[0012] The present invention also provides a method for preparing a dual-enzyme immobilized resin, which is characterized by comprising the following steps:

[0013] Step 1, centrifuging, resuspending and crushing the mixed fermentation dual enzyme solution obtained by mixing the fermentation broth prepared by fermentation of curcumin reductase and alcohol dehydrogenase to obtain a crude dual enzyme mixed solution containing curcumin reductase and alcohol dehydrogenase;

[0014] Step 2, adding a resin carrier to the dual-enzyme crude enzyme mixture, cross-linking the resin carrier with curcumin reductase and alcohol dehydrogenase to obtain a resin suspension adsorbed with curcumin reductase and alcohol dehydrogenase;

[0015] Step 3, filtering the resin suspension, washing the obtained precipitate, adding the cross-linking terminator, and continuing the reaction for a certain period of time to obtain a dual-enzyme immobilized resin suspension;

[0016] Step 4: The dual-enzyme immobilized resin suspension is filtered, washed, and dried to obtain a dual-enzyme immobilized resin loaded with curcumin reductase and alcohol dehydrogenase.

[0017] As a preferred embodiment of the present application, the concentration ratio of the bacteria in the curcumin reductase fermentation broth to the bacteria in the alcohol dehydrogenase fermentation broth in step 1 is 1: 1 to 10: 1. Specifically, the curcumin reductase fermentation broth and the alcohol dehydrogenase fermentation broth are mixed at a bacterial concentration ratio of 1: 1 to 10: 1, and then the fermentation broth is centrifuged, and the supernatant is discarded and the cells are collected to obtain a mixed fermentation double enzyme liquid.

[0018] As the preferred embodiment of the present application, the specific steps of obtaining the double enzyme crude enzyme mixture are as follows:

[0019] Add the mixed fermentation double enzyme solution obtained in step 1 to buffer A, shake until mixed evenly, and obtain a cell suspension;

[0020] The resuspended cell suspension was broken up using a high-pressure homogenizer to obtain a cell broken liquid, which was then centrifuged, the precipitate was removed, and the supernatant was collected to obtain a crude double enzyme mixture containing curcumin reductase and alcohol dehydrogenase.

[0021] As further preferred, the buffer A is a phosphate buffer, preferably a phosphate buffer of 20 to 2000 mmol / L. More preferably, it is a phosphate buffer of 50 to 500 mmol / L. More preferably, the buffer A is a phosphate buffer of 200 mmol / L, preferably a sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer. The pH of the phosphate buffer is preferably 4 to 8, more preferably 6 to 8.

[0022] As further preferred, the specific steps of obtaining the immobilized dual enzyme resin in step 2 are as follows:

[0023] The resin carrier is added to the crude double enzyme mixture obtained in step 1, so that the resin carrier undergoes a cross-linking reaction with the curcumin reductase and alcohol dehydrogenase in the crude double enzyme mixture, and the supernatant is removed by filtering, washing, and freeze-drying to obtain an immobilized double enzyme resin.

[0024] As a preferred embodiment of the present application, in step 2, the relative amount of curcumin reductase and the resin carrier contained in the dual-enzyme crude enzyme mixture is 10-1000 U / g, and the relative amount of alcohol dehydrogenase and the resin carrier contained in the dual-enzyme crude enzyme mixture is 1-500 U / g.

[0025] As a preference of the present application, in step 2, the time of the cross-linking reaction is 1 to 24 hours, and the temperature of the cross-linking reaction is 10 to 40°C.

[0026] As a preferred embodiment of the present application, the reaction time in step 2 is 4 to 12 hours, and the reaction temperature is 25 to 35°C.

[0027] As a preference of the present application, the terminator is a glycine solution.

[0028] As a preference of the present application, the concentration of the glycine solution is 0.5-3 mol / L, and the pH is preferably 5-10, and more preferably 7-9.

[0029] The invention also provides an application of a dual-enzyme immobilized resin in hydrogenation reduction of curcumin.

[0030] The invention also provides an application of a dual-enzyme immobilized resin in the preparation of tetrahydrocurcumin.

[0031] The present invention also provides a method for preparing tetrahydrocurcumin, which is characterized by comprising the following steps: performing a reduction reaction on curcumin under the catalysis of the dual-enzyme immobilized resin to obtain tetrahydrocurcumin.

[0032] As a preferred embodiment of the present application, the concentration of curcumin is 0.1-100 g / L.

