Nanometer flower type immobilized enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological catalysis and nanometer materials, and particularly relates to a nanometer flower type immobilized enzyme which is a double enzyme-inorganic metal ion hybrid nanometer flower, and the double enzyme is glucose dehydrogenase and carbonyl reductase; the inorganic metal ions exist in the form of zinc phosphate. Researches find that the immobilized enzyme has high catalytic efficiency, stability and practical application capability, overcomes the problems of poor stability, low catalytic activity, difficult recovery and poor reusability of the current free enzyme, and can reduce and relieve the inhibition effect of a substrate on the enzyme activity of the free enzyme.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis and nanomaterials, and specifically relates to a nanoflower-shaped immobilized enzyme and a preparation method and application thereof. Background Art

[0002] In the fields of chemistry and medicine, single enantiomer chiral alcohols are essential key intermediates for the synthesis of many chiral drugs. Among them, (S)-4-chloro-3-hydroxybutyric acid ethyl ester [(S)-CHBE] plays an important role in drug synthesis due to its unique chemical structure and biological activity. It is a commonly used organic synthesis intermediate that can be used to synthesize a variety of drugs and bioactive molecules, and has a wide range of applications in the fields of medicine, pesticides and chemistry. (S)-CHBE can be used as an angiotensin-converting enzyme inhibitor for the treatment of hypertension; it can also inhibit the synthesis of cholesterol, thereby reducing cholesterol levels in the blood. Therefore, the development of efficient, environmentally friendly and economical (S)-CHBE synthesis methods is of great significance for promoting the development of the fields of medicine and fine chemicals.

[0003] Traditional chemical synthesis methods often have complicated steps, low yields, and serious environmental pollution, which limit their large-scale application. In contrast, biocatalysis has gradually become a research hotspot for the synthesis of (S)-CHBE due to its advantages such as high efficiency, good selectivity, and environmental friendliness. In the process of biosynthesis of (S)-CHBE, carbonyl reductase plays an important catalytic role, but its catalytic process is highly dependent on coenzymes. However, the high price of coenzymes has become a major obstacle to the industrial application of enzyme catalysis technology. In order to overcome this problem, current research has found that the cost of enzyme catalysis production can be reduced by introducing a coenzyme regeneration system. Despite this, in the process of enzyme catalysis production, the instability of free enzymes and their difficult reuse characteristics are still key issues that need to be solved urgently.

[0004] In recent years, organic-inorganic hybrid nanoflower immobilization technology, as an innovative enzyme immobilization method, is gradually becoming a research hotspot. This technology uses proteases, metal cations and phosphate anions to form a petal-like complex - organic-inorganic hybrid nanoflowers through a self-assembly process. Nanoflower enzymes have a high specific surface area and can reduce the mass transfer resistance between enzymes, substrates and products. Compared with free enzymes, nanoflower enzymes show higher activity, better stability and reusability. However, this technology still has some issues that need to be further studied. On the one hand, not all metal ions have a promoting effect on enzymes during the immobilization process. Among them, the effect of Zn 2+There are still few reports on enzymes involved in the formation of nanoflowers; on the other hand, there are still few studies on the co-immobilization of dual enzyme molecules to form nanoflowers, and there is still a blank in the study of the co-immobilization of carbonyl reductase and coenzyme regeneration enzyme by forming nanoflowers. In the study of co-immobilization of multiple enzymes, the order and proportion of immobilization of different enzymes will have different effects on the overall catalytic effect and performance of the enzymes, especially the competition for substrates or mutual inhibition between dual enzymes, which requires us to conduct comprehensive research and reasonable design in the research process of organic-inorganic hybrid nanoflowers. Summary of the invention

[0005] The purpose of the present invention is to study and develop a new dual enzyme-inorganic hybrid nanoflower immobilization technology to address the deficiencies of the prior art, co-immobilize glucose dehydrogenase and carbonyl reductase to achieve efficient synthesis of (S)-CHBE. This technology can not only solve the problem of high coenzyme cost, but also improve the catalytic activity and stability of the enzyme, eliminate the inhibitory effect of substrates and products on the enzyme, and promote the application and development of enzyme catalysis in the industrial field.

[0006] To this end, the inventors have conducted extensive research and found that compared with other metal ions, zinc ions will weaken the enzyme activity when forming nanoflower enzymes with other enzymes. However, surprisingly, the co-immobilization of zinc ions with glucose dehydrogenase and carbonyl reductase will promote the improvement of enzyme activity, and the promotion effect is significantly better than other metal ions, thus obtaining the present invention.

[0007] Therefore, the first aspect of the present invention provides a nanoflower-type immobilized enzyme, which is a dual enzyme-inorganic metal ion hybrid nanoflower, wherein the dual enzyme is glucose dehydrogenase and carbonyl reductase; and the inorganic metal ion exists in the form of zinc phosphate.

