Immobilized Rhizopus oryzae lipase and controllable preparation method and application thereof

Abstract

The present invention discloses an immobilized Rhizopus oryzae lipase and a controllable preparation method and application thereof, and belongs to the technical field of immobilized lipase preparation. The present invention prepares an amorphous ZIF‑8 structure by adjusting the concentration of metal organic ligands, and uses an in-situ self-encapsulation method to fix the lipase ROL on the MOF carrier aZIF‑8 to prepare the immobilized lipase ROL@aZIF‑8. The results show that compared with the free lipase ROL, its enzyme activity is improved, and the immobilized lipase ROL@aZIF‑8 has higher thermal stability, pH tolerance and storage time stability. At the same time, when the added enzyme concentration is 4 mg / mL, the immobilized lipase presents a maximum enzyme activity of 5.69U / mg. The present invention helps to provide a potential approach for immobilized lipase, which is worthy of expansion in the catalytic applications and industrial biocatalysis of different types of MOFs and lipases.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized lipase preparation, relates to a method for immobilizing Rhizopus oryzae lipase using an amorphous ZIF-8 carrier material, and specifically relates to an immobilized Rhizopus oryzae lipase and a controllable preparation method and application thereof. Background Art

[0002] Rhizopus oryzae lipase (ROL) has good catalytic specificity for the sn-1.3-position of triglycerides, can produce structural oils with certain special functions, is an excellent catalyst for biodiesel, and has important applications in food, chemical industry, and bioenergy. However, despite the advantages of high catalytic efficiency, mild reaction conditions, and environmental friendliness, free ROL has disadvantages such as poor stability, easy inactivation, and inability to operate continuously, which limit its practical application in the food industry. Compared with free enzymes, the stability of lipases after immobilization treatment is greatly improved, the tolerance to heat, pH, and other environments is improved, and the sensitivity to inhibitors is reduced, which significantly improves the utilization efficiency of lipases and greatly reduces production costs. Therefore, finding a biocompatible carrier and immobilization method suitable for lipases has always been a difficult task and challenge that researchers must face.

[0003] Metal-organic frameworks (MOFs) materials have attracted extensive attention due to their unique performance advantages such as large specific surface area, high porosity, pore controllability, easy functionalization and modification. Zeolitic imidazolate framework-8 (ZIF-8) is a second-generation porous coordination polymer based on Zn 2+ Ions and imidazole linkers are self-assembled through coordination bonds and are a type of MOFs material with high specific surface area, excellent chemical and thermal stability, and negligible cytotoxicity. 2+ The concentration ratio of ions and imidazole linkers can change the morphology and properties of ZIF-8. The immobilized lipase prepared by ZIF-8 material shows the characteristics of easy recovery and reuse, high tolerance, high pH value and good thermal stability in the production process.

[0004] At present, four methods are usually used to prepare immobilized lipase based on ZIF-8 materials: surface adsorption, covalent cross-linking, pore encapsulation and in-situ self-encapsulation. Surface adsorption and covalent cross-linking methods mainly fix enzyme molecules on the surface of ZIF-8 carriers through hydrogen bonds, covalent bonds, etc.; pore encapsulation and in-situ self-encapsulation are to enclose enzyme molecules inside ZIF-8, "put on" ZIF-8 "armor" for enzyme molecules, and effectively improve the stability of enzymes with the rigid framework of ZIF-8 structure. Among them, the pore encapsulation method prepares immobilized enzymes by incubating lipase molecules with synthesized ZIF-8 carriers, allowing enzyme molecules to pass through ZIF-8 channels and enter the carrier; the in-situ self-encapsulation method is to add enzyme molecules to the reaction solution synthesized by ZIF-8 carriers, and directly encapsulate enzyme molecules inside MOFs while self-assembling to form carriers. Studies have shown that compared with immobilized lipase prepared by surface adsorption and covalent cross-linking methods, immobilized lipase based on ZIF-8 structure prepared by pore encapsulation and in situ self-encapsulation showed better stability because the enzyme molecules were inside the carrier; in the process of preparing immobilized lipase by pore encapsulation, the protein structure of the enzyme molecules is prone to unfolding when passing through the pores of the ZIF-8 carrier, causing the enzyme molecules to lose activity, while the in situ self-encapsulation method can effectively avoid the inactivation problem of enzyme molecules due to changes in protein structure.