[0033] As a preferred embodiment of the present application, the concentration of curcumin is 1 to 30 g / L, more preferably 10 to 20 g / L, and most preferably 20 g / L.

[0034] Curcumin reductase requires reduced coenzyme II (NADPH) as a coenzyme in the process of hydrogenation reduction of curcumin to produce tetrahydrocurcumin, hydrogenates C=C, and produces the coenzyme factor NADP + . In order to make the hydrogenation reaction proceed continuously, it is necessary to regenerate the coenzyme factor. Usually, dehydrogenases such as alcohol dehydrogenase, formaldehyde dehydrogenase or glucose dehydrogenase are used as auxiliary catalysts, and the corresponding cheap substrates such as alcohol, formic acid or glucose are used to achieve the regeneration of the coenzyme factor. Therefore, curcumin reductase is used in biocatalytic processes, and usually requires the assistance of a coenzyme regeneration system, that is, it is necessary to construct a dual enzyme system. In view of this, a dual enzyme immobilization method is established in a simple, convenient and easy-to-operate manner, while achieving high enzyme activity recovery rate and high cyclic stability, which has certain application value. As a preferred embodiment of the present application, NADP with a mass concentration of 0.1mmol / L to 10mmol / L is added during the reduction reaction. + , isopropanol with a mass concentration of 1 to 20 wt % and / or Tween 80 with a mass concentration of 1 to 20 wt %.

[0035] As a preferred embodiment of the present application, the reduction reaction is carried out in buffer B, which is a phosphate buffer, preferably a 20-2000mmol / L phosphate buffer, with a pH value of 4.5-6.5; more preferably a 50-500mmol / L phosphate buffer; and further preferably a 200mmol / L phosphate buffer. The phosphate buffer is preferably a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, with a pH value of 6.0.

[0036] As a preference of the present application, the pH of the buffer solution B is preferably 4-8, more preferably 6-8.

[0037] As a preference of the present application, the reaction temperature of the reduction reaction is 20 to 50° C., and the reaction time of the reduction reaction is 1 to 24 hours.

[0038] As a preference of the present application, the reaction temperature of the reduction reaction is 20 to 50° C., and the reaction time of the reduction reaction is 1 to 24 hours.

[0039] As a preference of the present application, the reaction temperature of the reduction reaction is 30 to 50° C., and the reaction time of the reduction reaction is 1 to 24 hours.

[0040] As a preference of the present application, the reaction temperature of the reduction reaction is 45° C., and the reaction time of the reduction reaction is 6 to 24 hours.

[0041] As a preferred embodiment of the present application, after the reduction reaction is completed, the immobilized dual-enzyme resin can be recovered by filtration and used again for the preparation of tetrahydrocurcumin, thereby realizing the recycling of the immobilized dual-enzyme resin.

[0042] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0043] (1) The immobilized dual enzyme resin prepared by the method of the present invention is simple, has an enzyme activity recovery rate of more than 92%, and a protein loading capacity of 93%, which is nearly double the tetrahydrocurcumin yield of only 48.66% in the prior art.

[0044] (2) The immobilized dual-enzyme resin prepared by the method of the present invention has high enzyme activity, high cyclic stability, effectively improves the recycling of coenzymes, and has excellent characteristics of high mechanical strength, good biocompatibility, strong environmental tolerance, not easy to inactivate, and high stability;

[0045] (3) The immobilized dual enzyme resin obtained by the present invention can be uniformly dispersed in a solution by bubbling or stirring mixing to be used for catalytic preparation of tetrahydrocurcumin, which has the advantages of high product selectivity, simple process operation, low solid waste and product loss, high product yield, and effectively reducing reaction costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the catalytic reaction equation of the present invention.

[0047] Figure 2 The figure is a liquid chromatogram of the reaction solution for preparing tetrahydrocurcumin by catalytic reduction of curcumin using dual enzyme immobilized resin.

[0048] Figure 3 This is the appearance diagram of the preparation of dual-enzyme immobilized resin.

[0049] Figure 4 The effect of cross-linking reaction time with resin on the recovery rate of enzyme activity.

[0050] Figure 5 The effect of temperature on the catalytic activity of dual enzyme immobilized resin.

