Immobilized double-enzyme resin as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to immobilized bi-enzyme resin as well as a preparation method and application thereof. The immobilized bi-enzyme resin comprises a resin carrier, and dihydrofolate reductase and alcohol dehydrogenase which are connected to the resin carrier. According to the method, dihydrofolate reductase and alcohol dehydrogenase are immobilized on the resin carrier through a covalent binding method, so that the enzyme activity recovery rate of the immobilized bi-enzyme resin is relatively high, and the efficient utilization of the enzyme used in the preparation of (6S)-tetrahydrofolic acid can be realized. The immobilized bi-enzyme resin obtained by the invention can be uniformly dispersed in a solution in a stirring and mixing manner for catalytically preparing tetrahydrofolic acid, the operation process is simple, the loss of solid wastes and products is low, the product yield is high, the process flow can be effectively simplified, and the production cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to an immobilized dual-enzyme resin and a preparation method and application thereof. Background Art

[0002] (6S)-tetrahydrofolate, abbreviated as (6S)-THF, has a chemical name of (6S)-5,6,7,8-tetrahydropteroyl-L-glutamic acid, and is an important intermediate for the preparation of L-5-methyltetrahydrofolate calcium. L-5-methyltetrahydrofolate is a natural active folic acid that can be used as a folic acid supplement and can also be used to treat megaloblastic anemia, rheumatoid arthritis, psoriasis and other pro-immune diseases. Therefore, (6S)-tetrahydrofolate is an important intermediate for the production of folic acid products.

[0003] In practice, (6S)-tetrahydrofolate can be prepared from dihydrofolate. For example, patent CN 116574768 A discloses a one-pot enzymatic method for preparing L-5-MTHF. Through this technical solution, some dihydrofolate reductases can use synthetic folic acid as a substrate and efficiently catalyze its reduction reaction to synthesize (6S)-THF, such as dihydrofolate reductase from Lactobacillus salivarius (LasDHFR), dihydrofolate reductase from Lactobacillus rhamnosus (LarDHFR), dihydrofolate reductase from Bifidobacterium bifidum (BbiDHFR), etc. However, in this technical solution, the free enzyme cannot be separated from the system, which will cause enzyme waste.

[0004] Immobilized enzyme technology refers to a biotechnology that binds free enzymes to a certain space or an insoluble carrier, thereby limiting the free movement of free enzymes, allowing them to play a catalytic role for a long time and be recycled. Immobilized enzymes can be recycled and reused, thereby effectively reducing production costs. At present, immobilized enzyme technologies mainly include: adsorption method, embedding method, cross-linking method and covalent bonding method, but there are few studies on resin covalent cross-linked aggregates of dihydrofolate reductase in the prior art. An immobilized enzyme for preparing (6S)-tetrahydrofolate from dihydrofolate is urgently needed to solve the above problems. Summary of the invention

[0005] The present invention aims to overcome the problem that the enzyme used in the preparation of (6S)-tetrahydrofolate from dihydrofolate in the prior art cannot be reused, resulting in high production costs. An immobilized dual enzyme resin and a preparation method and application thereof are proposed to overcome the above defects.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: In a first aspect, the present invention discloses an immobilized dual-enzyme resin, which comprises a resin carrier, and a dihydrofolate reductase and an alcohol dehydrogenase connected to the resin carrier.

[0007] In the present invention, dihydrofolate reductase (LasDHFR) derived from Lactobacillus salivarius is the basis for achieving efficient catalytic reaction, and alcohol dehydrogenase (LbADH) derived from Lactobacillus brevis is a coenzyme. Since the immobilized enzyme prepared by physical adsorption method has poor stability and cyclicity, the present invention selects covalent binding method to covalently immobilize dihydrofolate reductase and alcohol dehydrogenase through a resin carrier, thereby preparing an immobilized enzyme that can efficiently catalyze folic acid reduction reaction and can be recycled and reused.

[0008] Furthermore, the resin carrier is one or more of epoxy resin, amino resin, and ion exchange resin.

[0009] Furthermore, the epoxy resin is one or more of LX-1000EPA epoxy resin, LX-103B epoxy resin, ES-103B epoxy resin, and ES-109 epoxy resin.

[0010] In a second aspect, the present invention discloses a method for preparing an immobilized dual enzyme resin, characterized in that it comprises the following steps: S1, fermenting to prepare dihydrofolate reductase fermentation broth and alcohol dehydrogenase fermentation broth, and mixing them evenly to obtain a dual-enzyme fermentation broth; S2, adding a buffer solution to the dual-enzyme fermentation broth, mixing evenly, and performing cell disruption and centrifugation to obtain a dual-enzyme crude enzyme mixture; S3. Add the resin carrier as described in any one of claims 1 to 3 to the dual-enzyme crude enzyme mixture to allow the resin carrier to undergo a cross-linking reaction with the dihydrofolate reductase and the alcohol dehydrogenase. After filtering, washing, and freeze-drying, an immobilized dual-enzyme resin is obtained.

[0011] When the resin carrier is added in step S3, the relative amount of dihydrofolate reductase contained in the crude enzyme mixture of the double enzyme and the resin carrier is preferably 1×10 -5 -1×10 -2 U / g, the relative amount of alcohol dehydrogenase and resin carrier contained in the dual enzyme crude enzyme mixture is preferably 10-1000 U / g. The reaction time of the cross-linking reaction is preferably 1-24 h, and the reaction temperature is preferably 10-40°C Furthermore, the cell concentration ratio of dihydrofolate reductase and alcohol dehydrogenase in the dual enzyme fermentation broth in step S1 is 2-4: 1. In practice, when the cell density addition ratio of LasDHFR: LbADH is 3: 1, the enzyme activity efficiency is the best.

[0012] Furthermore, the buffer solution in step S2 is HEPES buffer solution. The preferred concentration of HEPES buffer solution is 0.4 mol / L, and the pH value is preferably 6-9, preferably 7.5.

[0013] In a third aspect, the present invention discloses the use of the above-mentioned immobilized dual enzyme resin in the preparation of asymmetric hydrogenation reduction of folic acid.

[0014] In a fourth aspect, the present invention discloses the use of the above-mentioned immobilized dual enzyme resin in the preparation of (6S)-tetrahydrofolate.

[0015] In a fifth aspect, the present invention discloses a method for preparing (6S)-tetrahydrofolate based on the above-mentioned immobilized dual enzyme resin, comprising the following steps: S1, configuration containing NADP + , DTT, isopropanol, folic acid reaction system; S2. Adding the immobilized dual-enzyme resin into the above reaction system, and subjecting folic acid to a reduction reaction under the catalysis of the immobilized dual-enzyme resin to obtain (6S)-tetrahydrofolate.

[0016] When configuring the reaction system, nicotinamide adenine dinucleoside phosphate (NADP + ) is 0.01 mmol / L-1.0mmol / L, dithiothreitol is referred to as DTT, the concentration of isopropanol is 1-5% (v / v), the addition amount of the immobilized dual enzyme resin in step S2 is 1-50%, and the reaction temperature of the reduction reaction is 25-45°C.

[0017] Therefore, the present invention has the following beneficial effects: (1) The present invention immobilizes dihydrofolate reductase and alcohol dehydrogenase on a resin carrier by covalent bonding. The immobilized dual enzyme resin of the present invention has a high enzyme activity recovery rate and can achieve efficient utilization of the enzymes used in the preparation of (6S)-tetrahydrofolate.

[0018] (2) The immobilized dual enzyme resin obtained by the present invention is used in the process of catalytically preparing tetrahydrofolate. The product has high optical purity, which is convenient for the subsequent preparation of further folic acid products.

[0019] (3) The immobilized dual enzyme resin obtained by the present invention can be evenly dispersed in a solution by stirring and mixing for catalytic preparation of tetrahydrofolate. The operation process is simple, solid waste and product loss are low, and the product yield is high. The process flow can be effectively simplified and the production cost can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The HPLC spectrum of THF contained in the catalyst solution of the present invention.

[0021] Figure 2It is the standard curve of THF in the present invention.

[0022] Figure 3 Schematic diagram of the effect of reaction temperature on yield during the preparation of tetrahydrofolate.

[0023] Figure 4 Schematic diagram of the effect of different enzyme ratios of immobilized enzyme on yield.

[0024] Figure 5 The figure is a comparison of the conversion rates of (6S)-tetrahydrofolate at different substrate concentrations. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further clearly and completely described below through specific examples and in combination with the accompanying drawings. It should be noted that the embodiments described in the present invention are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating processes are given, but they are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the embodiments, unless otherwise specified, can be obtained from commercial channels.

[0026] Among the resins used below, ES-103B, ES-109 resin and some primary amino resins were purchased from Tianjin Nankai Hecheng Technology Co., Ltd., and the remaining resins were purchased from Xi'an Lanxiao Technology New Materials Co., Ltd. unless otherwise specified.

[0027] The concentration of (6S)-tetrahydrofolate synthesized by immobilized dual enzyme resin is analyzed by liquid chromatography. The specific analysis method is as follows: An Infinity 1260 HPLC system (Agilent, USA) was used, with a DAD detector, a detection wavelength of 290 nm, and an Agilent C18 column (250×4.6 mm, 5 μm). The mobile phase was a pH 6.3 phosphate buffer solution at a flow rate of 1.0 mL / min.

[0028] First, a dual-enzyme crude enzyme mixture is prepared, corresponding to steps S1 and S2 in the second aspect of the present invention. The main process is: construct an engineered strain Escherichia coli E. coli IEF-LasDHFR expressing dihydrofolate reductase (LasDHFR) from Lactobacillus bulgaricus, and an engineered strain Escherichia coli E. coli IEF-LbADH expressing alcohol dehydrogenase (LbADH) from Lactobacillus brevis. After the above strains are fermented separately, the respective fermentation broths are obtained, the cells are collected by centrifugation, and the cells are crushed, and then the supernatant is obtained by centrifugation as the original crude enzyme solution made by the immobilized enzyme. The specific operation steps are as follows: Step 1, cultivation of seed solution: The colony of the freshly activated Escherichia coli engineered bacteria was inoculated into LB medium containing 50 mg / L kanamycin, and cultured overnight at 37°C and 200 rpm to obtain the seed solution. The final concentration of the LB medium was: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, the solvent was deionized water, and the pH was 6.8-7.0.

[0029] Step 2, shake flask fermentation culture: The seed solution was inoculated into a basal fermentation medium with 50 mg / L kanamycin at a 1% inoculum and cultured at 37°C and 200 rpm until the OD 600 The final concentration of IPTG is 0.6-0.8; the final concentration of IPTG is 0.5 mmol / L, and the culture is continued under shaking at 24°C and 200 rpm for 6-8 hours to obtain the fermentation liquid. The final concentration of the basic fermentation medium is composed of: yeast powder 12 g / L, peptone 15 g / L, glycerol 10 g / L, Na2HPO4·12H2O 8.9 g / L, KH2PO43.4 g / L, NH4Cl 2.67 g / L, Na2SO40.71 g / L, MgSO4·7H2O 0.3 g / L, and pH is 6.8-7.0.

[0030] Step 3, fermentation culture in a fermenter: The freshly cultured seed liquid was inoculated at a volume concentration of 1% into a basic fermentation medium containing a volume concentration of 0.05% (v / v) defoamer and 50 mg / L kanamycin, and batch-fed fermentation was carried out in a 3.0 L fermenter; the initial fermentation temperature was 37 °C, and when the OD of the fermentation liquid reached 600When the pH reaches 8.0, the fermentation temperature is lowered to 24 °C, and glycerol feed medium and lactose feed medium are added. The inducer lactose is added at a rate of 3 g / (L·h) to achieve the induced expression of the target protein. During the fermentation process, the pH of the fermentation liquid is maintained at about 6.8 by automatically adding ammonia water, the air flow is maintained at 2.0 L / (min·L fermentation medium), and the dissolved oxygen level of the fermentation liquid is maintained at >20% by coupled stirring. When the glycerol feed is completed, the OD of the fermentation liquid is 600 After the growth stops, the fermentation is stopped and the fermentation liquid is collected.

[0031] The basic culture medium used in this step is composed of 12 g / L yeast powder, 15 g / L peptone, 10 g / L glycerol, 8.9 g / L Na2HPO4·12H2O, 3.4 g / L KH2PO4, 2.67 g / L NH4Cl, 0.71 g / L Na2SO4, 0.3 g / L MgSO4·7H2O, and deionized water. The pH is adjusted to about 6.80 with ammonia water and the volume is fixed. 0.05%-0.1% defoamer is added. The glycerol feed medium used is composed of 100 g / L glycerol and 7.5 g / L MgSO4·7H2O. The lactose feed medium used is composed of 100 g / L milk.

[0032] Step 4, preparation of original crude enzyme solution: The fermentation broth was centrifuged at 4200 × g and 4 °C for 10 min, the supernatant was discarded, the cell pellet was collected, and resuspended in 0.4 mol / L HEPES buffer (pH 7.5). The OD 600 The whole cell heavy suspension was broken by high pressure homogenization, and then centrifuged at 8000×g for 10 min at 4°C to collect the supernatant as the original crude enzyme solution for immobilized enzyme.

[0033] Step 5, measurement of enzyme activity of the double enzyme crude enzyme mixture: Take 1 mL of crude enzyme mixture containing dihydrofolate reductase and alcohol dehydrogenase (2:1), add 2.2 mmol / L folic acid, 50 μmol / L NADP + , 6.4 mmol / L DTT, 3% isopropanol (v / v) were reacted in a thermostatic metal oscillator at 37°C for 1 hour at a speed of 1200 rpm. After 1.0 hour of reaction, the sample was taken out and the reaction was terminated by 5% ammonia. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the reaction for 1 minute to synthesize 1 micromole of (6S)-tetrahydrofolate under specific conditions.

[0034] Then, the crude double enzyme mixture is mixed with the resin carrier to undergo a cross-linking reaction, corresponding to step S3 of the second aspect of the present invention, and the specific operation is as follows: Take 5 g of resin and add 45 mL of 0.4 mol / L HEPES buffer (pH 7.5) at 200 rpm for 30 min at room temperature, let stand at room temperature for 30 min, wash with water and filter with filter paper to obtain the pretreated resin. Weigh 0.3 g of pretreated resin and put it into an EP tube, add 1 mL of E. coli IEF-LasDHFR and E. coli IEF-LbADH (cell mixing ratio 2:1, total cell density OD 600 = 60) was mixed with the supernatant of the crushing liquid, and the reaction was shaken at 800 rpm in a metal bath at 28°C for 12 h. After filtration, washing and freeze-drying, the immobilized dual-enzyme resin was obtained. The supernatant after the immobilization of the resin was collected to measure the immobilization efficiency and protein loading capacity.

[0035] The resin in the immobilized dual enzyme resin was optimized by experiments: Nine 5.0 mL catalytic reaction systems (2.2 mmol / L folic acid, 50 μmol / L NADP) were set up respectively. + , 6.4mmol / L DTT, 0.4 mol / L HEPES, pH 7.5) in a 5.0mL centrifuge tube, add the above 0.3g of immobilized enzymes using different resins to 8 catalytic reaction systems respectively, and place them in a thermostatic metal oscillator at 37°C and 1200 rpm for reaction. After 1.0 h of reaction, take out the sample and terminate the reaction with 5% ammonia water. The total enzyme activity in the crude enzyme solution used to prepare the immobilized enzyme is recorded as 100%, and the immobilization efficiency (enzyme activity recovery rate) is the ratio of the total enzyme activity of the immobilized enzyme to the total enzyme activity of the crude enzyme solution, that is: Immobilization efficiency = immobilized enzyme activity / crude enzyme activity × 100% Protein loading = (protein content of crude enzyme solution - protein content of immobilized supernatant) / protein content of crude enzyme solution × 100% The steps for measuring protein content are as follows: (1) Preparation of dye reagent: Weigh 10 mg of Coomassie Brilliant Blue G-250 and dissolve it in 5 mL of 95% ethanol aqueous solution. Then add 10 mL of 85% H3PO4. Finally, add deionized water to make the final volume 100 mL. Filter the mixture with filter paper to remove insoluble matter and store it in a brown bottle for later use.

[0036] (2) Protein concentration determination: Dilute the 1.0 g / L bovine serum albumin standard sample to a final concentration of 100, 50, 25, and 12.5 mg / L. Take 180 μL of each diluted sample and the sample to be tested and mix with the above 20 μL dye reagent in a 96-well plate to avoid the introduction of bubbles, and measure A within 10 min. 595Draw a standard curve and calculate the protein concentration in the samples.

[0037] Table 1. Screening performance of dual enzyme immobilized resin The samples were used for HPLC analysis of the products and the dual enzyme immobilization efficiency was calculated. The results are shown in Table 1. Las DHFR- Lb ADH@EPA is the best immobilized resin. The HPLC spectrum of THF contained in the catalyst solution is as follows: Figure 1 As shown, the standard curve of THF in this group is as follows Figure 2 shown.

[0038] The optimal reaction temperature for preparing (6S)-tetrahydrofolate by immobilized dual enzyme resin was determined by experiments: Prepare 2 mL of catalytic reaction system, the final concentration of each component is 2.2 mmol / L folic acid, 50.0 μmol / L NADP + , 3.0% (v / v) isopropanol, 6.4 mmol / L DTT, 0.4 mol / L HEPES, pH 7.5. Add 0.3g of the above LasDHFR-LbADH@EPA and place it in a thermostatic metal oscillator at 25℃, 30℃, 35℃, 40℃, and 45℃ for reaction at a speed of 1200 rpm. Each sample is reacted in parallel 3 times. After 1 hour of reaction, take out the sample, terminate the reaction with 5% ammonia water, use the sample for HPLC analysis, and calculate the enzyme activity. The results are shown in Figure 3 As shown, the optimum temperature of LasDHFR-LbADH@EPA is around 35°C.

[0039] The optimization of the enzyme ratio in the preparation of (6S)-tetrahydrofolate by immobilized dual enzyme resin was carried out through experiments: The supernatant of LasDHFR and LbADH cell lysate was used to study the production of THF by dual enzyme coupling. The amount of dual enzymes added was adjusted, that is, the cell density addition ratio of LasDHFR: LbADH was 2: 1, 3: 1, 4: 1, and 3: 2, and the cell lysate was prepared while controlling the total cell density OD 600 =45; the reaction system contained a final concentration of 50.0 μmol / L NADP + , 1.0 g / L DTT, 3.0% (v / v) isopropanol and FA with a final concentration of 1.0 g / L. The reaction was carried out in a thermostatic metal shaker at 37 °C with a rotation speed of 1200 rpm. Three parallel reactions were performed for each sample. After 1 h of reaction, the sample was taken out and the reaction was terminated with 5% ammonia water. The sample was used for HPLC analysis and the immobilization efficiency was calculated.

[0040] The results are as follows Figure 4 As shown, when the dual enzyme feed ratio is 3:1, the enzyme activity efficiency is the best, and the conversion rate reaches 65%. This ratio can be used for catalytic production in practice.

[0041] The optimal reaction time and feed amount of immobilized dual enzyme resin for preparing (6S)-tetrahydrofolate were optimized through experiments: Prepare 50 mL of catalytic reaction system, the final concentration of each group is: 50 μmol / L NADP + , 1.0 g / L DTT, 3.0% (v / v) isopropanol and folic acid (FA) at a final concentration of 1.0 g / L, 3.0 g / L, and 5.0 g / L, and then 7.5 g of the immobilized dual enzyme resin LasDHFR-LbADH@EPA was added, and the reaction was carried out in a water bath at 37 °C with magnetic stirring, and sampling was performed at intervals. Samples were taken out after 0.5 h, 1 h, and 1.5 h of reaction, and the reaction was terminated with 5% ammonia water. The samples were used for HPLC analysis, and the immobilization efficiency was calculated.

[0042] The results are as follows Figure 5 As shown in the figure, when the feed amount is 1 g / L and 3 g / L folic acid, the immobilized dual enzyme reaction takes 2.5 h to completely convert FA into THF, but when the feed amount is 5 g / L folic acid, the yield is 3.2 g / L THF. Therefore, in practice, the folic acid feed amount can be selected to be 1 g / L~3 g / L, and the reaction time can be more than 2.5 h.

[0043] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. An immobilized dual enzyme resin, characterized in that: The invention comprises a resin carrier, and dihydrofolate reductase and alcohol dehydrogenase connected to the resin carrier.

2. The immobilized dual enzyme resin according to claim 1, characterized in that: The resin carrier is one or more of epoxy resin, amino resin and ion exchange resin.

3. An immobilized dual enzyme resin according to claim 2, characterized in that: The epoxy resin is one or more of LX-1000EPA epoxy resin, LX-103B epoxy resin, ES-103B epoxy resin, and ES-109 epoxy resin.

4. A method for preparing an immobilized dual enzyme resin, characterized in that: The following steps are involved: S1, fermenting to prepare dihydrofolate reductase fermentation broth and alcohol dehydrogenase fermentation broth, and mixing them evenly to obtain a dual-enzyme fermentation broth; S2, adding a buffer solution to the dual-enzyme fermentation broth, mixing evenly, and performing cell disruption and centrifugation to obtain a dual-enzyme crude enzyme mixture; S3. Add the resin carrier as described in any one of claims 1 to 3 to the dual-enzyme crude enzyme mixture to allow the resin carrier to undergo a cross-linking reaction with the dihydrofolate reductase and alcohol dehydrogenase, and obtain an immobilized dual-enzyme resin after filtering, washing, and freeze-drying.

5. The method for preparing an immobilized dual enzyme resin according to claim 4, characterized in that: In step S1, the bacterial concentration ratio of dihydrofolate reductase and alcohol dehydrogenase in the dual-enzyme fermentation broth is 2-4:

1.

6. The method for preparing an immobilized dual enzyme resin according to claim 4, characterized in that: The buffer solution in step S2 is HEPES buffer.

7. Use of the immobilized dual enzyme resin according to any one of claims 1 to 3 in the preparation of asymmetric hydrogenation reduction of folic acid.

8. Use of the immobilized dual enzyme resin according to any one of claims 1 to 3 in the preparation of (6S)-tetrahydrofolate.

9. A method for preparing (6S)-tetrahydrofolate, based on the immobilized dual enzyme resin according to any one of claims 1 to 3, characterized in that: The steps include: S1, configuration containing NADP + , DTT, isopropanol, folic acid reaction system; S2. Adding the immobilized dual-enzyme resin into the above reaction system, and subjecting folic acid to a reduction reaction under the catalysis of the immobilized dual-enzyme resin to obtain (6S)-tetrahydrofolate.

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