[0005] However, due to the small volume of the ZIF-8 carrier pores, the substrate or product passes through the immobilized enzyme pores, resulting in mass transfer resistance. Therefore, the catalytic activity of the immobilized enzyme currently prepared by the in-situ self-encapsulation method has a certain loss. Chinese patent CN108396023B discloses a method of preparing a magnetic MOF material by grinding and used for enzyme fixation. The scheme shows that the material is prepared first, and then the enzyme is immobilized after the material is formed. However, since the enzyme is always on the surface of the material, the stability is affected to a certain extent, and the process adds a magnetic substance Fe3O4 during the preparation of the material, which may reduce the enzyme loading of the immobilized enzyme and increase the mass transfer resistance of the substrate or product. Chinese patent CN111471663A discloses a method for immobilizing Pseudomonas fluorescens lipase with a metal organic framework material. The process also prepares the material first and then immobilizes the enzyme, that is, the lipase is fixed on the surface of Uio-66 (Zr), so the stability will also be affected. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide an immobilized Rhizopus oryzae lipase and a controllable preparation method and application thereof, which can effectively solve the problem that the immobilized enzyme prepared by the prior art has low catalytic activity and affected stability.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The invention discloses a controllable preparation method of immobilized Rhizopus oryzae lipase, comprising: adding a zinc acetate solution to a 2-methylimidazole solution and then adding a solution containing the Rhizopus oryzae lipase, stirring the solution for reaction for 20 to 40 minutes, washing and collecting precipitates, and performing vacuum freeze-drying treatment to prepare the immobilized Rhizopus oryzae lipase, namely, the immobilized lipase ROL@aZIF-8.

[0009] Preferably, the concentration of the zinc acetate solution is 20 mmol / L, the concentration of the 2-methylimidazole solution is 33.8 mmol / L, and the concentration of the solution containing Rhizopus oryzae lipase is 5 mg / mL.

[0010] Further preferably, the volume ratio of the zinc acetate solution, the 2-methylimidazole solution and the solution containing Rhizopus oryzae lipase is 1:1:0.08.

[0011] Preferably, the stirring reaction is carried out at a rotation speed of 300 to 500 r / min.

[0012] Preferably, the precipitate is washed with deionized water for 3 to 5 times, and the collected precipitate is subjected to vacuum freeze-drying treatment at -80°C for 12 hours.

[0013] The invention also discloses immobilized Rhizopus oryzae lipase prepared by the controllable preparation method.

[0014] Preferably, the immobilized Rhizopus oryzae lipase has higher enzyme activity than the free enzyme at a temperature range of 30 to 70° C. and a pH value of 7.5 to 10.0.

[0015] Preferably, when the added enzyme concentration of the immobilized Rhizopus oryzae lipase is 4 mg / mL, the maximum enzyme activity reaches 5.69 U / mg.

[0016] The invention also discloses the application of the immobilized Rhizopus oryzae lipase in the preparation of industrial biocatalysts.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discloses immobilized Rhizopus oryzae lipase and a controllable preparation method thereof. 2-methylimidazole and a zinc acetate solution are mixed, and then a solution containing Rhizopus oryzae lipase is immediately added thereto for stirring reaction. The prepared amorphous MOF carrier aZIF-8 material has a smaller mass transfer resistance of substances entering and leaving the carrier than that of the ZIF-8 material. The immobilized lipase ROL@aZIF-8 is prepared by immobilizing the Rhizopus oryzae lipase (ROL) by an in-situ self-encapsulation method.

[0019] Furthermore, the ZIF-8 structure can be prepared in a controllable manner through a certain concentration ratio of 2-methylimidazole to zinc acetate. Therefore, the present invention preferably uses a zinc acetate solution with a concentration of 20 mmol / L and a 2-methylimidazole solution with a concentration of 33.8 mmol / L. It has been found through experiments that a zinc nitrate solution can also be used, but after fixation, it is found that the enzyme activity of the original enzyme cannot be retained to the maximum extent. By adjusting the concentration, the structure is changed from a complete structure to an amorphous structure, and then a suitable immobilization concentration for Rhizopus oryzae lipase is found. Therefore, after the ZIF-8 structure-immobilized enzyme prepared by the present invention using the zinc acetate solution and the 2-methylimidazole solution at the above concentrations is selected, the enzyme activity is significantly improved.