[0051] Figure 6 The effect of pH on the catalytic activity of dual enzyme immobilized resin. DETAILED DESCRIPTION

[0052] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0053] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0054] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the existence of other devices / apparatuses before or after the combination device / apparatus or the insertion of other devices / apparatuses between these explicitly mentioned two devices / apparatuses, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0055] In the following embodiments, the resin carriers used include ES-103B, ES-109 resin and ESR-3 of Tianjin Nankai Hecheng Technology Co., Ltd., EP-109F and EP-115F resins of Zhejiang Zhengguang Industrial Co., Ltd., and LX-1000EPA, LX-1000HA and LX-103B resins of Xi'an Lanxiao Technology New Materials Co., Ltd.

[0056] Example 1 Preparation of the original crude enzyme solution of curcumin reductase

[0057] In this embodiment, a recombinant E. coli E. coil IFE-AgvcurA containing a curcumin reductase gene was constructed according to the method in CN 116064435A. The recombinant E. coli E. coil IFE-AgvcurA and a recombinant strain E. coli IEF-LbADH expressing an alcohol dehydrogenase from Lactobacillus brevis preserved in a laboratory were fermented to obtain a fermentation broth. After the cells were collected by centrifugation, they were homogenized and crushed under high pressure. The supernatant was obtained by centrifugation again as the original crude enzyme solution for preparing the immobilized enzyme. The specific steps are as follows:

[0058] 1. Preparation of curcumin reductase fermentation broth and alcohol dehydrogenase fermentation broth:

[0059] 1.1 Preparation of seed solution: The recombinant E. coli IFE-AgvcurA containing the curcumin reductase gene and the recombinant strain E. coli IEF-LbADH expressing the alcohol dehydrogenase from Lactobacillus brevis were streaked and activated on LB plates containing 50 μg / mL kanamycin, and single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200rpm to the middle of logarithmic growth to obtain seed solution A producing curcumin reductase and seed solution B producing alcohol dehydrogenase. The final concentration of the LB medium is composed of: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, the solvent is deionized water, and the pH is 6.8-7.0;

[0060] 1.2 Shake flask fermentation: Inoculate the seed solution A and seed solution B prepared in step 1.1 into the basic fermentation medium with 50 mg / L kanamycin at a 1% inoculation rate, and culture at 37°C and 200 rpm until OD 600 1.0; add IPTG with a final concentration of 0.5mmol / L, place at 24°C and 200rpm to continue shaking induction culture for 10 hours, and obtain curcumin reductase fermentation broth and alcohol dehydrogenase fermentation broth respectively. The final concentration of the basic fermentation medium is composed of: yeast powder 12g / L, peptone 15g / L, glycerol 10g / L, Na2HPO4·12H2O8.9g / L, KH2PO4 3.4g / L, NH4Cl 2.67g / L, Na2SO4 0.71g / L, MgSO4·7H2O 0.3g / L, and pH is 6.8-7.0.

[0061] 2. Preparation of double enzyme crude enzyme mixture:

[0062] The curcumin reductase fermentation broth and the alcohol dehydrogenase fermentation broth were mixed evenly according to the cell density of the fermentation broth of 4:1, and centrifuged at 4400 rpm for 15 min using a cabinet centrifuge, the supernatant was discarded, and the cells were collected to obtain a mixed fermentation double enzyme broth;

[0063] The fermentation broth was centrifuged, and the bacteria were resuspended in 100 mL of 200 mmol / L phosphate buffer (pH 6.0) to obtain a cell suspension. The cells were broken by a high-pressure cell homogenizer to obtain a cell suspension and centrifuged. The precipitate was removed and the supernatant was collected to obtain a crude double enzyme mixture containing curcumin reductase and alcohol dehydrogenase.

[0064] Example 2 A method for preparing dual enzyme immobilized resin

[0065] Step 1, 20 mL of the crude double enzyme mixed solution containing curcumin reductase and alcohol dehydrogenase obtained in Example 1 was centrifuged at a speed of 4400 rpm for 30 min in a cabinet centrifuge, the precipitate was removed and the supernatant was collected to obtain a crude double enzyme mixed solution containing curcumin reductase and alcohol dehydrogenase;

[0066] Step 2: Add 5 g of ES-109 resin to 20 mL of the crude enzyme mixture of the double enzymes, and perform cross-linking reaction between ES-109 and the curcumin reductase and alcohol dehydrogenase in the crude enzyme mixture of the double enzymes at 1000 rpm in a constant temperature metal oscillator at 28°C for 12 hours. After the reaction is completed, filter out the supernatant, wash with 10 BV of deionized water three times, and filter and dry again to obtain an immobilized resin;