[0008] The study found that the immobilized enzyme has high catalytic efficiency, stability and practical application capabilities, overcoming the current problems of poor stability, low catalytic activity, difficulty in recovery and poor reusability of free enzymes. At the same time, it can reduce and eliminate the inhibitory effect of the substrate on the enzymatic activity of the free enzyme.

[0009] According to the present invention, the dual enzyme-inorganic metal ion hybrid nanoflower uses zinc phosphate as an inorganic carrier, and co-fixes glucose dehydrogenase and carbonyl reductase through the coordination effect between metal ions and enzyme protein molecules.

[0010] In some embodiments of the present invention, -NH2 of the enzyme protein molecule and the metal ion Zn 2+ A complex is formed through coordination, and the complex forms a crystal nucleus with the phosphate radical. The complex further forms a petal-shaped aggregate with the crystal nucleus. The petal-shaped aggregate grows into branches around the petals to form a nano-flower-shaped immobilized enzyme.

[0011] In some embodiments of the present invention, the loading amount of the dual enzyme in the dual enzyme-inorganic metal ion hybrid nanoflower is 50wt%-60wt%.

[0012] The second aspect of the present invention provides a method for preparing a nanoflower-shaped immobilized enzyme, which uses zinc phosphate as an inorganic carrier, and co-immobilizes glucose dehydrogenase and carbonyl reductase through the coordination effect between metal ions and enzyme proteins to form a dual-enzyme-inorganic metal ion hybrid nanoflower.

[0013] According to the method of the present invention, the preparation method comprises:

[0014] Step A, adding glucose dehydrogenase and carbonyl reductase into phosphate buffer to obtain a double enzyme mixed solution;

[0015] Step B, adding zinc sulfate solution to the double enzyme mixed solution, and mixing to obtain a double enzyme-zinc sulfate mixed solution;

[0016] Step C, incubating the dual enzyme-zinc sulfate mixture, collecting the precipitate by centrifugation after the incubation is completed, and washing the precipitate with ultrapure water to obtain dual enzyme-inorganic metal ion hybrid nanoflowers.

[0017] In some embodiments of the present invention, the total content of glucose dehydrogenase and carbonyl reductase in the phosphate buffer is 0.2-1.0 mg / mL; the mass ratio of glucose dehydrogenase to carbonyl reductase is 3:1-1:3, preferably 3:1, 2:1, 1:1, 1:2, 1:3.

[0018] The research results show that when the mass ratio of glucose dehydrogenase to carbonyl reductase is 1:1, the yield of the product (S)-4-chloro-3-hydroxybutyric acid ethyl ester is the highest.

[0019] In some embodiments of the present invention, the pH of the phosphate buffer is 7.4.

[0020] In some embodiments of the present invention, Zn in the dual enzyme-zinc sulfate mixture 2+ The concentration is 1-6mM.

[0021] In some embodiments of the present invention, the incubation temperature is 2-6° C., and the incubation time is 1-12 h.

[0022] The third aspect of the present invention provides the use of the nanoflower-shaped immobilized enzyme as described in the first aspect of the present invention or the nanoflower-shaped immobilized enzyme prepared by the preparation method as described in the second aspect of the present invention in the synthesis of (S)-4-chloro-3-hydroxybutyric acid ethyl ester.

[0023] Preferably, the reaction system for synthesizing (S)-4-chloro-3-hydroxybutyric acid ethyl ester is: 20 mM 4-chloroacetoacetic acid ethyl ester, 20 mM glucose, 50 mM pH=6 citric acid / sodium citrate buffer, 0.3 mM NADPH, 0.3 mM NADP + .

[0024] Preferably, the reaction conditions for synthesizing (S)-4-chloro-3-hydroxybutyric acid ethyl ester are: reaction temperature of 25° C. and reaction time of 12 h.

[0025] In some embodiments of the present invention, in the above-mentioned reaction system for synthesizing (S)-4-chloro-3-hydroxybutyric acid ethyl ester, the reaction is carried out at 25° C. for 12 hours to obtain 4-chloro-3-hydroxybutyric acid ethyl ester ((S)-CHBE).

[0026] The term "water" in the present invention, unless otherwise specified or limited, refers to deionized water, distilled water, purified water or ultrapure water.

