Lipase activated by MOF in-situ encapsulated proline as well as preparation method and application of lipase

Abstract

The invention discloses lipase activated by MOF (Metal Organic Framework) in-situ encapsulated proline as well as a preparation method and application of the lipase. The lipase is activated in advance through proline, so that a'cover 'outside the center of a catalytic domain of the lipase is opened in advance, and the high-catalytic-activity spatial conformation of the lipase is fixed by utilizing MOF in-situ encapsulation, so that the enzyme catalyst has high catalytic activity (which is 2.5 times of that of CRL-coated CuBDC). The obtained enzyme catalyst can keep the enzyme activity at a high temperature (7.7 times of the enzyme activity of free enzyme at 90 DEG C), the reaction pH tolerance range is widened (4-11), meanwhile, the enzyme can be recycled and reused, and a new method is provided for solving the problem of activity regulation of lipase in-situ encapsulation. The lipase catalyst obtained by the invention can realize high-efficiency single-enzyme catalytic synthesis of isoamyl acetate, greatly improves the catalytic reaction efficiency of lipase, and provides a new thought for a technology for catalytic synthesis of esters in an immobilized enzyme system.

Description

Technical Field

[0001] The present invention belongs to the field of lipase immobilization, and particularly relates to a method for pre-activating lipase with proline and then in-situ encapsulating it with MOF to improve enzyme activity, as well as the encapsulated and activated lipase obtained by this method and its applications. Background Art

[0002] Lipase, as one of the important industrial enzyme preparations, can catalyze the hydrolysis, alcoholysis, esterification, transesterification and reverse synthesis reactions of triglycerides and some water-insoluble esters, and is widely used in fields such as food, biofuels, and chemical processing products. However, the spatial structure of free lipase is extremely easy to change under harsh environments such as high temperature, strong acid, strong alkali, and organic solvent systems, thereby reducing its catalytic performance. In addition, the application scenarios of free lipase are limited due to its inability to be recycled and reused. To address this, lipase needs to be immobilized. There are various carrier materials for lipase immobilization. Metal-organic frameworks (MOFs) are one of the promising enzyme immobilization support materials at present due to their excellent properties such as large specific surface area, high porosity, adjustable topological structure, and abundant binding sites. The methods for immobilizing enzymes with MOFs can be roughly divided into three categories, namely: adsorption method, covalent method, and encapsulation method. In the first two methods, the MOF-based carrier material is pre-synthesized during the enzyme immobilization process, and then the enzyme is immobilized. Although some of the original enzyme activity can be retained, the immobilized enzyme synthesized by this method is mostly adsorbed on the surface of the carrier material, so its tolerance to the external environment is generally poor. The encapsulation method can immobilize the enzyme inside the MOF carrier material, and the growth of MOF and the immobilization of the enzyme occur simultaneously, protecting it from the influence of the external environment. However, the encapsulation method also has certain limitations. Although the framework protection of MOF improves the tolerance of free enzyme, its growth around the enzyme increases the mass transfer resistance during the catalytic reaction process, resulting in limited activity of the immobilized enzyme. Therefore, how to retain the enzyme activity after encapsulating lipase is a major difficulty.