[0067] Step 3, adding the immobilized resin obtained in step 2 to 30 mL of 3 mol / L glycine solution (pH 8.5), and reacting the remaining uncrosslinked part of ES-109 with glycine crosslinking reaction at 1000 rpm in a metal oscillator at 28° C. for 8 h to obtain a dual-enzyme immobilized resin suspension;

[0068] Step 4: Filter the suspension of the dual enzyme immobilized resin to remove the supernatant, wash it with 10 BV of deionized water for 3 times, and filter it again to dry to obtain the dual enzyme immobilized resin loaded with curcumin reductase and alcohol dehydrogenase. Figure 3 The dual enzyme immobilized resin was stored at 4°C for future use.

[0069] Example 3 Application of dual enzyme immobilized resin in the preparation of tetrahydrocurcumin by hydrogenation reduction of curcumin.

[0070] The catalytic reaction equation is as follows Figure 1 shown.

[0071] Take 0.3 g of the immobilized curcumin reductase dual enzyme resin obtained in step 4 of Example 2 into a 5 mL centrifuge tube, add 10 μL of 20 mmol / L NADP + , 100 μL isopropanol, 100 μL Tween 80, 0.005 g curcumin, add 790 μL 0.1 mol / L phosphate buffer solution (pH 6.0), and react at 45°C for 1 h at a speed of 1000 rpm in a constant temperature metal oscillator. After the reduction reaction is completed, 0.2 mL of the reaction solution is added to 0.8 mL of ethanol, and the mixture is shaken in a vortex mixer (the speed of the vortex mixer is 8000 rpm) for 1 min until it is evenly mixed. After centrifugation, the upper organic phase is taken for HPLC analysis.

[0072] The HPLC analysis conditions were as follows: Agilent 1260 Infinity III HPLC, Agilent C18 chromatographic column, mobile phase of acetonitrile: 0.1% acetic acid aqueous solution = 48:52, detection wavelength of 280 nm, flow rate of 1 mL / min, column temperature of 30° C., injection volume of 10 μL.

[0073] Liquid chromatography Figure 2 As shown, tetrahydrocurcumin has keto form and enol form in the solution, with retention times of 4-5 min and 8-9 min respectively, and curcumin has a retention time of 10-11 min.

[0074] Example 4

[0075] The difference between this embodiment and embodiment 2 is that:

[0076] In this example, LX-103B is used as the resin carrier in step 2 to replace ES-109, and the rest is the same as in Example 2.

[0077] Example 5

[0078] The difference between this embodiment and embodiment 2 is that:

[0079] In this example, LX-1000EPA is used as the resin carrier in step 2 instead of ES-109, and the rest is the same as in Example 2.

[0080] Example 6

[0081] The difference between this example and Example 2 is that in this comparative example, EP-109F is used as the resin carrier in step 2 instead of ES-109, and the rest is the same as in Example 2.

[0082] Example 7

[0083] The difference between this example and Example 2 is that in this comparative example, EP-115F is used as the resin carrier in step 2 instead of ES-109, and the rest is the same as in Example 2.

[0084] Example 8

[0085] The difference between this example and Example 2 is that in this comparative example, ES-103B is used as the resin carrier in step 2 instead of ES-109, and the rest is the same as in Example 2.

[0086] Example 9

[0087] Step 1, the crude enzyme solution of curcumin reductase and alcohol dehydrogenase obtained in Example 1 is centrifuged at a speed of 4400 rpm for 30 min in a cabinet centrifuge, the precipitate is removed and the supernatant is collected to obtain a crude enzyme mixture containing curcumin reductase and alcohol dehydrogenase;

[0088] Step 2, adding multiple portions of ES-109 resin to the crude enzyme mixture obtained in step 1 to form multiple groups (each portion contains 1g of ES-109 resin and 4mL of crude enzyme mixture), and performing cross-linking reaction of ES-109 with curcumin reductase and alcohol dehydrogenase at 28°C in a constant temperature metal oscillator at a speed of 1000rpm for 4, 6, 8, 10, and 12 hours. After the reaction is completed, the supernatant is filtered off by suction, washed with 10BV of deionized water 3 times, and filtered again by suction to obtain an immobilized resin;

[0089] Step 3, adding the immobilized resin obtained in step 2 to a 3 mol / L glycine solution (pH=8.5), and performing a cross-linking reaction between the remaining uncross-linked part of ES-109 and glycine at 28°C for 8 h in a metal oscillator at a rotation speed of 1000 rpm to obtain a dual-enzyme immobilized resin suspension;

[0090] Step 4: Filter the dual-enzyme immobilized resin suspension, remove the supernatant, wash with 10 BV of deionized water for 3 times, and filter and dry again to obtain the dual-enzyme immobilized resin loaded with curcumin reductase and alcohol dehydrogenase.