[0027] The loading amount of the dual enzyme in the dual enzyme-inorganic metal ion hybrid nanoflower of the present invention = (initial enzyme protein concentration in the solution - residual enzyme protein concentration in the solution after fixation) / initial enzyme protein concentration in the solution × 100%.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) Zinc phosphate is used as an inorganic carrier for the first time in the present invention. Through the coordination between metal ions and enzyme proteins, glucose dehydrogenase and carbonyl reductase are successfully co-immobilized to form a dual enzyme-inorganic hybrid nanoflower. This nanoflower significantly improves the catalytic activity and stability of the enzyme, and effectively reduces the problem of protein self-degradation. At the same time, the zinc ions contained in the nanoflower can produce a synergistic effect with the glucose dehydrogenase and carbonyl reductase immobilized on the nanoflower, thereby improving the catalytic activity of the enzyme, and the enzyme stability is also significantly improved. In addition, the high specific surface area of ​​the dual enzyme-inorganic hybrid nanoflower increases the contact opportunity between the enzyme molecule and the substrate, greatly improving the catalytic efficiency. This innovative immobilization method has not only made a significant breakthrough in the field of enzyme immobilization technology, but also demonstrated great potential in industrial enzyme catalytic applications.

[0030] (2) The novel nanoflower prepared by the present invention has achieved a significant breakthrough in the field of enzyme immobilization technology, especially in terms of removing substrate inhibition. Specifically, the novel nanoflower immobilized enzyme system of the present invention shows the following significant beneficial results compared to the traditional free dual enzyme: the activity of the traditional free enzyme is easily inhibited when the substrate concentration is high, resulting in a significant decrease in catalytic efficiency. Experimental data show that the catalytic activity of the unimmobilized free enzyme is significantly inhibited when the concentration of ethyl 4-chloroacetoacetate reaches 40mM. However, the novel nanoflower immobilized enzyme prepared by the present invention still maintains a high conversion rate of 71% when the substrate concentration reaches 100mM. This shows that the nanoflower structure effectively improves the enzyme's tolerance to the substrate, removes the inhibitory effect of the substrate on the enzyme, and enables the enzyme to still efficiently perform its catalytic function at high substrate concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0032] Figure 1 Effects of different metal ions on the immobilization efficiency and (S)-CHBE yield of dual enzyme-inorganic metal ion hybrid nanoflowers.

[0033] Figure 2 For different Zn 2+ Effect of concentration on the immobilization efficiency and (S)-CHBE yield of dual enzyme-inorganic metal ion hybrid nanoflowers.

[0034] Figure 3 This graph shows the effect of different incubation temperatures on the immobilization efficiency of dual enzyme-inorganic metal ion hybrid nanoflowers and the yield of (S)-CHBE.

[0035] Figure 4 This graph shows the effect of different incubation times on the immobilization efficiency of dual enzyme-inorganic metal ion hybrid nanoflowers and the yield of (S)-CHBE.

[0036] Figure 5 This graph shows the effect of different enzyme protein concentrations on the immobilization efficiency of dual enzyme-inorganic metal ion hybrid nanoflowers and the yield of (S)-CHBE.

[0037] Figure 6 This graph shows the effect of different dual-enzyme addition ratios on the immobilization efficiency of dual-enzyme-inorganic metal ion hybrid nanoflowers and the yield of (S)-CHBE.

[0038] Figure 7 This is a scanning electron microscope image of Zn3(PO4)2 crystal.

[0039] Figure 8 This is a scanning electron microscope image of dual-enzyme-inorganic metal ion hybrid nanoflowers.

[0040] Fig. 9 : The X-ray photoelectron energy spectrum of the dual enzyme-inorganic metal ion hybrid nanoflower, where a is the total spectrum; b is the Zn 2p spectrum, c is the P 2p spectrum, d is the O1s spectrum, e is the C1s spectrum and f is the N1s spectrum.

[0041] Fig.10 X-ray diffraction patterns of Zn3(PO4)2 crystals (a) and dual enzyme-inorganic metal ion hybrid nanoflowers (b).

[0042] Fig.11 Fourier transform infrared spectra of Zn3(PO4)2 (a), glucose dehydrogenase (b), carbonyl reductase (c) and dual enzyme-inorganic metal ion hybrid nanoflowers (d).

[0043] Fig.12 This is the thermogravimetric analysis curve of dual enzyme-inorganic metal ion hybrid nanoflowers.

[0044] Fig.13 This is a comparison chart of the optimal reaction temperatures of the free dual-enzyme system and the dual-enzyme-inorganic metal ion hybrid nanoflowers.

[0045] Fig.14 This is a comparison chart of the optimal reaction pH of the free dual enzyme system and the dual enzyme-inorganic metal ion hybrid nanoflowers.

[0046] Fig.15 This is a comparison chart of the low-temperature stability of the free dual-enzyme system and the dual-enzyme-inorganic metal ion hybrid nanoflowers.

[0047] Fig.16 This is a comparison chart of the tolerance of the free dual enzyme system and the dual enzyme-inorganic metal ion hybrid nanoflowers to the substrate 4-chloroacetoethyl acetate.