[0020] The immobilized Rhizopus oryzae lipase prepared according to the above method of the present invention has improved enzyme activity compared with free lipase ROL, and the immobilized lipase ROL@aZIF-8 has higher thermal stability, pH tolerance and storage stability, that is, when the temperature range is 30-70°C and the pH value is 7.5-10.0, its enzyme activity is higher than that of the free enzyme, and within the storage time range of 0-30 days, the residual activity of the immobilized lipase ROL@aZIF-8 decreases less, while the residual activity of the free lipase ROL decreases relatively more, and the enzyme activity of the immobilized lipase ROL@aZIF-8 is higher than that of the free lipase ROL. At the same time, when the added enzyme concentration is 4 mg / mL, the immobilized lipase presents a maximum enzyme activity of 5.69U / mg.

[0021] Based on the above-mentioned activity characteristics of immobilized lipase, a potential approach is provided for the immobilization of lipase, which is worthy of expansion in the catalytic applications and industrial biocatalysis of different types of MOFs materials and lipases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Scanning electron microscope (SEM) images of the MOF carrier aZIF-8 and immobilized lipase ROL@aZIF-8 prepared by the present invention; wherein A is the MOF carrier aZIF-8; B is the immobilized lipase ROL@aZIF-8;

[0023] Figure 2 Transmission electron microscope (TEM) images of the MOF carrier aZIF-8 and the immobilized lipase ROL@aZIF-8 prepared by the present invention; wherein A is the MOF carrier aZIF-8; B is the immobilized lipase ROL@aZIF-8;

[0024] Figure 3The scanning transmission electron microscope-energy spectrum elemental surface scanning analysis diagram of the MOF carrier aZIF-8 and the immobilized lipase ROL@aZIF-8 prepared by the present invention; wherein A is the MOF carrier aZIF-8; B is the immobilized lipase ROL@aZIF-8;

[0025] Figure 4 X-ray diffraction (XRD) spectra of the immobilized lipase ROL@aZIF-8, MOF carrier aZIF-8 and simulated ZIF-8 prepared in the present invention;

[0026] Figure 5 N2 adsorption and desorption curves of immobilized lipase ROL@aZIF-8 and MOF carrier aZIF-8 prepared in the present invention;

[0027] Figure 6 Thermogravimetric (TGA) analysis diagram of the immobilized lipase ROL@aZIF-8 and MOF carrier aZIF-8 prepared in the present invention;

[0028] Figure 7 Fourier transform infrared spectroscopy (FTIR) analysis diagram of free lipase ROL, immobilized lipase ROL@aZIF-8 and MOF carrier aZIF-8 prepared by the present invention;

[0029] Figure 8 The water contact angle analysis diagram of the immobilized lipase ROL@aZIF-8 and the MOF carrier aZIF-8 prepared by the present invention; wherein A is the MOF carrier aZIF-8; B is the immobilized lipase ROL@aZIF-8;

[0030] Fig. 9 The effect of temperature on the catalytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8;

[0031] Fig.10 The effect of pH on the catalytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8;

[0032] Fig.11 The effect of storage time on the catalytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8;