[0091] Take 0.3g of the dual enzyme immobilized resin obtained in step 4 above in a 5mL centrifuge tube, add 10μL 20mmol / LNADP+, 100μL isopropanol, 100μL Tween 80, 0.005g curcumin, add 790μL 0.1mol / L phosphate buffer solution (pH6), and react at 45°C for 1h at a speed of 1000rpm in a constant temperature metal oscillator. After the reduction reaction is completed, take 0.2mL of the reaction solution, add 0.8mL of ethanol, shake in a vortex mixer (vortex mixer speed is 8000rpm) for 1min until mixed evenly, and take the upper organic phase after centrifugation for liquid chromatography analysis results.

[0092] Comparison of enzyme activities of dual enzyme immobilized resins with different cross-linking times Figure 4 As shown, the results show that the optimal cross-linking time is 8h.

[0093] Example 10

[0094] 1) Determination of enzyme activity of double enzyme crude enzyme mixture:

[0095] Take 790 μL of the crude enzyme mixture prepared in Example 1, add 10 μL of 20 mmol / L NADP+ , 100 μL isopropanol, 100 μL Tween 80, 0.005 g curcumin, placed in a 45°C constant temperature metal shaker, the speed is 1000 rpm, and the reaction is carried out for 1 hour. Take 0.2 mL of the reaction solution and add 0.8 mL of ethanol and shake it in a vortex mixer (vortex mixer speed 8000 rpm) for 1 min until the mixture is uniform. After centrifugation, the upper organic phase is taken for liquid chromatography analysis to determine the concentration of substrate and product.

[0096] One unit of enzyme activity (U) is defined as the amount of enzyme required to synthesize 1 micromole of the product tetrahydrocurcumin per minute under the above reaction conditions. The enzyme activity of the dual enzyme crude enzyme mixture was calculated based on the product concentration, and the formula is as follows:

[0097]

[0098] 2) Determination of enzyme activity of dual enzyme immobilized resin:

[0099] According to the preparation methods in Examples 3 to 8, the dual enzyme immobilized resin was obtained. 0.3 g of the obtained dual enzyme immobilized resin was placed in a 5 mL centrifuge tube, and 10 μL of 20 mmol / L NADP was added. + , 100 μL isopropanol, 100 μL Tween 80, 0.005 g curcumin, add 790 μL 0.1 mol / L phosphate buffer solution (pH 6.0), and react at 45°C for 1 h at a speed of 1000 rpm in a constant temperature metal oscillator. After the reduction reaction is completed, 0.2 mL of the reaction solution is added to 0.8 mL of ethanol, and the mixture is shaken in a vortex mixer (the speed of the vortex mixer is 8000 rpm) for 1 min until the mixture is uniformly mixed. After centrifugation, the upper organic phase is taken for liquid chromatography analysis. Then, according to the obtained liquid chromatogram, the product concentration is calculated by area normalization method.

[0100] One unit of enzyme activity (U) is defined as the amount of enzyme required to synthesize 1.0 micromole of the product tetrahydrocurcumin per minute under the above reaction conditions.

[0101] The enzyme activity recovery rate of the immobilized dual enzyme resin was calculated as follows:

[0102]

[0103] The enzyme activity recovery rates of immobilized dual enzyme resins with different resin materials are shown in Table 1.

[0104] 3) Determination of protein loading of dual enzyme immobilized resin:

[0105] The clear liquid containing the crude dual enzyme of curcumin reductase and alcohol dehydrogenase in the upper layer collected in step 1 of obtaining the dual enzyme immobilized resin according to the preparation method in Examples 3 to 8 was retained, and the filtrate after the cross-linking reaction between the resin and the crude dual enzyme solution of curcumin reductase and alcohol dehydrogenase was completed was retained. After diluting both groups of samples by 100 times, 20 μL was taken and mixed with 200 μL of the microplate reader working solution, and the samples were allowed to stand for 5 minutes and then analyzed using a microplate reader, and then the readings were substituted into the standard curve for analysis.

[0106] The protein loading capacity of the immobilized dual enzyme resin was calculated as follows:

[0107]

[0108] The protein loading capacity of immobilized dual enzyme resins with different resin materials is shown in Table 2.

[0109] Comparative Example 1

[0110] The difference between this comparative example and Example 2 is that in this comparative example, ESR-3 is used as the resin carrier in step 2 instead of ES-109, and the rest is the same as in Example 2.

[0111] Comparative Example 2

[0112] The difference between this comparative example and Example 2 is that in this comparative example, LX-1000HA is used as the resin carrier in step 2 instead of ES-109, and the rest is the same as in Example 2.

[0113] Comparative Example 3

[0114] The difference between this comparative example and Example 2 is that in this comparative example, dihydrofolate reductase fermentation broth is used instead of curcumin reductase fermentation broth in step 2, and the rest is the same as in Example 2.

[0115] The recovery rate of the prepared dihydrofolate reductase dual enzyme resin enzyme activity and the protein loading capacity were determined. The results are shown in Table 3.

[0116] Table 1 Recovery rate of enzyme activity of different immobilized curcumin reductase dual enzyme resins

[0117]

[0118] From the data analysis in Table 1, we can see that the enzyme activity recovery rate of the dual enzyme immobilized resin prepared by using ES-109 and LX-103B as the resin carrier is the highest, which is 924% and 66.5% respectively. Among them, ES-109 has the best immobilization effect.

[0119] Table 2 Protein loading of different immobilized curcumin reductase dual enzyme resins

[0120]

[0121] From the data analysis in Table 2, we can see that the protein loading capacity of the dual enzyme immobilized resin prepared by using LX-1000EPA, LX-103B and ES-109 as the resin carrier is the highest, which is 82.1%, 83.1% and 93.0% respectively. Considering the enzyme activity recovery rate and protein loading capacity, LX-103B and ES-109 are both good choices. ES-109 is the best.

[0122] Table 3 Enzyme activity recovery and protein loading of different immobilized dihydrofolate reductase dual enzyme resins

[0123] Vector Name LX-1000EPA LX-103B EP-109F EP-115F ES-103B ES-109 Enzyme activity recovery rate (%) 26.1 14 7 12.2 11.5 16.4 Protein loading (%) 72.5 66.9 56 62.8 71.6 67.2

[0124] From the data analysis in Table 3, it can be seen that the dihydrofolate reductase dual enzyme resin prepared by the carrier LX-1000EPA has the best enzyme activity recovery rate. Although dihydrofolate reductase is also a reductase, the enzyme activity recovery rate of the dual enzyme resin enzyme prepared by this method is only 26.1%, and the protein loading is 72.5%, indicating that this method has a poor effect on dihydrofolate reductase.

[0125] Comparative Example 4

[0126] The difference between this comparative example and Example 2 is that in this comparative example, ketone reductase fermentation broth is used instead of curcumin reductase fermentation broth in step 2, and the rest is the same as in Example 2.

[0127] The enone reductase dual enzyme resin activity recovery rate and protein loading capacity were determined, and the results are shown in the following table.

[0128] Table 4 Enzyme activity recovery and protein loading of different immobilized enone reductase dual enzyme resins

[0129] Vector Name LX-1000EPA LX-103B EP-109F EP-115F ES-103B ES-109 Enzyme activity recovery rate (%) 24.6 34 17.4 22 16.8 26.5 Protein loading (%) 70.9 84.4 44.3 43.6 48.3 54.8

[0130] From the data analysis in Table 4, it can be seen that the enone reductase dual enzyme resin prepared by carrier LX-103B has the best enzyme activity recovery rate, with an enzyme activity recovery rate of only 34% and a protein loading of 84.4%, indicating that this method has a poor effect on enone reductase.

[0131] Example 11 Enzymatic properties test of dual enzyme immobilized resin

[0132] 1) Determination of optimal temperature. Take 0.3 g of the dual enzyme immobilized resin obtained in Example 2 into a 5 mL centrifuge tube, add 10 μL of 20 mmol / L NADP +, 100 μL isopropanol, 100 μL Tween 80, 0.005 g curcumin, add 790 μL 0.1 mol / L phosphate buffer solution (pH 6), and place in a constant temperature metal oscillator at a speed of 1000 rpm at 30°C, 35°C, 40°C, 45°C, and 50°C for reaction for 1 hour. After the reduction reaction is completed, take 0.2 mL of the reaction solution and add 0.8 mL of ethanol in a vortex mixer (the vortex mixer speed is 8000 rpm) and shake for 1 minute until mixed evenly. After centrifugation, take the upper organic phase for liquid chromatography analysis. The comparison results of enzyme activity at different temperatures are shown in Figure 2. Figure 5 As shown, the optimum reaction temperature is 45°C.

[0133] 2) Determination of optimal pH. Take 0.3 g of the immobilized dual enzyme resin obtained in Example 2 into a 5 mL centrifuge tube and add 10 μL of 20 mmol / L NADP + , 100μL isopropanol, 100μL Tween 80, 0.005g curcumin, add 790μL 0.1mol / L phosphate buffer solution, the pH values ​​of which are 4.5, 5.0, 5.5, 6.0 and 6.5 respectively. The reaction solution was placed in a constant temperature metal oscillator with a speed of 1000rpm and a temperature of 45°C for 1h. After the reaction, 0.2mL of the reaction solution was added with 0.8mL of ethanol and shaken in a vortex mixer (vortex mixer speed 8000rpm) for 1min until mixed evenly. After centrifugation, the upper organic phase was taken for liquid chromatography analysis. The comparison results of enzyme activity under different pH conditions are shown in Figure 6 As shown, the optimum pH value is 6.0.

[0134] Example 12 Application of dual enzyme immobilized resin in the preparation of tetrahydrocurcumin

[0135] 1) Catalytic reaction with 1 g / L substrate curcumin

[0136] Take 5 g of immobilized curcumin reductase dual enzyme resin on ES-109 carrier into a 50 mL round bottom flask and add 0.031 g NADP + , 1mL isopropanol, 1mL Tween 80, 0.02g curcumin, add 0.1mol / L phosphate buffer solution (pH6.0) to 20mL, stir with a stirrer in a 45°C water bath, the speed is 200rpm, and react for 6h. After the reduction reaction is completed, take 0.2mL of the reaction solution and add 0.8mL of ethanol in a vortex mixer (the speed of the vortex mixer is 8000rpm) to shake for 1min until the mixture is uniform, and take the upper organic phase after centrifugation for liquid chromatography analysis. The concentration of tetrahydrocurcumin was measured to be 0.94g / L, and the conversion rate of substrate curcumin was 94%.

[0137] 2) Catalytic reaction with 10 g / L substrate curcumin

[0138] Take 5 g of immobilized curcumin reductase dual enzyme resin on ES-109 carrier into a 50 mL round bottom flask and add 0.031 g NADP + , 1mL isopropanol, 1mL Tween 80, 0.2g curcumin, add 0.1mol / L phosphate buffer solution (pH6.0) to 20mL, stir with a stirrer in a 45°C water bath, the speed is 200rpm, and the reaction is 12h. After the reduction reaction is completed, take 0.2mL of the reaction solution and add 0.8mL of ethanol in a vortex mixer (the vortex mixer speed is 8000rpm) to shake for 1min until the mixture is uniform, and take the upper organic phase for liquid chromatography analysis after centrifugation. The concentration of the product tetrahydrocurcumin is 8.2g / L, and the conversion rate of the substrate curcumin is 82%.

[0139] 3) Feeding amount of 20g / L

[0140] Take 5 g of immobilized curcumin reductase dual enzyme resin on ES-109 carrier into a 50 mL round bottom flask and add 0.031 g NADP + , 1mL isopropanol, 1mL Tween 80, 0.4g curcumin, add 0.1mol / L phosphate buffer solution (pH6.0) to 20mL, stir with a stirrer in a 45°C water bath, the speed is 200rpm, and react for 24h. After the reduction reaction is completed, take 0.2mL of the reaction solution and add 0.8mL of ethanol in a vortex mixer (the vortex mixer speed is 8000rpm) to shake for 1min until the mixture is uniform, and take the upper organic phase for liquid chromatography analysis after centrifugation. The concentration of the product tetrahydrocurcumin was measured to be 14.6g / L, and the conversion rate of the substrate curcumin was 73%.