[0048] Fig.17 This is a graph showing the reusability results of dual-enzyme-inorganic metal ion hybrid nanoflowers. DETAILED DESCRIPTION

[0049] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0050] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0051] Example

[0052] The present invention is described in detail below through specific examples. The experimental methods described below, unless otherwise specified, are all routine laboratory methods. The experimental materials described below, unless otherwise specified, can all be obtained from commercial channels.

[0053] Example 1: Preparation of dual enzyme-inorganic metal ion hybrid nanoflower immobilized enzyme and optimization of conditions

[0054] Glucose dehydrogenase and carbonyl reductase were dissolved in phosphate buffer at pH 7.4, and the enzyme concentrations were 1.0 mg / mL.

[0055] The prepared dual enzyme-inorganic metal ion hybrid nanoflowers were reacted in the following solution system: 20 mM ethyl 4-chloroacetoacetate, 20 mM glucose, 50 mM pH = 6 citric acid / sodium citrate buffer, 0.3 mM NADPH, 0.3 mM NADP + . The reaction temperature was 25°C, and after the reaction time was 12h, the concentration of 4-chloro-3-hydroxybutyric acid ethyl ester ((S)-CHBE) was measured. The determination method of the product (S)-CHBE is as follows: high performance liquid chromatography HPLC was used for detection, the chromatographic column was Agilent 5TC-C18 (filler size 5μm, column length 250×column inner diameter 4.6mm), the injection volume was 10μL, and the mobile phase was finally determined to be 80% water (0.1% formic acid) and 20% acetonitrile, the column temperature was set to 25°C, the flow rate was 0.8mL / min, and the VWD detector wavelength was 220nm. The retention time of (S)-CHBE was determined to be 14.9min.

[0056] (1) Selection of metal ions in hybrid nanoflowers

[0057] As inorganic components of hybrid nanoflowers, metal ions have multiple functions, including promoting structure formation, improving catalytic activity, enhancing immobilization efficiency, regulating functional properties, improving biocompatibility, and facilitating recycling and reuse. 2+ , Cu 2+ ,Co 2+ , Fe 3+ , Mn 2+ , Ni 2+ , Zn 2+As the inorganic part of hybrid nanoflowers. When preparing the metal ion solution, weigh CaCl2 0.5549g, CuCl2·2H2O 0.8524g, CoCl2·6H2O 1.18965g, FeCl3·6H2O 1.3515g, MnCl2·4H2O0.9896g, NiCl2·6H2O 1.1885g, ZnSO4·7H2O 1.4378g in a conical flask, add ultrapure water to 50mL, and prepare a metal ion solution (0.1M). Add 500μL of glucose dehydrogenase enzyme solution and 500μL of carbonyl reductase enzyme solution (enzyme concentration is 1.0mg / mL) to a 2mL centrifuge tube, mix well, and add 40μL of metal ion solution. The final concentration of metal ions in the mixed solution is 4mM. Then incubate in a 4℃ refrigerator for 6h. After removal, centrifugation was performed and the supernatant was retained for determination of the remaining protein concentration. The precipitate was washed three times with deionized water to obtain the hybrid nanoflower. Figure 1 As shown in Figure 2, the substrate conversion rate and enzyme immobilization efficiency of the dual enzyme-Zn nanoflowers were the highest. 2+ As the target metal ions, they form dual enzyme-inorganic hybrid nanoflowers with glucose dehydrogenase and carbonyl reductase.

[0058] (2) Effect of metal ion concentration on enzyme immobilization

[0059] Select different concentrations (1mM, 2mM, 3mM, 4mM, 5mM, 6mM) of Zn 2+ Solution, add 500 μL of glucose dehydrogenase and carbonyl reductase solution (enzyme concentration is 1.0 mg / mL) to a 2 mL centrifuge tube, mix well and add 40 μL of zinc sulfate solution of different concentrations. The mixed solution was incubated in a 4 ° C refrigerator for 3 hours, then centrifuged at 13000 rpm for 3 minutes in a high-speed centrifuge to collect the precipitate. Wash the precipitate 3 times with deionized water to remove the unfixed glucose dehydrogenase and carbonyl reductase, and the resulting precipitate is the glucose dehydrogenase and carbonyl reductase co-fixed hybrid nanoflower.

[0060] The results are as follows Figure 2 As shown in the figure, when the zinc ion concentration is lower than 4 mM, the immobilization efficiency of the dual enzyme-inorganic hybrid nanoflowers and the enzyme (S)-CHBE production increase with the increase of salt solution concentration. 2+ When the ion concentration reached 4 mM, the immobilization efficiency and (S)-CHBE yield reached their peak values. After that, the yield gradually decreased with the increase of metal ion concentration. 2+ The ion concentration is 4 mM.