[0033] Fig.12 The effect of different enzyme addition amounts on the catalytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0035] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0036] The present invention prepares an amorphous ZIF-8 material, i.e., a MOF carrier aZIF-8, by a certain ratio of metal ion and organic ligand concentration, and uses an in-situ self-encapsulation method to immobilize a lipase derived from Rhizopus oryzae in the MOF carrier aZIF-8 to obtain an immobilized lipase ROL@aZIF-8. Under the same conditions, the immobilized lipase ROL@aZIF-8 has a stronger ability to hydrolyze p-nitrophenylbutyrate (p-NPB). In further studies, compared with the free lipase ROL, the enzyme activity of the immobilized lipase ROL@aZIF-8 is improved, and the immobilized lipase ROL@aZIF-8 has higher thermal stability, pH tolerance and storage time stability. That is, when the temperature range is 30-70℃ and the pH value is 7.5-10.0, its enzyme activity is higher than that of the free enzyme. In the storage time range of 0-30 days, the residual activity of the immobilized lipase ROL@aZIF-8 decreases slightly, while the residual activity of the free lipase ROL decreases relatively greatly, and the enzyme activity of the immobilized lipase ROL@aZIF-8 is higher than that of the free lipase ROL. At the same time, when the added enzyme concentration is 4 mg / mL, the immobilized lipase ROL@aZIF-8 presents a maximum enzyme activity of 5.69U / mg. The results show that the immobilized lipase prepared by in situ self-encapsulation method based on aZIF-8 carrier can effectively improve the enzyme activity and stability of ROL.

[0037] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0038] 1. Immobilization of lipase

[0039] 1 mL of 20 mmol / L zinc acetate solution was added dropwise to 1 mL of 33.8 mmol / L 2-methylimidazole, and then 80 μL of 5 mg / mL ROL solution was immediately added, and the mixture was reacted at 300 r / min for 30 min. After the reaction, the precipitate was washed three times with deionized water, collected, freeze-dried (-80°C, 12 h), and stored at 4°C to obtain immobilized lipase ROL@aZIF-8.

[0040] At the same time, aZIF-8 prepared under the same conditions without adding enzyme was used as a blank control. The specific method is as follows:

[0041] 1 mL of 20 mmol / L zinc acetate solution was added dropwise to 1 mL of 33.8 mmol / L 2-methylimidazole and reacted at 300 r / min for 30 min. After the reaction, the precipitate was washed 3 times with deionized water, collected, freeze-dried in a vacuum (-80°C, 12 h), and stored at 4°C to obtain the MOF carrier aZIF-8. The TEM image is shown in FIG. Figure 2 As shown in A.

[0042] The immobilized lipase ROL@ZIF-8 prepared without changing the concentration ratio of 2-methylimidazole and zinc acetate was used as a control. The specific method is as follows:

[0043] 1 mL of 20 mmol / L zinc acetate solution was added dropwise to 1 mL of 80 mmol / L 2-methylimidazole, and then 80 μL of 5 mg / mL ROL solution was immediately added, and the mixture was reacted at 300 r / min for 30 min. After the reaction, the precipitate was washed 3 times with deionized water, collected, freeze-dried (-80°C, 12 h), and stored at 4°C to obtain immobilized lipase ROL@ZIF-8. The TEM image is shown in FIG. Figure 2 As shown in B.

[0044] Immobilized lipase ROL@aZIF-8 (1:3) prepared with different concentration ratios of 2-methylimidazole and zinc acetate was used as a control. The specific method is as follows:

[0045] 1 mL of 20 mmol / L zinc acetate solution was added dropwise to 1 mL of 60 mmol / L 2-methylimidazole, and then 80 μL of 5 mg / mL ROL solution was immediately added, and the mixture was reacted at 300 r / min for 30 min. After the reaction, the precipitate was washed three times with deionized water, collected, freeze-dried (-80°C, 12 h), and stored at 4°C to obtain immobilized lipase ROL@aZIF-8 (1:3).

[0046] 2. Determination of enzyme activity of immobilized lipase ROL@aZIF-8, ROL@ZIF-8 and ROL@aZIF-8(1:3)

[0047] The immobilized lipases ROL@aZIF-8, ROL@ZIF-8 and ROL@aZIF-8 (1:3) were weighed, and 1 mL of 2 mg / mL p-NPB solution dissolved in isopropanol and 1 mL of 0.01 mol / L (pH 7.5) phosphate buffer saline (PBS) were added, homogenized at 13000 r / min for 30 s, and then reacted at 200 r / min for 5 min. After the reaction, 1 mL of stop solution (NaOH 40 g, EDTA 93.5 g dissolved in 1 L deionized water) was added, and the supernatant was collected by centrifugation at 4 ° C, and the absorbance was measured at 405 nm. One unit of enzyme activity is defined as the amount of immobilized enzyme required to release 1 μmol of p-nitrophenol (p-NP) per minute, expressed as U / mg.