[0141] Comparative Example 5

[0142] Take 16 mL of the crude dual enzyme mixture prepared in Example 1 into a 50 mL round-bottom flask and add 0.031 g NADP + , 1mL isopropanol, 1mL Tween 80, 0.4g curcumin, add 0.1mol / L phosphate buffer solution (pH6.0) to 20mL, stir with a stirrer in a 45°C water bath, the speed is 200rpm, and react for 24h. After the reduction reaction is completed, take 0.2mL of the reaction solution and add 0.8mL of ethanol in a vortex mixer (the vortex mixer speed is 8000rpm) to shake for 1min until the mixture is uniform, and take the upper organic phase for liquid chromatography analysis after centrifugation. The concentration of the product tetrahydrocurcumin was measured to be 18.4g / L, and the conversion rate of the substrate curcumin was 92%.

[0143] In summary, the substrate addition concentration is preferably 1-20 g / L, and the curcumin is the highest when the substrate addition concentration is 1 g / L, reaching 94%. As the substrate addition concentration increases, the conversion rate gradually decreases. Considering the production efficiency and economic cost, the substrate addition concentration is preferably 10-20 g / L, and the optimal is 20 g / L. This method has a high conversion rate and good selectivity. It can catalyze 20 g / L of curcumin to produce 14.6 g / L of tetrahydrocurcumin within 24 hours, and has great application prospects.

[0144] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A dual enzyme immobilization resin, characterized in that: The dual-enzyme immobilized resin comprises a resin carrier and a curcumin reductase and an alcohol dehydrogenase connected to the resin carrier.

2. The dual enzyme immobilization resin according to claim 1, characterized in that: The resin carrier is any one of epoxy resin and primary amino resin or a combination of the two.

3. A dual enzyme immobilization resin according to claim 2, characterized in that: The resin carrier is an epoxy resin, and the epoxy resin contains an epoxy functional group of any one or a combination of two of formula (I) and formula (II): (I) (II).

4. A method for preparing the dual-enzyme immobilized resin according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, mixing the fermentation broth of curcumin reductase and the fermentation broth of alcohol dehydrogenase prepared by fermentation to obtain a mixed fermentation double enzyme liquid, centrifuging, resuspending and crushing the mixed fermentation double enzyme liquid to obtain a double enzyme crude enzyme mixture containing curcumin reductase and alcohol dehydrogenase; Step 2, adding a resin carrier to the crude enzyme mixture of the double enzyme, and performing a cross-linking reaction between the resin carrier and the curcumin reductase and alcohol dehydrogenase in the crude enzyme mixture of the double enzyme to obtain a resin suspension; Step 3, filtering the resin suspension, washing the resin suspension, adding the obtained precipitate to a cross-linking terminator, and continuing the reaction to obtain a dual-enzyme immobilized resin suspension covalently bound with curcumin reductase and alcohol dehydrogenase; Step 4: The dual-enzyme immobilized resin suspension is filtered, washed, and dried to obtain a dual-enzyme immobilized resin to which curcumin reductase and alcohol dehydrogenase are covalently bound.

5. The preparation method according to claim 4, characterized in that: In step 1, the concentration ratio of the bacterial cells in the curcumin reductase fermentation broth to the bacterial cells in the alcohol dehydrogenase fermentation broth is 1:1 to 10:

1.

6. The preparation method according to claim 4, characterized in that: The relative amount of curcumin reductase and resin carrier contained in the dual-enzyme crude enzyme mixture is 10-1000 U / g, and the relative amount of alcohol dehydrogenase and resin carrier contained in the dual-enzyme crude enzyme mixture is 1-500 U / g; the time of the cross-linking reaction is 1-24 hours, and the temperature of the cross-linking reaction is 10-40°C.

7. The preparation method according to claim 4, characterized in that: The terminator is a glycine solution, the concentration of the glycine solution is 0.5-3 mol / L, and the pH is 5-10.

8. Use of a dual enzyme immobilized resin as described in any one of claims 1 to 3 in the preparation of tetrahydrocurcumin.

9. A method for preparing tetrahydrocurcumin, characterized in that: The following steps are involved: Curcumin is subjected to a reduction reaction under the catalysis of the dual enzyme immobilized resin described in any one of claims 1 to 3 to obtain tetrahydrocurcumin.

10. The method for preparing tetrahydrocurcumin according to claim 9, wherein: In the reduction reaction, the concentration of curcumin is 0.1-100 g / L.

Images