[0061] (3) Effect of incubation temperature on enzyme immobilization

[0062] Select different incubation temperatures (4°C, 15°C, 25°C, 37°C, 50°C), add 500 μL of glucose dehydrogenase and carbonyl reductase solutions (enzyme concentration is 1.0 mg / mL) to 2 mL centrifuge tubes respectively, mix well and add 40 μL of 0.1 M zinc sulfate solution. Then incubate at the above temperature for 3 hours. Centrifuge the mixture at 13000 rpm for 3 minutes in a high-speed centrifuge to collect the precipitate. Wash the precipitate three times with deionized water to remove the unfixed glucose dehydrogenase and carbonyl reductase. The resulting precipitate is the glucose dehydrogenase and carbonyl reductase co-fixed hybrid nanoflower. The results are as follows Figure 3 As shown in the figure, with the gradual increase of temperature, the overall immobilization efficiency does not change much, but the yield of (S)-CHBE continues to decrease. The product yield is the highest when the incubation temperature is 4°C, so the incubation temperature of 4°C is selected to prepare the dual enzyme-inorganic hybrid nanoflowers.

[0063] (4) Effect of incubation time on enzyme immobilization

[0064] Different incubation times (1h, 2h, 3h, 6h, 9h, 12h) were selected, and the dual enzyme-inorganic hybrid nanoflowers were prepared according to the synthesis method of Example 1.3 under the above incubation time. The results are as follows Figure 4 As shown, with the increase of incubation time, the overall immobilization efficiency does not change much, all above 50%; while the yield of (S)-CHBE shows a trend of first increasing and then decreasing. When the incubation time is 3h, the yield reaches a peak of 17.256mM, and the immobilization efficiency at this time is 58.432%. Although the difference between the yield and immobilization efficiency of 3h and 6h is not large, considering the time cost, 3h is selected as the incubation time of hybrid nanoflowers.

[0065] (5) Effect of the amount of dual enzymes added on enzyme immobilization

[0066] Different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) of glucose dehydrogenase and carbonyl reductase were selected and immobilized enzymes were prepared according to the synthesis method of Example 1.3. Figure 5 As shown, as the concentration of the added protease increases, the yield of (S)-CHBE also increases. Taking comprehensive consideration, the concentration of glucose dehydrogenase and carbonyl reductase added was selected to be 1.0 mg / mL.

[0067] (6) Effect of the ratio of dual enzyme addition on enzyme immobilization

[0068] Different addition ratios of glucose dehydrogenase and carbonyl reductase (enzyme concentrations were all 1.0 mg / mL) (3:1, 2:1, 1:1, 1:2, 1:3) were selected, and immobilized enzymes were prepared according to the synthesis method of Example 1.3. Figure 6As shown in the figure, when the addition ratio of carbonyl reductase to glucose dehydrogenase was 1:1, the yield of (S)-CHBE was the highest. After comprehensive consideration, the addition ratio of the two enzymes was 1:1.

[0069] Example 2: Characterization of dual enzyme-inorganic metal ion hybrid nanoflower materials

[0070] Glucose dehydrogenase and carbonyl reductase were dissolved in phosphate buffer at pH 7.4, and the enzyme concentration was 1.0 mg / mL. 500 μL of glucose dehydrogenase and carbonyl reductase solution were added to 2 mL centrifuge tubes, respectively, and 40 μL of 0.1 M zinc sulfate solution was added after mixing. Then it was incubated in a refrigerator at 4 ° C for 3 hours. The mixed solution was centrifuged at 13000 rpm for 3 minutes with a high-speed centrifuge, and the precipitate was collected. The precipitate was washed 3 times with deionized water to remove the unfixed glucose dehydrogenase and carbonyl reductase. The resulting precipitate was the glucose dehydrogenase and carbonyl reductase co-fixed hybrid nanoflower.

[0071] The prepared hybrid nanoflowers were characterized, and the specific results are as follows:

[0072] Figure 7 The scanning electron microscope image of zinc phosphate crystals is shown. The image results show that zinc sulfate crystals are micrometer-scale particles with regular geometric shapes, mainly cubic and polyhedral structures. These cubic and polyhedral particles have relatively smooth surfaces and clear boundaries, showing the characteristics of crystals. The size of the particles is approximately between hundreds of nanometers and several microns, and they show a certain degree of aggregation and accumulation in the image.

[0073] Figure 8 The scanning electron microscope image of the prepared dual enzyme-inorganic metal ion hybrid nanoflower is shown. It can be seen from the image that the nanoflower is composed of a large number of lamellar structures, which are intertwined to form a complex three-dimensional network. The crystal morphology is similar to a flower, and the lamellar structure radiates from the center to the outside, forming a multi-layered flower-like morphology. The lamellar structure has high crystallinity and a large specific surface area, indicating that it has potential application value in the fields of catalysts and adsorption materials. The porous structure and dense stacking characteristics of the nanoflower further enhance its performance in material transport and enzyme activity.

[0074] Fig. 9 The X-ray photoelectron spectrum of the prepared dual enzyme-inorganic hybrid nanoflower is shown. As can be seen from the figure, the chemical elements of Zn, P, O, C, Na and Cl all appear in the overall spectrum of the hybrid nanoflower. Among them, Na is a necessary component of phosphate buffer, and Cl is a necessary chemical element for the formation of hybrid nanoflowers. Fig. 9 b is the spectrum of Zn2p. The results in the figure show that Zn 2+ is the main valence state of Zn in hybrid nanoflowers. Fig. 9The P 2p spectrum in c is at 134.58 eV binding energy and Fig. 9 The O1s spectrum in d shows that the main component of the hybrid nanoflower is PO4 at a binding energy of 530.28 eV. 3- , which proves the presence of zinc phosphate. Fig. 9 The binding energies of 284.78, 286.08 and 287.58 eV in the C1s spectrum in e indicate the existence of CH, CN and C=O chemical bonds, respectively. Fig. 9 The binding energy of the N1s spectrum at 398.28 eV in f indicates the existence of NC=O chemical bond. These analysis results show that the present invention successfully synthesized the dual enzyme-inorganic metal ion hybrid nanoflower.

[0075] Fig.10 It is the X-ray diffraction pattern of Zn3(PO4)2 and dual enzyme-inorganic hybrid nanoflower. As can be seen in the figure, all diffraction peaks of Zn3(PO4)2 are consistent with the peaks obtained by JCPDS card (00-037-0465). The powder X-ray diffraction pattern of dual enzyme-inorganic hybrid nanoflower shows that the prepared hybrid nanoflower is mainly composed of Zn3(PO4)2 crystals. These results show that the crystal structure of the dual enzyme-inorganic hybrid nanoflower obtained by the present invention has not changed due to the synthesis process.

[0076] Fig.11 The infrared spectra of four materials, Zn3(PO4)2, glucose dehydrogenase, carbonyl reductase and dual enzyme-inorganic metal ion hybrid nanoflower, are shown. First, Zn3(PO4)2 has a wavelength of 400-1200 cm -1 The absorption peaks are mainly attributed to the vibration of Zn-O and PO bonds. Glucose dehydrogenase and carbonyl reductase have obvious absorption peaks in the range of 3300-3500cm -1 1600-1700cm -1 1500-1600cm -1 The absorption peaks at 1000-1400 cm-1 reflect the stretching vibration of NH and OH bonds, amide I band and amide II band, respectively, indicating the presence of α-helical structure in the protein. -1 There are multiple peaks of medium intensity, which are related to the CN bond stretching vibration and COC vibration in the protein skeleton. The infrared spectrum results of Zn3(PO4)2-NFs show the main absorption peaks of Zn3(PO4)2 and enzyme at the same time, indicating that the enzyme on the dual enzyme-inorganic metal ion hybrid nanoflower maintains the original enzyme secondary structure and forms a complex with Zn3(PO4)2. The above results show that the present invention successfully synthesized the dual enzyme-inorganic metal ion hybrid nanoflower.

[0077] Fig.12The thermogravimetric analysis curve of the dual enzyme-inorganic hybrid nanoflower is shown. According to the information in the figure, in the first stage, the decomposition of the dual enzyme-inorganic hybrid nanoflower starts from 25°C and ends at about 150°C, which is mainly caused by the removal of water molecules in the hybrid nanoflower. In the second stage, the decomposition starts at about 150°C and ends at 750°C, which is caused by the loss of glucose dehydrogenase and carbonyl reductase. This result shows that the total weight of organic enzymes in the hybrid nanoflower is about 27.4%. This result also proves the high degree of hybridization of zinc phosphate and enzymes.

[0078] Example 3: Determination of enzymatic properties of dual enzyme-inorganic hybrid nanoflowers

[0079] (1) Overall enzyme activity determination method

[0080] This example detects the product conversion rate of the dual enzyme-inorganic hybrid nanoflower and the free dual enzyme system under the same total enzyme amount. The definition of the enzyme activity unit is: under the optimal reaction conditions, the amount of enzyme required to generate 1 μmol (S)-CHBE per minute is one enzyme activity unit (U), that is, 1U = 1 μmol / min. According to the optimal immobilization conditions obtained in Example 1, the dual enzyme-inorganic metal ion hybrid nanoflower was prepared, and the immobilized nanoflower and the free dual enzyme with the same enzyme amount were reacted in the solution system described in Example 1. The concentration of (S)-CHBE is determined as described in Example 1. The ratio of the product concentration to the substrate concentration before the reaction is expressed as the product conversion rate.

[0081] (2) Optimal reaction temperature of free enzyme and hybrid nanoflower

[0082] Temperature is one of the important factors that affect the catalytic efficiency of enzymes. Within a certain range, when the temperature is too low, the molecular motion slows down, the collision frequency between the enzyme and the substrate decreases, and the catalytic efficiency decreases. As the temperature gradually increases, the number of activated molecules in the reaction system increases, and the number of effective collisions between the enzyme molecules and the substrate also increases, thereby gradually increasing the activity of the enzyme. However, when the temperature is too high, the enzyme may denature, resulting in the destruction of its three-dimensional structure, the ineffectiveness of the active center, and the decrease in catalytic efficiency.

[0083] According to the overall enzyme activity determination method, by changing the reaction temperature (4, 16, 25, 30, 37, 45, 52, 60°C), the ratio of product concentration to substrate concentration was calculated to represent the substrate conversion rate of hybrid nanoflowers and free enzymes, and the optimal reaction temperature of free dual enzymes and hybrid nanoflowers was compared. The results are as follows Fig.13As shown in the figure, the optimal reaction temperature of the free dual enzyme and hybrid nanoflower is 25°C. When the temperature exceeds 25°C, the relative enzyme activity of both decreases. Compared with the free dual enzyme system, the dual enzyme-inorganic hybrid nanoflower shows a higher substrate conversion rate in the range of 4°C-60°C, indicating that the hybrid nanoflower has better temperature adaptability and a wider temperature adaptability range after immobilization.

[0084] (3) Optimal reaction pH of free enzyme and hybrid nanoflowers

[0085] pH is one of the important factors affecting enzyme activity and stability. Changes in pH will affect the ionization state of the enzyme, thereby changing its three-dimensional structure and the charge distribution of the active center, and further affecting the binding of the enzyme to the substrate and the catalytic efficiency.

[0086] According to the overall enzyme activity determination method, by changing the pH (3, 4, 5, 6, 7, 8) during the reaction, the product conversion rate of the free enzyme system and the immobilized enzyme system was calculated respectively, and the optimal reaction pH of the free dual enzyme and the hybrid nanoflower was determined and compared. 50mM citric acid / sodium citrate buffer was selected to prepare the above different pH values. The buffer preparation method was to weigh 10.507g citric acid and 14.705g trisodium citrate in a beaker, add ultrapure water to 1L, mix the two solutions in different proportions and adjust to different pH values. The results are shown in Figure 2. Fig.14 As shown in the figure, the optimal reaction of free bienzyme and hybrid nanoflower is 50mM citric acid / sodium citrate buffer at pH 6. In the buffer environment of pH 6-8, both free bienzyme and hybrid nanoflower maintain relatively good catalytic ability, but in the low pH environment, the catalytic activity of free bienzyme and hybrid nanoflower decreases to varying degrees. However, the overall enzyme activity of hybrid nanoflower at different pH is higher than that of free bienzyme.

[0087] (4) Storage stability of free enzymes and hybrid nanoflowers

[0088] The immobilized enzyme and free enzyme prepared in Example 1 were stored at 4°C to investigate the storage stability of the free enzyme and the dual enzyme-inorganic hybrid nanoflower. Fig.15 As shown, the dual enzyme-inorganic hybrid nanoflowers can still retain about 80% of their activity after 25 days of low-temperature storage, while the activity of the free dual enzymes is reduced to 70% after 5 days of storage. By comparing the changes in the enzyme activity of the two systems during low-temperature storage, the dual enzyme-inorganic hybrid nanoflowers show higher stability at low temperatures, and their long-term stability of enzyme activity is significantly better than that of the free dual enzyme system. This result shows that the dual enzyme-inorganic hybrid nanoflowers have better stability and application potential under low-temperature storage conditions, providing new ideas and methods for the long-term storage and practical application of enzymes.