[0003] The key to the catalytic performance of lipase lies in the opening and closing state of the "lid" outside its catalytic center. By adjusting the lipase immobilization strategy, the effects of hydrophobic interaction, covalent interaction, and adsorption on the catalytic performance of lipase are explored. Molecular dynamics simulation is used to analyze the conformational changes of the lipase "lid" and its effects on substrate binding and catalysis, and it is concluded that hydrophobic binding is the best orientation strategy (Angew. Chem. Int. Ed. 2020, 59, 47, 21080 - 21087). A hierarchical porous lipase immobilization carrier NU-1003 with interconnected mesoporous and microporous channels is prepared. The interfacial interaction of the hydrophobic pores is used to activate the open conformation of lipase, making the captured lipase catalytic center highly accessible. The activities in ester hydrolysis and para-selective catalysis are 1.57 times and 2.46 times that of native lipase (J. Am. Chem. Soc. 2024, 146, 25, 17189 - 17200). From the above, it can be seen that it is feasible to open the "lid" of the lipase a polypeptide chain by hydrophobic interaction to improve the catalytic performance of lipase. However, how to maintain the high-active conformation of lipase is another major problem, and it is necessary to encapsulate in situ when the enzyme is in the high-active spatial structure state to fix its spatial structure. An idea for quickly encapsulating enzymes, abbreviated as the AAOPE strategy, is proposed (Angew. Chem. Int. Ed. 2019, 58(5), 1463 - 7). This method promotes the formation of prenucleation clusters around proteins in the form of self-assembly of proteins, polyvinylpyrrolidone (PVP), and cysteine (Cys). Among them, PVP is used to cover and enrich Cys around the protein, promoting the formation of thiol bonds between Cys and the protein to accumulate Zn 2+ ions in the MOF, thereby accelerating the formation of prenucleation clusters and the growth of MOF around the protein. Experiments prove that the encapsulated protein can maintain its inherent conformation, and the structural tightness of the MOF endows the enzyme with excellent stability. In summary, pre-activating lipase and fixing its spatial structure are of great significance for the preparation of highly active MOF-encapsulated lipase in situ. Summary of the Invention

[0004] To solve the shortcomings and deficiencies of the prior art, the primary object of the present invention is to provide a preparation method for MOF in-situ encapsulating proline-activated lipase, specifically: using proline to pre-activate lipase to open the "lid" outside the center of its catalytic domain, and at the same time using the coordination effect of proline with Cu 2+ to attract it to form prenucleation clusters to promote the in-situ encapsulation process and fix the high-active conformation of lipase, so as to prepare a highly active and temperature-resistant enzyme catalyst.

[0005] To open the "lid" outside the catalytic center of lipase and improve its catalytic activity, by utilizing the induction effect of amino acids on the enzyme's spatial structure, the effects of different types of amino acids on the catalytic activity of lipase were first studied, and it was found that the moderate hydrophobic interaction force between proline and the α-helix "lid" of lipase could induce lipase to be in the optimal highly active state. In addition, both the hydroxyl oxygen and imino nitrogen of proline can coordinate with metals, which is beneficial to the subsequent formation of enzyme prenucleation clusters. Combining with the positive enzyme activity promotion effect of Cu 2+ on lipase, Cu-based MOF CuBDC was selected to encapsulate lipase in situ. The enzyme loading of the prepared proline-CRL@CuBDC can reach up to 139 mg / g at most, which is 41% higher than that of direct in-situ encapsulation, and the encapsulation rate can reach up to 96% at this time. The maximum enzyme activity can be 2.5 times that of directly encapsulated CRL@CuBDC, and the enzyme activity at 90 °C is 7.7 times that of free lipase.

[0006] Another object of the present invention is to provide a MOF in-situ encapsulated proline-activated lipase prepared by the above preparation method.

[0007] Another object of the present invention is to provide the application of the above MOF in-situ encapsulated proline-activated lipase as a catalyst in the catalytic synthesis of esters.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] A preparation method of a MOF in-situ encapsulated proline-activated lipase, comprising the following steps:

[0010] Mix the proline solution and the lipase solution to activate the lipase, then add the soluble copper salt solution and mix evenly, and then add the terephthalic acid compound solution. After mixing and stirring for a period of time, keep it at a constant temperature and stand still. Separate the solid and liquid, wash the solid phase and dry it to obtain the proline-CRL@CuBDC enzyme complex.

[0011] Preferably, the concentration of the proline solution is 50 - 400 mmol / L, and the solvent is water; the concentration of the soluble copper salt solution is 60 ± 5 mmol / L, and the solvent is water; the concentration of the terephthalic acid compound is 30 ± 5 mmol / L, and the solvent is water; the concentration of the lipase solution is 25 ± 5 mg / mL, and the solvent is Tris-HCl buffer solution with pH = 7 - 8.

[0012] Preferably, the soluble copper salt in the soluble copper salt solution is at least one of copper nitrate and copper acetate; the terephthalic acid compound in the terephthalic acid compound solution is at least one of terephthalic acid and disodium terephthalate.

[0013] Preferably, the mass ratio of proline in the proline solution to lipase in the lipase solution is 1.15 to 9.2:1; more preferably 2.3 to 6.9:1.

[0014] Preferably, the mixing and activation time of the proline solution and the lipase solution is 1.5 ± 0.5 h.

[0015] Preferably, the molar ratio of the soluble copper salt in the soluble copper salt solution to the terephthalic acid compound in the terephthalic acid compound solution is 1:1 to 4:1, more preferably 1:1 to 3:1; the ratio of the soluble copper salt in the soluble copper salt solution to the lipase in the lipase solution is 2 to 3 mmol:50 mg, 2.4 mmol:50 mg.

[0016] Preferably, the mixing and stirring time after adding the terephthalic acid compound solution is 0.5 to 2 h, more preferably 1 ± 0.5 h; the stirring speed is 50 to 200 r / min; more preferably 100 ± 50 r / min.

[0017] Preferably, the constant temperature standing means the temperature condition is 30 ± 2 °C; the standing time is 6 to 14 h; more preferably 12 ± 2 h.

[0018] Preferably, the washing liquid used for washing is deionized water and sodium dodecyl sulfate (SDS) detergent. In the washing process, first wash with deionized water 3 to 5 times, and then wash with SDS detergent 3 to 5 times; the drying temperature is 30 to 40 °C, and the drying time is 12 ± 2 h.

[0019] The present invention also provides a MOF in-situ encapsulated proline-activated lipase (proline-CRL@CuBDC) prepared by the above preparation method.

[0020] The present invention also provides the application of the above MOF in-situ encapsulated proline-activated lipase (proline-CRL@CuBDC) as a catalyst in the catalytic synthesis of esters.

[0021] Preferably, the application of a proline-CRL@CuBDC as a catalyst in the catalytic synthesis of isoamyl acetate.

[0022] More preferably, the specific steps are: mixing the substrate with proline-CRL@CuBDC and reacting at 40 to 80 °C for 8 to 48 h to achieve the catalytic synthesis of isoamyl acetate.

[0023] Further preferably, the molar ratio of alcohol to acid in the substrate is 1:1 to 16:1.

[0024] Further preferably, the substrate is a mixture of glacial acetic acid and isoamyl alcohol.

[0025] Further preferably, the enzyme concentration is 0.1 - 1.0 mg / mL, and the substrate concentration is 0.5 - 10 mmol / L.

[0026] The mechanism of the present invention is as follows:

[0027] As a hydrophobic cyclic imino acid, proline has a moderate hydrophilic index value (-1.6), which has the optimal spatial structure induction effect on lipase. It can be seen from the secondary spatial structure before and after lipase encapsulation that the α-helix of lipase continuously decreases, and the lid covering the center of the lipase catalytic domain is induced to open by proline, exposing the active center of lipase, thereby improving its catalytic activity. In addition, the hydroxyl oxygen and imino nitrogen of proline coordinate with Cu 2+ to promote the formation of pre-nucleation clusters of enzyme complexes, improve the encapsulation efficiency of lipase, and further facilitate the preparation of highly efficient lipase catalysts.

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

[0029] 1. The MOF in-situ encapsulation method adopted by the present invention can not only well retain the enzyme activity of lipase under extreme conditions (high temperature, strong acid and strong alkali solutions, various organic solvents), but also this in-situ encapsulation method can realize the recycling and reuse of lipase.

[0030] 2. The proline-CRL@CuBDC prepared by the present invention has a defective mesoporous and macroporous structure, which can reduce the mass transfer resistance of substrate and product in and out, and is beneficial to the progress of the enzyme-catalyzed process.

[0031] 3. The proline-CRL@CuBDC prepared by the present invention adopts an innovative path of pre-activation. Proline has excellent enzyme activation performance of 2.2 times, which greatly improves the catalytic performance of lipase after in-situ encapsulation.

[0032] 4. Proline can not only activate lipase here, but the coordination binding of its own carboxyl oxygen and imino nitrogen with Cu 2+ is also beneficial to the formation of pre-nucleation clusters during the subsequent enzyme encapsulation process, thereby promoting the in-situ encapsulation process of lipase. The enzyme loading of the prepared proline-CRL@CuBDC can reach 139 mg / g, which is 41% higher than that of direct in-situ encapsulation.

[0033] 5. The proline-CRL@CuBDC prepared by the present invention has excellent catalytic performance. The enzyme activity retention rate of the catalyst can reach up to 2.5 times that of the directly in-situ encapsulated CRL@CuBDC. Moreover, at 90 °C, the high-temperature stability of the catalyst is 7.7 times that of free lipase, and the pH tolerance range of the catalyst is broadened to 4 - 11. Meanwhile, proline-CRL@CuBDC also exhibits excellent organic solvent tolerance, storage stability, and recycling stability. In the application of catalytic synthesis of isoamyl acetate, its yield at 80 °C can reach 94%, which is 6.3 times that of free enzyme. Description of the Drawings

[0034] Figure 1 It shows the effects of different (a) stirring speeds, (b) stirring times, (c) crystallization times, and (d) metal ligand molar ratios on the XRD of CuBDC for the complexes obtained from Exploration Experiments 1 - 4 of the present invention.

[0035] Figure 2 In (a), it shows the remaining enzyme activities of different amino acids, namely proline, histidine, serine, arginine, tryptophan, lysine, alanine, and glycine, after activating lipase in Comparative Examples 3 - 10 and Comparative Example 13 of the present invention. In (b), it shows the remaining enzyme activities of the lipase catalysts encapsulated in-situ with proline, histidine, and serine and the CRL@CuBDC catalyst obtained in Example 1, Comparative Examples 11 - 13, and Comparative Example 2 of the present invention.

[0036] Figure 3 It is a schematic diagram of the preparation process of the proline-CRL@CuBDC enzyme complex obtained from Examples 1 - 5 of the present invention.

[0037] Figure 4 It shows the (a - f) SEM images, (g) mapping images, (h) TEM diffraction imaging, and (i) CLSM imaging of the CRL@CuBDC obtained in Comparative Example 2 and the proline-CRL@CuBDC materials obtained from Examples 1 - 5 of the present invention.

[0038] Figure 5 It shows the enzyme loading and encapsulation efficiency of the CRL@CuBDC obtained in Comparative Example 2 and the proline-CRL@CuBDC materials obtained from Examples 2 - 5 of the present invention, where the concentration of amino acid refers to the concentration in the entire system mixed solution.

[0039] Figure 6 It shows the synthesis yields of isoamyl acetate of the CRL obtained in Comparative Example 13, the CRL@CuBDC obtained in Comparative Example 2, and the proline-CRL@CuBDC materials obtained from Examples 2 - 4 of the present invention under (a) different solvent system conditions, (b) different alcohol-acid molar ratios, and (c) different temperature conditions.

[0040] Figure 7 For the CRL obtained in Comparative Example 13, the CRL@CuBDC obtained in Comparative Example 2, and the proline-CRL@CuBDC materials obtained in Examples 2 to 4 of the present invention, (a) organic solvent tolerance (proine 30 -CRL@CuBDC), (b) storage stability (proine 30 -CRL@CuBDC), (c) cycle stability (proine 30 -CRL@CuBDC), (d) high-temperature stability. Detailed implementation mode

[0041] The present invention will be further described in detail below with reference to the examples and the drawings, but the implementation modes of the present invention are not limited thereto.

[0042] For those not specified in the examples of the present invention, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. The raw materials, reagents, etc. not specified for the manufacturer can be obtained as conventional products through commercial purchase.

[0043] Exploration experiment 1

[0044] Table 1 Influence of different metal ligand molar ratios on CuBDC

[0045]

[0046] According to the above table, the copper nitrate solution and the disodium terephthalate solution are mixed and stirred at a volume ratio of 1:1. The stirring temperature of the above solution is 30°C. After stirring, it is kept at a constant temperature and left standing to achieve crystallization. The crystallization conditions are in a 30°C constant temperature incubator. After the reaction is completed, suction filtration is carried out to achieve solid-liquid separation. The solid phase is washed three times with deionized water to obtain MOF-CuBDC with different metal ligand molar ratios, and it is stored for later use at room temperature.

[0047] Exploration experiment 2

[0048] Table 2 Influence of different stirring speeds on CuBDC

[0049]

[0050] According to the above table, the copper nitrate solution and the disodium terephthalate solution are mixed and stirred at a volume ratio of 1:1. The stirring temperature of the above solution is 30°C. After stirring, it is kept at a constant temperature and left standing to achieve crystallization. The crystallization conditions are in a 30°C constant temperature incubator. After the reaction is completed, suction filtration is carried out to achieve solid-liquid separation. The solid phase is washed three times with deionized water to obtain MOF-CuBDC with different metal ligand molar ratios, and it is stored for later use at room temperature.

[0051] Exploration experiment 3

[0052] Table 3 Effects of Different Stirring Times on CuBDC

[0053]

[0054] Mix the copper nitrate solution and disodium terephthalate solution according to the volume ratio of 1:1 as shown in the above table and stir. The stirring temperature of the above solution is 30 °C. After stirring, keep it at a constant temperature and let it stand to achieve crystallization. The crystallization condition is in a 30 °C constant temperature incubator. After the reaction is completed, perform suction filtration to achieve solid-liquid separation. Wash the solid phase three times with deionized water to obtain MOF-CuBDC with different metal-ligand molar ratios, and store it for later use at room temperature.

[0055] Exploration Experiment 4

[0056] Table 4 Effects of Different Crystallization Times on CuBDC

[0057]

[0058] Mix the copper nitrate solution and disodium terephthalate solution according to the volume ratio of 1:1 as shown in the above table and stir. The stirring temperature of the above solution is 30 °C. After stirring, keep it at a constant temperature and let it stand to achieve crystallization. The crystallization condition is in a 30 °C constant temperature incubator. After the reaction is completed, perform suction filtration to achieve solid-liquid separation. Wash the solid phase three times with deionized water to obtain MOF-CuBDC with different metal-ligand molar ratios, and store it for later use at room temperature.

[0059] Comparative Example 1

[0060] Mix 40 mL of 60 mmol / L copper nitrate hexahydrate solution and 40 mL of 30 mmol / L disodium terephthalate solution, stir at 30 °C for 1 h, and the stirring speed is 100 r / min. Incubate in a 30 °C constant temperature incubator for 12 h. Then perform suction filtration for solid-liquid separation, leave the solid phase and wash it three times with deionized water to obtain CuBDC, and store it for later use at room temperature.

[0061] Comparative Example 2

[0062] Add 50 mg of lipase to 2 mL of Tris-HCl buffer solution with pH = 8 and dissolve it by ultrasonic treatment. Then mix it evenly with 40 mL of 60 mmol / L copper nitrate hexahydrate solution, and then add 40 mL of 30 mmol / L disodium terephthalate solution dropwise. Stir at 30 °C for 1 h and incubate in a 30 °C constant temperature incubator for 12 h. Then perform suction filtration to achieve solid-liquid separation, wash the solid phase three times with deionized water and 0.1 g / mL sodium dodecyl hexasulfonate (SDS) aqueous solution respectively to obtain the solid phase CRL@CuBDC, dry it at 30 °C for 12 h, grind the dried sample, and store it in a 4 °C refrigerator for later use.

[0063] Comparative Example 3

[0064] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L proline solution for 1.5 h.

[0065] Comparative Example 4

[0066] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L histidine solution for 1.5 h.

[0067] Comparative Example 5

[0068] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L serine solution for 1.5 h.

[0069] Comparative Example 6

[0070] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L arginine solution for 1.5 h.

[0071] Comparative Example 7

[0072] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L tryptophan solution for 1.5 h.

[0073] Comparative Example 8

[0074] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L lysine solution for 1.5 h.

[0075] Comparative Example 9

[0076] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L alanine solution for 1.5 h.

[0077] Comparative Example 10

[0078] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L glycine solution for 1.5 h.

[0079] Comparative Example 11

[0080] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L histidine solution for 1.5 h. Then it was mixed evenly with 40 mL of 60 mmol / L copper nitrate hexahydrate solution, and 40 mL of 30 mmol / L disodium terephthalate solution was added dropwise thereto. It was stirred at 30 °C for 1 h and cultured in a constant temperature incubator at 30 °C for 12 h. Then suction filtration was carried out to achieve solid-liquid separation, and the solid phase histidine was washed three times with deionized water and 0.1 g / mL sodium dodecyl hexasulfonate (SDS) aqueous solution respectively. 30 -CRL@CuBDC, dried at a constant temperature of 30 °C for 12 h, the dried sample was ground and stored in a refrigerator at 4 °C for standby.

[0081] Comparative Example 12

[0082] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L serine solution for 1.5 h. Then it was mixed evenly with 40 mL of 60 mmol / L copper nitrate hexahydrate solution, and 40 mL of 30 mmol / L disodium terephthalate solution was added dropwise thereto. It was stirred at 30 °C for 1 h and cultured in a constant temperature incubator at 30 °C for 12 h. Then suction filtration was carried out to achieve solid-liquid separation, and the solid phase serine was washed three times with deionized water and 0.1 g / mL sodium dodecyl hexasulfonate (SDS) aqueous solution respectively. 30 -CRL@CuBDC, dried at a constant temperature of 30 °C for 12 h, the dried sample was ground and stored in a refrigerator at 4 °C for standby.

[0083] Comparative Example 13

[0084] Purchased free lipase CRL.

[0085] Example 1

[0086] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 50 mmol / L proline solution for 1.5 h. Then it was mixed evenly with 40 mL of 60 mmol / L copper nitrate hexahydrate solution, and 40 mL of 30 mmol / L disodium terephthalate solution was added dropwise thereto. It was stirred at 30 °C for 1 h and cultured in a constant temperature incubator at 30 °C for 12 h. Then suction filtration was carried out to achieve solid-liquid separation, and the solid phase proline 5-CRL@CuBDC was washed three times with deionized water and 0.1 g / mL sodium dodecyl hexasulfonate (SDS) aqueous solution respectively, dried at a constant temperature of 30 °C for 12 h, the dried sample was ground and stored in a refrigerator at 4 °C for standby.

[0087] Example 2

[0088] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer solution with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 100 mmol / L proline solution for 1.5 h. Then it was mixed evenly with 40 mL of 60 mmol / L copper nitrate hexahydrate solution, and then 40 mL of 30 mmol / L disodium terephthalate solution was added dropwise thereto. It was stirred at 30 °C for 1 h and cultured in a constant temperature incubator at 30 °C for 12 h. Then suction filtration was carried out to achieve solid-liquid separation, and the solid phase proline was obtained by washing three times with deionized water and 0.1 g / mL sodium dodecyl hexasulfonate (SDS) aqueous solution respectively. 10 -CRL@CuBDC, dried at a constant temperature of 30 °C for 12 h, the dried sample was ground and stored in a refrigerator at 4 °C for later use.

[0089] Example 3

[0090] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer solution with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 200 mmol / L proline solution for 1.5 h. Then it was mixed evenly with 40 mL of 60 mmol / L copper nitrate hexahydrate solution, and then 40 mL of 30 mmol / L disodium terephthalate solution was added dropwise thereto. It was stirred at 30 °C for 1 h and cultured in a constant temperature incubator at 30 °C for 12 h. Then suction filtration was carried out to achieve solid-liquid separation, and the solid phase proline was obtained by washing three times with deionized water and 0.1 g / mL sodium dodecyl hexasulfonate (SDS) aqueous solution respectively. 20 -CRL@CuBDC, dried at a constant temperature of 30 °C for 12 h, the dried sample was ground and stored in a refrigerator at 4 °C for later use.

[0091] Example 4

[0092] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer solution with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 300 mmol / L proline solution for 1.5 h. Then it was mixed evenly with 40 mL of 60 mmol / L copper nitrate hexahydrate solution, and then 40 mL of 30 mmol / L disodium terephthalate solution was added dropwise thereto. It was stirred at 30 °C for 1 h and cultured in a constant temperature incubator at 30 °C for 12 h. Then suction filtration was carried out to achieve solid-liquid separation, and the solid phase proline was obtained by washing three times with deionized water and 0.1 g / mL sodium dodecyl hexasulfonate (SDS) aqueous solution respectively. 30 -CRL@CuBDC, dried at a constant temperature of 30 °C for 12 h, the dried sample was ground and stored in a refrigerator at 4 °C for later use.

[0093] Example 5

[0094] After dissolving 50 mg of lipase in 2 mL of Tris-HCl buffer with pH = 8 by ultrasonic treatment, it was mixed and stirred with 10 mL of 400 mmol / L proline solution for 1.5 h. Then it was mixed evenly with 40 mL of 60 mmol / L copper nitrate hexahydrate solution, and 40 mL of 30 mmol / L disodium terephthalate solution was added dropwise thereto. It was stirred at 30 °C for 1 h and cultured in a constant temperature incubator at 30 °C for 12 h. Then suction filtration was carried out to achieve solid-liquid separation, and the solid phase proline was obtained by washing three times with deionized water and 0.1 g / mL aqueous solution of sodium dodecyl hexasulfonate (SDS) respectively. 40 -CRL@CuBDC was dried at a constant temperature of 30 °C for 12 h, the dried sample was ground, and stored in a refrigerator at 4 °C for standby.

[0095] Test conditions:

[0096] I. Determination of lipase activity

[0097] Under suitable conditions, p-nitrophenyl caproate can be decomposed into yellow p-nitrophenol (pNP) by lipase and others. By detecting the absorbance of the product pNP, the lipase activity can be calculated. The specific steps are as follows:

[0098] Prepare 1.5 mL of buffer system (Tri-HCl, pH = 8.0, 100 mmol / L), in which the enzyme concentration is 0.1 mg / mL and the substrate concentration of p-nitrophenyl caproate is 10 mmol / L. It was cultured at 50 - 90 °C for 1.5 h. After the culture was completed, the reaction system was quenched for 10 min, and then the absorbance of p-nitrophenol at 410 nm was measured with an enzyme-labeling instrument. It was substituted into the pNP standard curve with the pNP concentration (μmol / L) as the abscissa and the absorbance at 410 nm as the ordinate drawn under the same conditions to calculate the content of p-nitrophenol. All tests were repeated three times, and the specific reaction temperature was recorded in the subsequent test results. In the determination of lipase activity, the amount of free enzyme added is equal to the amount of encapsulated enzyme in the enzyme complex.

[0099] The definition of lipase activity unit (U) is: the amount of enzyme required for 1 mg of free enzyme / immobilized enzyme to catalyze the hydrolysis of the substrate to produce 1.0 μmoL of p-nitrophenol at 50 °C and pH 8.0 for 1 min. Lipase activity formula:

[0100]

[0101] Among them, X is the lipase activity (U); C is the pNP concentration (μmol / L); V is the final volume of the reaction solution (L); T is the reaction time (min); M is the enzyme dosage (mg).

[0102] II. Calculation of enzyme encapsulation efficiency and enzyme loading

[0103] The BCA method was used to determine the lipase content, and the purchased free lipase was used to make the standard curve. The specific operation of the BCA method is as follows: Mix 20 μL of the standard enzyme sample or the sample to be tested with 200 μL of the BCA working solution (where the volume ratio of solution A to solution B = 50:1), react at 37 °C for 30 min, and then use a microplate reader to detect the absorbance at 562 nm. The standard curve and the concentration of the sample to be tested can be obtained according to the relationship between the absorbance and the concentration.

[0104] The encapsulation efficiency of the enzyme is defined as the mass percentage of the enzyme encapsulated in the carrier MOF to the amount of the original free enzyme added. The formula is as follows:

[0105]

[0106] