Functionalized temperature-sensitive surfactant for HRP in selective froth flotation crude extract and preparation method and application of functionalized temperature-sensitive surfactant

Abstract

The invention belongs to the technical field of functional materials, and discloses a functional temperature-sensitive surfactant for HRP (horse radish peroxidase) in selective froth flotation crude extract as well as a preparation method and application of the functional temperature-sensitive surfactant. A hydrophilic temperature-sensitive monomer acrylamide AAm, a hydrophobic monomer acrylonitrile AN and a monomer glycidyl methacrylate GMA with an epoxy group are used as polymeric monomers, RAFT polymerization is performed to obtain the temperature-sensitive surfactant P (AAm-co-AN-co-GMA), the surface tension is regulated and controlled by regulating and controlling the length and temperature of a hydrophobic chain segment, and the temperature-sensitive surface active agent P (AAm-co-AN-co-GMA) is obtained. The performance and the switchable foam stability regulation effect are tested. Based on the excellent foam stabilizing performance of the temperature-sensitive surfactant and the high specific binding capacity of the aptamer to a target enzyme, the aptamer is modified to the temperature-sensitive surfactant, and the selectively functionalized temperature-sensitive surfactant P (AAm-co-AN-co-GMA)-Apt is prepared. HRP in a crude extract prepared from fresh horseradish is taken as a research target, and high-affinity binding and selective froth flotation of P (AAm-co-AN-co-GMA)-Apt to HRP are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of functionalized materials, and in particular relates to a functionalized temperature-sensitive surfactant of horseradish peroxidase HRP in a selective foam flotation crude extract, and a preparation method and application thereof. Background Art

[0002] Enzyme biocatalysts are used in food, medicine, energy and other fields due to their high catalytic efficiency, strong substrate specificity and mild reaction conditions. Enzymes can be prepared from extracts of animals, plants and microorganisms. Compared with traditional enzyme separation technologies such as salting-out, ultrafiltration, chromatography and electrophoresis, foam flotation technology has the advantages of high separation efficiency, simple equipment, easy operation, low cost and low environmental pollution. In addition, foam separation technology is more suitable for the separation of target enzymes in low-concentration complex systems. It uses bubbles as carriers, injects bubbles into the solution to form a foam layer, and then separates the foam layer from the main liquid phase. During the flotation process, bubbles can provide a large amount of surface area, giving solutes the opportunity to adsorb on the gas-liquid interface. If a solute can be selectively adsorbed on the gas-liquid interface, its concentration in the foam layer will be much higher than that in the main liquid phase. Therefore, by controlling the adsorption characteristics of the solute at the gas-liquid interface, selective separation of the solute can be achieved. However, due to the low concentration and complex composition of the target enzyme in the enzyme extract, the foam flotation technology has problems such as insufficient selectivity and low efficiency in the separation of the target enzyme.

[0003] Aptamers are small oligonucleotide sequences or short polypeptides obtained through in vitro screening. They have high affinity and strong specificity and can bind to specific targets efficiently and quickly. Compared with traditional antibodies, aptamers have the advantages of small molecular weight, stable structure, easy synthesis and modification. Once the aptamer acts on the target enzyme, it will adaptively fold into a conformation that is complementary to the shape of the target enzyme, so that the target enzyme maintains its natural conformation and high enzyme activity, and the multiple interactions between the two will ensure the highly specific binding of the aptamer to the target enzyme.

[0004] Based on the different aggregation states of thermosensitive surfactants at the air-liquid interface at different temperatures, switchable regulation of high foam stability at high temperature and low foam stability at low temperature is achieved. Aptamers with specific binding ability are introduced on thermosensitive surfactants for selective foam flotation of HRP in complex crude extracts. Based on the multiple interaction forces between aptamers and HRP and the advantages of foam flotation technology, the problem of single driving force and poor selectivity of target enzyme separation in low-concentration and complex crude extract systems is solved. Summary of the invention

[0005] The present invention uses hydrophilic thermosensitive monomer acrylamide (AAm), hydrophobic monomer acrylonitrile (AN) and monomer glycidyl methacrylate (GMA) with epoxy groups as polymerization monomers, and performs RAFT polymerization to obtain a thermosensitive surfactant P (AAm-co-AN-co-GMA), and controls the surface tension by regulating the length and temperature of the hydrophobic segment, and tests its performance and the effect of switchable foam stability. Based on the excellent foam stabilizing performance of the thermosensitive surfactant and the highly specific binding ability of the aptamer to the target enzyme, the aptamer is modified onto the thermosensitive surfactant to obtain a selectively functionalized thermosensitive surfactant P (AAm-co-AN-co-GMA) -Apt. The HRP in the crude extract prepared from fresh horseradish is used as the research target, and the high affinity binding and selective foam flotation of P (AAm-co-AN-co-GMA) -Apt to HRP are achieved.

[0006] The method for preparing a selectively functionalized temperature-sensitive surfactant comprises the following steps:

[0007] (1) Preparation of RAFT chain transfer agent (BTPA)

[0008] Under stirring, 3-mercaptopropionic acid and carbon disulfide are slowly added dropwise to a potassium hydroxide aqueous solution, and the resulting mixed solution is reacted in an oil bath pot; then benzyl bromide is slowly added dropwise, and the oil pan is heated and refluxed overnight. After the reaction is completed, the mixed solution is cooled to room temperature, chloroform is added, and hydrochloric acid is added dropwise to separate the solution phases, and the upper aqueous phase is removed. Subsequently, the lower organic phase is washed with excess deionized water for multiple times until the upper aqueous phase is clarified; after the organic phase is concentrated under reduced pressure, an appropriate amount of dichloromethane is added dropwise, and the phase is placed in a refrigerator for recrystallization, and the precipitated yellow crude product is filtered and washed with water; then, the crude product is dissolved in chloroform, and the phase is concentrated under reduced pressure again, and dichloromethane is added for recrystallization and freezing, and the phase is washed with excess deionized water. The purification operation needs to be repeated 2 to 3 times; finally, the obtained product is placed in a vacuum drying oven, and a yellow solid is obtained after drying at room temperature, which is the chain transfer agent BTPA.

[0009] In step (1), the ratio of potassium hydroxide aqueous solution, 3-mercaptopropionic acid, carbon disulfide and benzyl bromide is 35-40 mL: 32.4 mmol: 4-6 mL: 36.2 mmol, wherein the concentration of potassium hydroxide aqueous solution is 2.07 M;

[0010] In step (1), the reaction temperature in the oil bath is 20 to 30° C. and the reaction time is 4 to 6 hours;

[0011] In step (1), the heating reflux temperature is 85 to 95° C. and the time is 12 to 14 hours.

[0012] (2) Preparation of thermosensitive surfactant P(AAm-co-AN-co-GMA)

[0013] The chain transfer agent BTPA prepared in step (1), glycidyl methacrylate (GMA), acrylamide (AAm), different proportions of acrylonitrile (AN) and azobisisobutyronitrile (AIBN) are dissolved in N,N'-dimethylformamide (DMF), and the mixed solution is transferred to a round-bottom flask. The mixed solution is purged and bubbled with nitrogen to remove oxygen, and the mixture is sealed and reacted under a nitrogen atmosphere. After the reaction is completed, the round-bottom flask is placed in an ice-water bath to quench the reaction. The obtained mixed solution is dropped into methanol for precipitation, and a solid product is obtained by centrifugation. The solid product is vacuum dried and dissolved in deionized water, and unreacted monomers and small molecules are further dialyzed to remove the mixture. The solution is collected and freeze-dried to obtain a surfactant P (AAm-co-AN-co-GMA).

[0014] In step (2), the ratio of BTPA, glycidyl methacrylate, acrylamide, acrylonitrile, azobisisobutyronitrile and N,N'-dimethylformamide is 1.2mmol:2mmol:30mmol:7-9mmol:121μmol:20-25mL;

[0015] In step (2), the nitrogen purge time is 30 to 60 minutes; the temperature of the sealed reaction is 70 to 80° C., and the sealed reaction time is 3 to 5 hours; the ice water quenching time is 10 to 20 minutes; the volume of methanol used for the precipitated product is 100 to 150 mL; the dialysis bag used in the present invention has a molecular weight cutoff of 10,000 D, and the dialysis time is 48 to 60 hours.

[0016] (3) Aptamer Functionalization of Thermosensitive Surfactant P(AAm-co-AN-co-GMA)

[0017] The thermosensitive surfactant P (AAm-co-AN-co-GMA) solution obtained in step (2) is evenly mixed with the horseradish peroxidase aptamer (Apt) solution. After the mixed solution is subjected to constant temperature reaction, glycine is added to the reaction solution to continue the reaction. After the reaction is completed, low-temperature centrifugation is performed to obtain a crude product. The obtained crude product is dissolved in water again, and the dissolution-precipitation cycle is repeated 2 to 4 times to remove the residual glycine and Apt. The obtained product is the selectively functionalized thermosensitive surfactant P (AAm-co-AN-co-GMA) -Apt.

[0018] In step (3), the dosage ratio of P(AAm-co-AN-co-GMA) solution, horseradish peroxidase aptamer solution and glycine is 400 μL:200 μL:1.2 mg; wherein the concentration of P(AAm-co-AN-co-GMA) solution is 1 mg / mL, the concentration of horseradish peroxidase aptamer solution is 10 μM, and the solvent is deionized water;

[0019] In step (3), the isothermal reaction temperature is 30-40°C and the reaction time is 22-24 hours; glycine is added and the reaction is continued for 5-8 hours.

[0020] In step (3), the aptamer sequence of horseradish peroxidase is: 5'-NH2-GTC CGC AAG TTG TCG CGCGAT AAG CTT ATG GCT GGT TGA-3'.

[0021] Based on the thermosensitive surfactant P (AAm-co-AN-co-GMA), the present invention introduces a highly selective horseradish peroxidase aptamer to successfully prepare a selectively functionalized thermosensitive surfactant P (AAm-co-AN-co-GMA)-Apt material, which is then used for the selective foam flotation of HRP in a crude extract.

[0022] Based on the excellent foam stabilizing performance of the thermosensitive surfactant and the highly specific binding ability of the aptamer to the target enzyme, the present invention modifies the aptamer onto the thermosensitive surfactant to prepare the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt. Taking HRP in the crude extract as the research target, the high affinity binding of P(AAm-co-AN-co-GMA)-Apt to HRP and the selective foam flotation are achieved.

[0023] The beneficial effects of the present invention are:

[0024] Based on the highly specific binding ability of the aptamer to the target enzyme, a thermosensitive surfactant with selective recognition function for horseradish peroxidase (HRP) was constructed to achieve selective adsorption of HRP at the air-liquid interface; the surface activity of the thermosensitive surfactant can be regulated by changing the temperature, and the high foam stability at high temperature and the low foam stability at low temperature can be controllably adjusted, thereby achieving selective foam flotation, efficient separation and purification, and rapid recovery of HRP in the HRP crude extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis process of thermosensitive surfactant P (AAm-co-AN-co-GMA).

[0026] Figure 2(a) FT-IR spectra of thermosensitive surfactants P1, P2 and P3, (b) 1 (c) H NMR spectrum and GPC test graph.

[0027] Figure 3 (a) Transmittance changes of 30 mg / mL P1, P2 and P3 solutions, (b) T values ​​of different concentrations of P1, P2 and P3 solutions cp .

[0028] Figure 4 (a) Surface tension of thermosensitive surfactants P1, P2 and P3 solutions as a function of concentration; (b) Contact angles of thermosensitive surfactants P1, P2 and P3.

[0029] Figure 5 Schematic diagram of the synthesis of P(AAM-co-AN-co-GMA)-Apt.

[0030] Figure 6 (a) FT-IR spectra of Apt, P(AAm-co-AN-co-GMA) and P(AAm-co-AN-co-GMA)-Apt, (b) thermogravimetric analysis of P(AAm-co-AN-co-GMA) and P(AAm-co-AN-co-GMA)-Apt.

[0031] Figure 7 (a) Reaction heat release and (b) molar enthalpy change of the titration of HRP solution with P(AAm-co-AN-co-GMA)-Apt solution, (c) specific binding of P(AAm-co-AN-co-GMA)-Apt to HRP and nonspecific binding to trypsin, bromelain, lipase, soy protein, and bovine serum albumin.

[0032] Figure 8 (a) The surface tension of P(AAm-co-AN-co-GMA) solution and P(AAm-co-AN-co-GMA)-Apt solution changes with concentration, (b) t 1 / 2 and FS.

[0033] Fig. 9(a) IR spectra of free HRP and P(AAm-co-AN-co-GMA)-Apt, (b) thermogravimetric analysis of P(AAm-co-AN-co-GMA)-Apt and P(AAm-co-AN-co-GMA)-Apt-HRP complex, (c) gel electrophoresis analysis of HRP in crude extract. M: standard protein molecular weight; lane 1: commercial HRP; lane 2: HRP crude extract; lane 3: defoaming solution; lane 4: further low-temperature precipitation of defoaming solution, (d) CD spectra of free HRP and HRP obtained by foam flotation.

[0034] Fig.10 Effects of different P(AAm-co-AN-co-GMA)-Apt concentrations (a), HRP concentrations (b), gas flow rate (d), temperature (e) and foam separation time (f) on HRP froth flotation performance, (c) Langmuir model and Freundlich model.

[0035] Fig.11 (a) Schematic diagram of further processing of foam liquid based on the thermal response properties of P(AAm-co-AN-co-GMA)-Apt, and (b) purification multiples under different experimental conditions.

[0036] Fig.12 (a) Reusability of HRP in crude extract of P(AAm-co-AN-co-GMA)-Apt selective froth flotation, (b) recovery rate of P(AAm-co-AN-co-GMA)-Apt after repeated use. DETAILED DESCRIPTION

[0037] The present invention is further illustrated below by means of specific examples and the technical solution of the present invention is described in detail in conjunction with the accompanying drawings. Obviously, the examples cited are not all embodiments of the present invention. Without departing from the essential content of the present invention, any obvious improvement, replacement or change that can be made by those skilled in the art shall fall within the protection scope of the present invention.

[0038] Embodiment 1:

[0039] (1) Preparation of RAFT chain transfer agent (BTPA), the process is as follows Figure 1 As shown in (a)

[0040] Under stirring, 3-mercaptopropionic acid (2.8mL, 32.4mmol) and carbon disulfide (CS2, 5mL) were slowly added dropwise to a potassium hydroxide aqueous solution (2.07M, 36mL). The resulting mixed solution was reacted in an oil bath at 25°C for 6h, and then benzyl bromide (6.2g, 36.2mmol) was slowly added dropwise. The oil bath was heated to 90°C and refluxed overnight for 12h. After the reaction, the mixed solution was cooled to room temperature and transferred to a 500mL beaker. Chloroform (80mL) was added, and hydrochloric acid was added dropwise until the solution was phase-separated, and the upper aqueous phase was removed. Subsequently, the lower organic phase was washed several times with excess deionized water until the upper aqueous phase was clear. After the organic phase was concentrated under reduced pressure, an appropriate amount of dichloromethane was added dropwise, and it was placed in a refrigerator for recrystallization. The precipitated yellow crude product was filtered and washed with water. Next, the crude product is dissolved in chloroform, concentrated under reduced pressure again, recrystallized by adding dichloromethane, and frozen to precipitate, and washed with excess deionized water. The purification operation needs to be repeated 2 to 3 times. Finally, the obtained product is placed in a vacuum drying oven and dried at room temperature for 48 hours to obtain a yellow solid, which is the chain transfer agent BTPA.

[0041] (2) Preparation of thermosensitive surfactant P(AAm-co-AN-co-GMA), the process is as follows Figure 1 as shown in (b).

[0042] Chain transfer agent BTPA (0.0318g, 1.2mmol), glycidyl methacrylate (GMA, 0.284g, 2mmol), acrylamide (AAm, 2.14g, 30mmol), different proportions of acrylonitrile (AN, 0.371g, 7mmol; 0.425g, 8mmol; 0.478g, 9mmol) and azobisisobutyronitrile (AIBN, 20mg, 121μmol) were dissolved in N'N-dimethylformamide (DMF, 22mL), and the mixed solution was transferred to a round-bottom flask. The mixed solution was purged and bubbled with nitrogen for 30min to remove oxygen, and the round-bottom flask was placed in an oil bath at 70℃. The mixed solution was sealed and stirred for reaction for 3h under nitrogen atmosphere. After the reaction was completed, the round-bottom flask was placed in an ice-water bath for quenching for 10min, and the resulting mixed solution was dropped into methanol (100mL) for precipitation, and the solid product was obtained by centrifugation. The solid product was vacuum dried and dissolved in deionized water. After further dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 10,000 D, the solution was collected and freeze-dried to obtain the surfactant P (AAm-co-AN-co-GMA).

[0043] In step (2), the feed amounts of AAm and GMA were fixed, and the AN feed amount was adjusted to 7, 8 and 9 mmol to prepare three thermosensitive surfactants P (AAm-co-AN-co-GMA) with different AN hydrophobic segment lengths, which were recorded as P1, P2 and P3, respectively.

[0044] like Figure 2 As shown in the FT-IR spectrum of Figure (a), P1, P2 and P3 are found at 3350cm -1 and 3199cm -1 The NH stretching vibration absorption peak of AAm is shown at 1664 cm -1 The C=O stretching vibration absorption band appears at 2242 cm -1 The characteristic absorption band of AN C≡N stretching vibration appears at 913cm -1 and 838cm -1 The characteristic absorption band attributed to the stretching vibration of the epoxy group on GMA was found. And with the increase of AN feed amount, the C≡N characteristic absorption peak of AN (2242cm -1 ) intensity gradually increases, proving that the AN hydrophobic segment of the thermosensitive surfactant gradually increases from P1 to P3. Figure 2 (b) is the 1H NMR spectrum of P1, P2 and P3, and it is found that the trends of the three spectra are basically the same. Finally, GPC analysis was performed to determine the molecular weight and distribution of P1, P2 and P3. Figure 2 As shown in (c), the GPC elution lines of P1, P2 and P3 are all single-peaked, and the molecular weights of P1, P2 and P3 are 11136Da, 12768Da and 14132Da, respectively. The molecular weight distribution (PDI) is relatively narrow, indicating that the molecular weight of the thermosensitive surfactant is monodisperse.

[0045] Depend on Figure 3 (a) shows that the thermosensitive surfactants P1, P2, and P3 exhibit good thermosensitive state. The surfactant solution can be changed from turbid to clear by increasing the temperature, and become turbid again by lowering the temperature, thereby realizing the recycling of thermosensitive surfactants. At a concentration of 30 mg / mL, the turbidity points of the thermosensitive surfactants P1, P2, and P3 are 20.8, 23.6, and 26.2°C, respectively. It can be seen that from P1 to P3, as the hydrophobic segment AN increases, the turbidity point gradually increases. The turbidity points of P1, P2, and P3 solutions at different concentrations were further measured. Figure 3 As shown in (b), the T of the thermosensitive surfactant cp The UCST of surfactants P1, P2 and P3 showed an inverted "U"-shaped trend with concentration changes, and were 27.1, 29.6 and 33.2 °C, respectively, indicating that the turbidity point of thermosensitive surfactants can be controlled by regulating the length of the hydrophobic segment.

[0046] Depend on Figure 4From (b), we can see that from P1 to P3, with the increase of AN hydrophobic segments, the contact angle of the thermosensitive surfactant increases from 59.58° to 95.19°, and the hydrophobicity gradually increases. The ability to reduce surface tension should also increase positively, that is, the value of γmin gradually decreases. Figure 4 (a) The relationship between the surface tension and concentration of thermosensitive surfactants P1, P2 and P3 solutions with different AN hydrophobic chain lengths at 30°C was studied. Figure 4 (a)), with the increase of concentration, the surface tension of P1, P2 and P3 solutions all showed a trend of first decreasing and then remaining basically unchanged. The γmin values ​​of P1, P2 and P3 aqueous solutions were 60.66, 52.46 and 63.62 mN / m, respectively, and P2 solution had a lower surface tension.

[0047] (3) Functionalization of the thermosensitive surfactant P(AAm-co-AN-co-GMA) using the aptamer. Figure 5 shown.

[0048] The thermosensitive surfactant P(AAm-co-AN-co-GMA) solution (1 mg / mL, 400 μL) was mixed with the horseradish peroxidase aptamer solution (Apt, 10 μM, 200 μL) and the mixture was reacted at 35°C for 24 hours. After the reaction, 1.2 mg of glycine was added to the reaction solution and the reaction was continued for 6 hours. After the reaction was completed, the crude product was separated by low-temperature centrifugation, and the crude product was dissolved in water again. The residual glycine and Apt were removed by dissolution-precipitation cycles for 2 to 4 times, and the product obtained was the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt.

[0049] The FT-IR spectrum of P(AAm-co-AN-co-GMA)-Apt is shown in Figure 6 As shown in (a), 3412cm -1 and 1691cm -1 The characteristic absorption peak of 1638cm is attributed to the stretching vibration absorption peak of NH and C=O, while -1 The absorption peak at 3412 cm is the NH deformation vibration absorption peak. Comparing the FT-IR spectrum of P(AAm-co-AN-co-GMA), it can be seen that when Apt is grafted onto the thermosensitive surfactant P(AAm-co-AN-co-GMA), the absorption peak at 3412 cm in the FT-IR spectrum of P(AAm-co-AN-co-GMA)-Apt is 1.346 cm -1 and 1691cm -1 The stretching vibration absorption peaks of amino NH and carbonyl C=O are enhanced, and at 1638cm -1The NH deformation vibration absorption peak on the amide bond is also enhanced accordingly, indicating that when Apt is modified to P(AAm-co-AN-co-GMA)-Apt, the intensity of the absorption peaks overlapping the two positions is further enhanced. Based on the above results, it can be concluded that Apt is successfully combined with the thermosensitive surfactant to obtain the P(AAm-co-AN-co-GMA)-Apt material.

[0050] The compositions of P(AAm-co-AN-co-GMA) and P(AAm-co-AN-co-GMA)-Apt materials were further characterized by thermogravimetric analysis. Figure 6 As shown in (b), in the first stage 25-220°C, the thermal decomposition of the two materials is mainly caused by water loss, which is due to the evaporation of water in the low temperature range. Then, in the second stage 220-480°C, the materials began to decompose further, and the weight losses of P(AAm-co-AN-co-GMA) and P(AAm-co-AN-co-GMA)-Apt in this stage were 57.87% and 60.95%, respectively. By comparing the weight loss percentages of the two, it can be seen that the weight loss of Apt is about 3.08%, which further confirms the successful preparation of P(AAm-co-AN-co-GMA)-Apt material.

[0051] Embodiment 2:

[0052] Affinity and selectivity of the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt for HRP.

[0053] The binding stoichiometry between P(AAm-co-AN-co-GMA)-Apt and HRP was quantitatively studied using ITC to verify that the aptamer on P(AAm-co-AN-co-GMA)-Apt has affinity for HRP. Figure 7 As shown in (a) and (b), there is an obvious heat change in the process of titrating the P(AAm-co-AN-co-GMA)-Apt solution with the HRP solution, proving that there is a strong binding interaction between the two. The test data were fitted based on the Independent model to obtain the corresponding dissociation constant Kd, stoichiometric ratio n, enthalpy change ΔH, entropy change ΔS and other parameters. The large negative enthalpy change (ΔH = -22.66 kJ / mol) and positive entropy change (ΔS = 34.66 J / mol·K) indicate that the conformation of the aptamer on P(AAm-co-AN-co-GMA)-Apt has changed, and it selectively binds to HRP through hydrophobic interactions and hydrogen bonds. The Gibbs free energy change (ΔG) and dissociation constant (Kd) are -32.99 kJ / mol and 1.66×10 -6M. Combined with the above results, a possible binding mechanism was proposed. First, the affinity aptamer on P(AAm-co-AN-co-GMA)-Apt adaptively folded into a specific conformation when it came into contact with HRP. Second, the aptamer specifically bound to HRP through hydrogen bonds and hydrophobic interactions.

[0054] The specificity of P(AAm-co-AN-co-GMA)-Apt to HRP was further evaluated, and non-specific experiments were performed on commonly used proteins (trypsin, bromelain, lipase, soy protein and bovine serum albumin). As a new type of ligand, aptamers have the ability to bind to target molecules in a targeted manner. Compared with traditional physical adsorption and covalent binding, aptamers can more efficiently achieve specific adsorption of target substances. Figure 7 As shown in (c), 96% of HRP can bind to P(AAm-co-AN-co-GMA)-Apt to form a P(AAm-co-AN-co-GMA)-Apt-HRP complex, while the binding of the other five proteins to P(AAm-co-AN-co-GMA)-Apt does not exceed 10%. Therefore, P(AAm-co-AN-co-GMA)-Apt can specifically bind to HRP.

[0055] Embodiment 3:

[0056] Bubble-stabilizing properties of selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt

[0057] Figure 8 (a) is the surface tension test of the thermosensitive surfactant P(AAm-co-AN-co-GMA) and the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt. After the thermosensitive surfactant was selectively functionalized, the value of γmin was reduced from 52.46 mN / m to 50.22 mN / m, proving that P(AAm-co-AN-co-GMA)-Apt has good surface activity. From the inflection point of the γ-lg C curve, it can be determined that the cmc of P(AAm-co-AN-co-GMA)-Apt is 11.6 mg / mL, while the cmc of P(AAm-co-AN-co-GMA) is 18.3 mg / mL. The performance of P(AAm-co-AN-co-GMA)-Apt in stabilizing foam was analyzed by half-life and foam stability index. Figure 8 (b) shows that the t 1 / 2and FS were 75min and 56.02%, functionalization, foam properties and application of P(AAm-co-AN-co-GMA) thermosensitive surfactants compared with the t 1 / 2 (68 min) and FS (52.63%), it was found that the selective functionalization of the thermosensitive surfactant enhanced the stability of the foam, that is, P(AAm-co-AN-co-GMA)-Apt had excellent foam stability.

[0058] Embodiment 4:

[0059] Foam flotation of HRP in fresh horseradish using selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt

[0060] (1) Preparation of crude horseradish peroxidase (HRP) extract from fresh horseradish

[0061] Rinse the fresh horseradish with deionized water, chop it and weigh 100g of fresh horseradish, add it to the PBS buffer solution (pH=7.5, 50mM, 100mL) pre-cooled at 4℃, and homogenize it 5 times with a homogenizer, each time for 1min, with an interval of 2min. After the homogenization, the mixed solution was filtered, and the filtrate was ultrasonically oscillated 3 times in a 4℃ ice water bath, each time for 15min, with an interval of 10min. After the ultrasonication, the extract was centrifuged at 4℃ for 10min, and after removing the precipitate, the supernatant was centrifuged again for 10min. The final solution was the crude HRP extract.

[0062] (2) Selective Foam Flotation of HRP in Horseradish Extract by P(AAM-co-AN-co-GMA)-Apt

[0063] 50 mL of P(AAm-co-AN-co-GMA)-Apt with different concentrations and 50 mL of HRP crude extract with different concentrations were mixed evenly, and the mixed solution was mixed in a 30°C constant temperature water bath for 30 min, and then transferred to a glass flotation column kept at a constant temperature of 30°C, and nitrogen entered the glass column through a gas distributor at a certain flow rate. Therefore, the P(AAm-co-AN-co-GMA)-Apt-HRP complex is adsorbed on the gas-liquid interface, and as the bubbles rise, they further enter the foam phase from the water phase, and the bubbles with the P(AAm-co-AN-co-GMA)-Apt-HRP complex flow out from the top of the glass column and are stored in the foam collector.

[0064] (3) Evaluation of the specificity and affinity of P(AAM-co-AN-co-GMA)-Apt for HRP

[0065] In order to prove that P(AAm-co-AN-co-GMA)-Apt successfully and selectively adsorbed HRP in the crude extract, a P(AAm-co-AN-co-GMA)-Apt-HRP composite was obtained. The composition of the composite material was characterized by infrared spectroscopy and thermogravimetric analysis. Fig. 9 (a) FT-IR spectrum shows that the free HRP has a peak at 1530 cm -1 A characteristic absorption peak appeared at 1530 cm-1, which corresponds to the combined absorption peak of the bending vibration of the NH bond and the stretching vibration of the CN bond of the peptide bond skeleton on HRP. The FT-IR spectra of P(AAm-co-AN-co-GMA)-Apt and P(AAm-co-AN-co-GMA)-Apt-HRP complex are almost the same. The only difference is that the FT-IR spectrum of the complex also has a characteristic absorption peak at 1530 cm-1, which corresponds to the combined absorption peak of the bending vibration of the NH bond and the stretching vibration of the CN bond of the peptide bond skeleton on HRP. This phenomenon shows that HRP is selectively adsorbed on the gas-liquid interface, thus forming the P(AAm-co-AN-co-GMA)-Apt-HRP complex material.

[0066] Secondly, the P(AAm-co-AN-co-GMA)-Apt-HRP complex was further analyzed by thermogravimetric analysis, as shown in Fig. 9 As shown in (b), the thermal decomposition in the first stage (25-220°C) was mainly caused by water loss of the material. In the second stage (220-450°C) of thermal decomposition, the weight loss of P(AAm-co-AN-co-GMA)-Apt was 61.97%, while the weight loss of P(AAm-co-AN-co-GMA)-Apt-HRP complex was 64.56%. Comparing the weight loss percentages of the thermal decomposition of the two, it can be seen that the weight loss percentage of HRP is about 2.59%, which once again proves that HRP is selectively bound to the P(AAm-co-AN-co-GMA)-Apt material through foam flotation.

[0067] The effect of HRP in the crude extract of foam flotation was analyzed by gel electrophoresis SDS-PAGE. Fig. 9As shown in (c), there are many impurity proteins in the crude HRP extract (lane 2). After P(AAm-co-AN-co-GMA)-Apt specifically binds to HRP in the crude extract and performs foam flotation, the impurity proteins in the defoaming solution are reduced (lane 3). After further purification using the thermal response performance of P(AAm-co-AN-co-GMA)-Apt, there is only an HRP band of about 44.3 kDa on the protein lane (lane 4), which is consistent with the band of commercial HRP (lane 1), indicating that P(AAm-co-AN-co-GMA)-Apt selectively binds to HRP in the crude extract through foam separation.

[0068] The CD spectra of free HRP and HRP obtained by selective foam flotation of P(AAm-co-AN-co-GMA)-Apt from 190nm to 240nm were further recorded by circular dichroism (CD) spectrometer to prove that the conformation of HRP remained basically unchanged during the adsorption at the air-liquid interface and the cooling sedimentation process. Fig. 9 As shown in (d), as a common feature of HRP, the negative peaks at 208nm and 222nm correspond to the α-helical structure, while the positive peak at about 196nm corresponds to the β-folded structure. Through the spectral structure of HRP, it was found that the HRP obtained by foam flotation also showed a similar spectral structure, and the trends of the two spectral lines were basically the same, indicating that the aptamer on P(AAm-co-AN-co-GMA)-Apt adaptively folded to form a shape complementary to HRP, which can better maintain the original conformation of HRP during adsorption at the gas-liquid interface and cooling sedimentation.

[0069] Embodiment 5:

[0070] Optimization of Experimental Conditions for Selective Foam Flotation of HRP in Crude Horseradish Extract by P(AAM-co-AN-co-GMA)-Apt

[0071] The effect of HRP in the crude extract of selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt foam flotation under different experimental conditions was comprehensively discussed from four aspects: protein enrichment ratio (Ep), protein recovery rate (Rp), enzyme activity enrichment ratio (Ee) and enzyme activity recovery rate (Re), so as to obtain the optimal experimental conditions.

[0072] Effects of different P(AAm-co-AN-co-GMA)-Apt concentrations on the selective adsorption performance Fig.10As shown in (a), when the concentration of P(AAm-co-AN-co-GMA)-Apt was 2-8 mg / mL, the recovery rates Rp and Re gradually increased to 87.07% and 93.05%, while the enrichment ratios Ep and Ee slightly decreased to 1.41 and 1.78; when the concentration was greater than 8 mg / mL, the recovery rates Rp and Re began to decrease, while the enrichment ratios Ep and Ee did not change much.

[0073] Effect of different initial HRP concentrations in crude extract on foam flotation Fig.10 As shown in (b), when the initial HRP concentration is in the range of 0.2-0.8 mg / mL, as the HRP concentration in the crude extract increases, the recovery rates Rp and Re increase significantly, while the changes in the enrichment ratios Ep and Ee are not very obvious; when the HRP concentration is 0.8 mg / mL, the recovery rates Rp and Re reach the maximum values ​​of 87.18% and 96.29%, and the enrichment ratios Ep and Ee are 1.65 and 1.85.

[0074] The adsorption behavior of P(AAm-co-AN-co-GMA)-Apt-HRP complex at the air-liquid interface was further analyzed by Langmuir and Freundlich adsorption isotherm models. 2 =0.9384) than the Freundlich model (R 2 =0.8905) is more suitable to describe the adsorption behavior of P(AAm-co-AN-co-GMA)-Apt-HRP complex at the air-liquid interface ( Fig.10 (c) ), P(AAm-co-AN-co-GMA)-Apt-HRP is adsorbed as a monolayer at the air-liquid interface.

[0075] Effects of different gas flow rates on HRP froth flotation Fig.10 As shown in (d), in the gas flow rate range of 50-100mL / min, the recovery rates Rp and Re increase significantly with the increase of N2 flow rate, while the enrichment ratios Ep and Ee gradually decrease with the increase of flow rate; when the gas flow rate is 100mL / min, Rp and Re increase to 87.12% and 93.52%, Ep and Ee decrease to 1.64 and 1.76; when the gas flow rate is greater than 100mL / min, the rate of increase of the recovery rate slows down, while the enrichment ratio still decreases.

[0076] Effects of different temperatures on the selective adsorption of HRP Fig.10As shown in (e), as the temperature increases from 25°C to 30°C, the enrichment ratios Rp and Re increase to 87.15% and 94.52%, and Ep and Ee increase to 1.68 and 1.78. Continuing to increase the temperature has little effect on the recovery rate (Rp, Re) and enrichment ratio (Ep, Ee).

[0077] Effects of different adsorption times on HRP foam flotation Fig.10 As shown in (f), within 5-20 minutes, as the flotation time increases, the recovery rates Rp and Re increase significantly, while the enrichment ratios Ep and Ee change slightly. At 20 minutes, the recovery rates Rp and Re reach their maximum values ​​of 90.20% and 98.70%, and the enrichment ratios Ep and Ee are 1.80 and 2.15.

[0078] Based on the analysis of the HRP effect in froth flotation under the above different experimental conditions, the optimal flotation experimental conditions were obtained: the concentration of P(AAm-co-AN-co-GMA)-Apt was 8 mg / mL, the initial concentration of HRP was 0.8 mg / mL, the gas flow rate was 100 mL / min, the flotation temperature was 30°C, and the flotation time was 20 min. Under the optimal experimental conditions, the recoveries Rp and Re were 90.20% and 98.70%, and the enrichment ratios Ep and Ee were 1.80 and 2.15.

[0079] Embodiment 6:

[0080] Further purification of HRP foam solution by selective foam flotation based on the thermal response of P(AAm-co-AN-co-GMA)-Apt

[0081] Thermosensitive surfactants have better bubble stability above Tcp, while bubbles stable below Tcp have poorer stability, and low temperature will accelerate the bursting of bubbles. Therefore, based on the thermal response performance of P(AAm-co-AN-co-GMA)-Apt, the foam liquid obtained after HRP foam flotation can be further treated. First, the temperature is lowered to 10°C to desorb the P(AAm-co-AN-co-GMA)-Apt-HRP complex adsorbed at the gas-liquid interface from the gas-liquid interface, and low-temperature centrifugation is performed to allow the P(AAm-co-AN-co-GMA)-Apt-HRP complex to fully aggregate and settle ( Fig.11 After removing the supernatant, the temperature was raised to dissolve the aggregated complex again. After further treatment using the thermal response of P(AAm-co-AN-co-GMA)-Apt, the purification factor of HRP in the obtained solution increased to 9.75 ( Fig.11 (b)).

[0082] Embodiment 7:

[0083] Cycling performance of P(AAm-co-AN-co-GMA)-Apt

[0084] According to previous studies, the interaction between protein and aptamer mainly includes hydrogen bonds and van der Waals forces, which can be destroyed by washing with acidic solution and high ionic strength. In this study, HRP was released from the P(AAm-co-AN-co-GMA)-Apt-HRP complex by adding 0.1M HAc solution, and further obtained reusable P(AAm-co-AN-co-GMA)-Apt by cooling, centrifugation and drying. The P(AAm-co-AN-co-GMA)-Apt was used again for the foam flotation of HRP in the crude extract. After 6 cycles, P(AAm-co-AN-co-GMA)-Apt could still effectively promote the selective foam flotation of HRP, with Ep and Rp of 1.65 and 83.6%, respectively ( Fig.12 At the same time, about 81.28% of P(AAm-co-AN-co-GMA)-Apt( Fig.12 (b)). Therefore, the froth flotation of HRP in the crude liquid can be effectively and selectively promoted by recovering P(AAm-co-AN-co-GMA)-Apt.

[0085] The embodiments described are preferred implementations of the present invention, and all other embodiments obtained by those skilled in the art without making any obvious improvements, substitutions or modifications belong to the protection scope of the present invention.

Claims

1. A functionalized thermosensitive surfactant for selective froth flotation of HRP in crude extract, characterized in that: The hydrophilic thermosensitive monomer acrylamide AAm, the hydrophobic monomer acrylonitrile AN and the monomer glycidyl methacrylate GMA with an epoxy group are used as polymerization monomers to carry out RAFT polymerization to obtain a thermosensitive surfactant P(AAm-co-AN-co-GMA), and then the aptamer is modified onto the thermosensitive surfactant, which is recorded as P(AAm-co-AN-co-GMA)-Apt.

2. The functionalized thermosensitive surfactant of HRP in the crude extract of selective froth flotation according to claim 1, characterized in that: The steps include: (1) Preparation of RAFT chain transfer agent BTPA: Under stirring, 3-mercaptopropionic acid and carbon disulfide are slowly added dropwise to a potassium hydroxide aqueous solution, and the resulting mixed solution is reacted in an oil bath pot; then benzyl bromide is slowly added dropwise, and the oil pan is heated and refluxed overnight. After the reaction is completed, the mixed solution is cooled to room temperature, chloroform is added, and hydrochloric acid is added dropwise until the solution is phase-separated, and the upper aqueous phase is removed, and then the lower organic phase is washed with excess deionized water for multiple times until the upper aqueous phase is clear; after the organic phase is concentrated under reduced pressure, an appropriate amount of dichloromethane is added dropwise, and the product is placed in a refrigerator for freezing and recrystallization, and the precipitated yellow crude product is filtered and washed with water; then, the crude product is dissolved in chloroform, and concentrated under reduced pressure again, and dichloromethane is added for recrystallization and freezing, and the product is washed with excess deionized water, and the purification operation needs to be repeated several times; finally, the obtained product is placed in a vacuum drying oven, and dried at room temperature to obtain a yellow solid, which is the chain transfer agent BTPA; (2) Preparation of thermosensitive surfactant P (AAm-co-AN-co-GMA): The chain transfer agent BTPA prepared in step (1), glycidyl methacrylate (GMA), acrylamide (AAm), acrylonitrile (AN) and azobisisobutyronitrile (AIBN) in different proportions are dissolved in N,N'-dimethylformamide (DMF), and the mixed solution is transferred to a round-bottom flask, the mixed solution is purged and bubbled with nitrogen to remove oxygen, and the mixture is sealed and reacted under a nitrogen atmosphere. After the reaction is completed, the round-bottom flask is placed in an ice-water bath to quench the reaction, the obtained mixed solution is dropped into methanol for precipitation, and a solid product is obtained by centrifugation. The solid product is vacuum dried and then dissolved in deionized water, and unreacted monomers and small molecules are further removed by dialysis, and the solution is collected and freeze-dried to obtain a surfactant P (AAm-co-AN-co-GMA); (3) Aptamer functionalization of thermosensitive surfactant P(AAm-co-AN-co-GMA): The thermosensitive surfactant P (AAm-co-AN-co-GMA) solution obtained in step (2) is evenly mixed with the horseradish peroxidase aptamer (Apt) solution. After the mixed solution is subjected to constant temperature reaction, glycine is added to the reaction solution to continue the reaction. After the reaction is completed, low-temperature centrifugation is performed to obtain a crude product. The crude product is dissolved in water again, and the dissolution-precipitation cycle is repeated several times to remove the residual glycine and Apt. The obtained product is the selectively functionalized thermosensitive surfactant P (AAm-co-AN-co-GMA) -Apt.

3. The preparation method according to claim 2, characterized in that: In step (1), the ratio of potassium hydroxide aqueous solution, 3-mercaptopropionic acid, carbon disulfide and benzyl bromide is 35-40 mL: 32.4 mmol: 4-6 mL: 36.2 mmol, wherein the concentration of potassium hydroxide aqueous solution is 2.07 M.

4. The preparation method according to claim 2, characterized in that: In step (1), the reaction temperature in the oil bath is 20-30° C. for 4-6 h; the heating reflux temperature is 85-95° C. for 12-14 h.

5. The preparation method according to claim 2, characterized in that: In step (2), the ratio of BTPA, glycidyl methacrylate, acrylamide, acrylonitrile, azobisisobutyronitrile and N,N'-dimethylformamide is 1.2mmol:2mmol:30mmol:7-9mmol:121μmol:20-25mL.

6. The preparation method according to claim 2, characterized in that: In step (2), the nitrogen purge time is 30 to 60 minutes; the temperature of the sealed reaction is 70 to 80° C., and the sealed reaction time is 3 to 5 hours; the ice water quenching time is 10 to 20 minutes; the volume of methanol used for the precipitated product is 100 to 150 mL; the dialysis bag used in the present invention has a molecular weight cutoff of 10,000 D, and the dialysis time is 48 to 60 hours.

7. The preparation method according to claim 2, characterized in that: In step (3), the usage ratio of P(AAm-co-AN-co-GMA) solution, horseradish peroxidase aptamer solution and glycine is 400 μL:200 μL:1.2 mg; The concentration of the P(AAm-co-AN-co-GMA) solution is 1 mg / mL, the concentration of the horseradish peroxidase aptamer solution is 10 μM, and the solvent is deionized water.

8. The preparation method according to claim 2, characterized in that: In step (3), the isothermal reaction temperature is 30-40°C and the reaction time is 22-24 hours; glycine is added and the reaction is continued for 5-8 hours.

9. The preparation method according to claim 2, characterized in that: In step (3), the aptamer sequence of horseradish peroxidase is: 5'-NH2-GTC CGC AAG TTG TCG CGC GAT AAG CTT ATG GCT GGT TGA-3'.

10. The functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt for selective froth flotation of HRP in a crude extract according to claim 1 is used for selective froth flotation of HRP in a crude extract.

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