[0048] The data in Table 1 below confirm that the enzyme activity of ROL@aZIF-8 is greatly improved compared with ROL@ZIF-8 and ROL@aZIF-8 (1:3).

[0049] Table 1 Comparison of enzyme activities of ROL@aZIF-8 and ROL@ZIF-8

[0050]

[0051] 3. Characterization of immobilized lipase ROL@aZIF-8 and its carrier

[0052] The surface morphology and size of immobilized lipase ROL@aZIF-8 and MOF carrier aZIF-8 were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The MOF carrier aZIF-8 structure and the immobilized lipase ROL@aZIF-8 prepared by it showed irregular disordered structures (such as Figure 1 A and B in the figure show a structure with a diameter of approximately 200-300 nm.

[0053] Scanning transmission electron microscope-energy spectrum element surface scanning analysis ( Figure 3 ) confirmed that Zn and N elements were distributed in the MOF carrier aZIF-8 material. Figure 3The yellow color in the figure indicates the distribution of the Zn element; the red color indicates the distribution of the N element, and the scale in the figure is 250 nm. Through the detection data of elemental analysis and inductively coupled plasma optical emission spectrometry (ICP-OES), as shown in Table 2 below, it can be found that the N / Zn ratio in the immobilized lipase ROL@aZIF-8 is 1.53 and 1.94, proving that ROL is successfully immobilized in aZIF-8, that is, in the amorphous aZIF-8 material, an average of one zinc ion is coordinated with 1.5 nitrogen atoms.

[0054] Table 2 Element percentages of aZIF-8 and ROL@aZIF-8

[0055] Material Weight(mg) N (wt%) Zn N / Zn ROL@aZIF-8 2.3440 16.33 39.06 1.94 aZIF-8 2.2110 11.98 36.46 1.53

[0056] See also Figure 4 XRD analysis shows that the synthesized aZIF-8 nanoparticles have strong peaks at 2θ=11.05°, 12.14°, 17.25° and 18.43°, which are significantly different from the simulated ZIF-8 diffraction peak distribution law, indicating that compared with ZIF-8, the crystal structure of the MOF carrier aZIF-8 has changed significantly, proving that it has an amorphous structure. At the same time, the spectrum of the synthesized immobilized lipase ROL@aZIF-8 nanocomposite is consistent with the spectrum of the synthesized aZIF-8, indicating that the effect of ROL encapsulation on the aZIF-8 material can be ignored. See Figure 5 , N2 adsorption analysis curve, the synthesized ZIF-8 nanomaterials exhibit a typical type I adsorption isotherm with a high level of N2 adsorption, indicating the microporous nature of the crystals. The data in Table 3 below confirm the presence of mesopores in aZIF-8 and ROL@aZIF-8.

[0057] Table 3 BET surface area and pore size of immobilized lipase ROL@aZIF-8 and aZIF-8

[0058] Material <![CDATA[BET surface area (m 2 g -1 )]]> <![CDATA[Pore volume (cm 3 g -1 )]]> ROL@aZIF-8 26.261 0.2177 aZIF-8 83.897 0.189

[0059] It can be seen that the collapse of micropores into mesopores leads to a decrease in specific surface area and pore volume, thereby reducing the nitrogen adsorption capacity. Figure 6 Thermogravimetric analysis showed that the heating rate in air was 10℃·min -1When the temperature is increased, the curve from room temperature to 800 ° C is recorded. The free lipase ROL curve shows two weight losses at 30 ° C ~ 150 ° C and 250 ° C ~ 500 ° C, corresponding to the removal of structural water, decomposition and calcination of the enzyme. The decomposition temperature of aZIF-8 starts at around 350 ° C, showing lower thermal stability. The second stage of immobilized lipase ROL@aZIF-8 starts at 250 ° C and ends at around 350 ° C. About 5wt% of the weight loss occurs in the second stage, which is attributed to the decomposition of protein molecules. And the weight loss at around 410 ° C is small.

[0060] In addition, FT-IR analysis showed that both aZIF-8 and ROL@aZIF-8 have -1 、758cm -1 、953cm -1 and 1308cm -1 A specific absorption peak appears at 1350-1500cm -1 The band at 900-1350cm corresponds to the stretching of the entire ring. -1 The band at 800cm is attributed to the in-plane bending of the ring. -1 The following bands are designated as out-of-plane bending. 421cm -1 The peak at is a typical Zn-N stretching mode, indicating the coordination of zinc ions and nitrogen atoms ( Figure 7 ). The immobilized lipase ROL@aZIF-8 was in the range of 1600-1700 cm -1 The specific absorption peak of the protein amide bond appears at , and the corresponding band cannot be observed in the infrared spectrum of the MOF carrier aZIF-8. This indicates that ROL has been successfully immobilized on the carrier ( Figure 7 ). From the water contact angle graph, it can be seen that the hydrophobicity of amorphous aZIF-8 is reduced, and the hydrophobicity of the aZIF-8 structure with ROL fixed shows a weakening trend ( Figure 8 ).

[0061] 4. Effect of temperature on the activity of immobilized lipase ROL@aZIF-8

[0062] The effect of temperature on the hydrolysis activity of free lipase ROL and immobilized lipase ROL@aZIF-8 was studied in the temperature range of 30-70℃. The results showed that ( Fig. 9), when the temperature was 30-50°C, the hydrolysis activity of free lipase ROL and immobilized lipase ROL@aZIF-8 increased with the increase of temperature. Subsequently, when the temperature was further increased from 50°C to 70°C, the hydrolysis activity of free lipase ROL and immobilized lipase ROL@aZIF-8 decreased. Therefore, the optimal temperature of free lipase ROL and immobilized lipase ROL@aZIF-8 was 50°C, and the immobilized lipase showed better activity than the free lipase. At the same time, after immobilization, when the temperature was between 30 and 70°C, the hydrolysis activity of immobilized lipase ROL@aZIF-8 was still higher than that of free lipase ROL. This may be because the amorphous aZIF-8 structure has a protective effect on lipase ROL.

[0063] 5. Effect of pH value on the activity of immobilized lipase ROL@aZIF-8

[0064] Effect of pH on the hydrolysis activity of free lipase ROL and immobilized lipase ROL@aZIF-8 Fig.10 As shown. The optimum pH value of free lipase ROL and immobilized lipase ROL@aZIF-8 is 9.0. Above or below the optimal pH value, the hydrolysis activity of free lipase ROL and immobilized lipase ROL@aZIF-8 decreases. Free lipase ROL and immobilized lipase ROL@aZIF-8 have high hydrolysis activity under alkaline conditions. When the pH value is 7.5-10.0, the hydrolysis activity of immobilized lipase ROL@aZIF-8 is higher than that of free lipase. These results indicate that the stability of the enzyme is improved under pH changes due to the formation of multi-point bonding (hydrophobic interaction, van der Waals interaction and hydrogen bonding) between aZIF-8 and ROL enzyme molecules, which creates a microenvironment that protects the enzyme molecules and enhances the rigidity of the enzyme.

[0065] 6. Effect of storage time on the activity of immobilized lipase ROL@aZIF-8

[0066] One of the most important reasons for producing immobilized lipase is its storage time stability in industrial applications, as well as achieving high efficiency and cost reduction of industrial products. The stability of immobilized lipase and free lipase ROL was determined by studying the activity decay of immobilized lipase and free lipase ROL after incubation for 25 days at 4°C in 0.01 mol / L phosphate buffer with a pH value of 7.5 ( Fig.11). The results show that the residual activity of immobilized lipase ROL@aZIF-8 decreased slightly, down to 1.49 U / mg, while the residual activity of free lipase ROL decreased relatively significantly, and the enzyme activity of ROL@aZIF-8 was higher than that of free lipase ROL. The improvement in storage stability may be due to the fact that aZIF-8 wrapped around the enzyme molecules played a protective role, preventing protein hydrolysis and aggregation, or weakening the degree of distortion of the chemical environment (buffer solution) on the enzyme active site, thereby slowing down the decrease in lipase activity. This result emphasizes that the lipase-embedded MOF composite material has high chemical and structural stability.