[0089] (5) Substrate adaptability of free enzymes and hybrid nanoflowers

[0090] Since the substrate ethyl 4-chloroacetoacetate has substrate inhibition on carbonyl reductase, it affects the reaction rate. Therefore, different substrate concentrations (20, 40, 60, 80, 100 mM) were selected to evaluate its effect on the enzyme activity of the free dual enzyme system and the immobilized dual enzyme nanoflower system. The immobilized enzyme and free dual enzyme system obtained in Example 1 were taken, and the yields of the two enzyme systems were determined according to the optimal reaction conditions of Example 1. The results are shown in Figure 2. Fig.16 As shown, ethyl 4-chloroacetoacetate has a substrate inhibition effect on both catalytic systems. As the substrate concentration increases, the yield of the free dual enzyme system continues to decrease. When the substrate concentration reaches 100mM, the yield of the free dual enzyme system is only 27.97%. However, as the substrate concentration of the dual enzyme-inorganic hybrid nanoflower system increases, the yield only decreases slightly, and is generally maintained at a high level. When the substrate concentration reaches 100mM, the yield of the dual enzyme-inorganic hybrid nanoflower is still 71.28%. The results show that the immobilized dual enzyme nanoflower system exhibits higher catalytic efficiency and substrate tolerance.

[0091] (6) Reusability of hybrid nanoflowers

[0092] Take an appropriate amount of the immobilized enzyme obtained in Example 1, and determine the relative enzyme activity of the immobilized enzyme according to the optimal reaction conditions, controlling the reaction time and substrate concentration to be the same each time. After reacting for 2 hours, centrifuge at 13000rpm for 2 minutes in a high-speed centrifuge, then quickly separate the supernatant from the solid precipitate to terminate the reaction, and determine the concentration of (S)-CHBE by HPLC after the supernatant passes through the membrane. The precipitate is washed repeatedly with deionized water 3 times, and the above operation is repeated to continuously determine the overall enzyme activity of the hybrid nanoflower. Determine the ratio of the product concentration to the substrate concentration after each reaction to calculate the overall enzyme activity. The results are as follows Fig.17 As shown, the immobilized enzyme still maintained more than 80% of its relative activity after being reused 5 times, and still maintained more than 60% of its relative activity after being reused 8 times, which indicates that the immobilized enzyme has good reusability.

[0093] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A nanoflower-type immobilized enzyme, which is a dual enzyme-inorganic metal ion hybrid nanoflower, wherein: The dual enzymes are glucose dehydrogenase and carbonyl reductase; and the inorganic metal ions exist in the form of zinc phosphate.

2. The nanoflower-shaped immobilized enzyme according to claim 1, characterized in that: The dual enzyme-inorganic metal ion hybrid nanoflower uses zinc phosphate as an inorganic carrier, and co-fixes glucose dehydrogenase and carbonyl reductase through the coordination effect between metal ions and enzyme protein molecules.

3. The nanoflower-shaped immobilized enzyme according to claim 1, characterized in that: The -NH2 of the enzyme protein molecule and the metal ion Zn 2+ A complex is formed through coordination, and the complex forms a crystal nucleus with the phosphate radical. The complex further forms a petal-shaped aggregate with the crystal nucleus. The petal-shaped aggregate grows into branches around the petals to form a nano-flower-shaped immobilized enzyme.

4. The nanoflower-shaped immobilized enzyme according to any one of claims 1 to 3, characterized in that: The loading amount of the dual enzyme in the dual enzyme-inorganic metal ion hybrid nanoflower is 50wt%-60wt%.

5. A method for preparing a nanoflower-shaped immobilized enzyme, which uses zinc phosphate as an inorganic carrier and co-immobilizes glucose dehydrogenase and carbonyl reductase through the coordination effect between metal ions and enzyme proteins to form a dual-enzyme-inorganic metal ion hybrid nanoflower.

6. The preparation method according to claim 4, characterized in that: The preparation method comprises: Step A, adding glucose dehydrogenase and carbonyl reductase to a phosphate buffer to obtain a dual enzyme mixed solution; Step B, adding zinc sulfate solution to the double enzyme mixed solution, and mixing to obtain a double enzyme-zinc sulfate mixed solution; Step C, incubating the dual enzyme-zinc sulfate mixture, collecting the precipitate by centrifugation after the incubation is completed, and washing the precipitate with ultrapure water to obtain dual enzyme-inorganic metal ion hybrid nanoflowers.

7. The preparation method according to claim 6, characterized in that: The total content of glucose dehydrogenase and carbonyl reductase in the phosphate buffer is 0.2-1.0 mg / mL; the mass ratio of glucose dehydrogenase to carbonyl reductase is 3:1-1:3 respectively.

8. The preparation method according to claim 6, characterized in that: The pH of the phosphate buffer is 7.4; and / or, Zn in the dual enzyme-zinc sulfate mixture is 2+ The concentration is 1-6mM.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The incubation temperature is 2-6°C, and the incubation time is 1-12h.

10. Use of the nanoflower-shaped immobilized enzyme as described in any one of claims 1 to 4 or the nanoflower-shaped immobilized enzyme prepared by the preparation method as described in any one of claims 4 to 9 in the synthesis of (S)-4-chloro-3-hydroxybutyric acid ethyl ester.

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