[0067] 7. Effect of different enzyme addition amounts on the activity of immobilized lipase ROL@aZIF-8

[0068] The hydrolysis activity of immobilized lipase ROL@aZIF-8 prepared under different enzyme addition concentration conditions was studied. As the enzyme concentration increased from 3 mg / mL to 4 mg / mL, the ability of immobilized lipase ROL@aZIF-8 to catalyze the hydrolysis of substrates gradually increased ( Fig.12 ). The enhancement of the enzyme activity of the immobilized lipase ROL@aZIF-8 may be due to the increase in the amount of enzyme encapsulated during the preparation process, which leads to more contact between the substrate and the enzyme molecules, thereby improving the catalytic efficiency. As the enzyme concentration increases from 4 mg / mL to 7 mg / mL, the ability of the immobilized lipase ROL@aZIF-8 to catalyze the hydrolysis of the substrate gradually weakens. The weakening of the enzyme activity of the immobilized lipase ROL@aZIF-8 may be due to the fact that during the preparation process, the enzyme molecules may aggregate with the increase in concentration, and are less encapsulated by the carrier, resulting in a decrease in the enzyme immobilization rate in the immobilized lipase, thereby reducing the catalytic efficiency. These research results confirm that the amount of enzyme added has an important regulatory effect on the catalytic activity of the immobilized lipase ROL@aZIF-8.

[0069] The above results of the present invention show that an amorphous ZIF-8 structure is prepared by a certain ratio of metal ion and organic ligand concentration, and the lipase ROL is fixed in the MOF carrier aZIF-8 by an in-situ self-encapsulation method to prepare the immobilized lipase ROL@aZIF-8. The results show that compared with the free lipase ROL, its enzyme activity is improved, and the immobilized lipase ROL@aZIF-8 has higher thermal stability, pH tolerance and storage time stability. At the same time, when the added enzyme concentration is 4 mg / mL, the immobilized lipase ROL@aZIF-8 presents a maximum enzyme activity of 5.69U / mg. The scheme of the present invention helps to provide a potential way for immobilizing lipase, which is worthy of expansion in the catalytic applications and industrial biocatalysis of different types of MOFs and lipases.

[0070] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A controllable preparation method of immobilized Rhizopus oryzae lipase, characterized in that: include: The zinc acetate solution was added to the 2-methylimidazole solution and then the solution containing Rhizopus oryzae lipase was added, and the mixture was stirred for 20-40 minutes. The precipitate was washed and collected, and vacuum freeze-dried to obtain the immobilized Rhizopus oryzae lipase, i.e., the immobilized lipase ROL@aZIF-8. The concentration of zinc acetate solution is 20 mmol / L, the concentration of 2-methylimidazole solution is 33.8 mmol / L, and the concentration of solution containing Rhizopus oryzae lipase is 5 mg / mL; the volume ratio of zinc acetate solution, 2-methylimidazole solution and solution containing Rhizopus oryzae lipase is 1:1:0.08; The collected precipitates were freeze-dried in a vacuum oven at -80 °C for 12 h.

2. The controllable preparation method of immobilized Rhizopus oryzae lipase according to claim 1, characterized in that: The stirring reaction is carried out at a rotation speed of 300~500 r / min.

3. The controllable preparation method of immobilized Rhizopus oryzae lipase according to claim 1, characterized in that: Wash the precipitate with deionized water 3 to 5 times.

4. The immobilized Rhizopus oryzae lipase prepared by the controllable preparation method according to any one of claims 1 to 3, characterized in that: When the added enzyme concentration of the immobilized Rhizopus oryzae lipase was 4 mg / mL, the maximum enzyme activity reached 5.69 U / mg.

5. The immobilized Rhizopus oryzae lipase according to claim 4, characterized in that The enzyme activity of the immobilized Rhizopus oryzae lipase was higher than that of the free enzyme at a temperature range of 30-70 ℃ and a pH value of 7.5-10.

0.

6. Use of the immobilized Rhizopus oryzae lipase according to any one of claims 4 to 5 in the preparation of industrial biocatalysts.

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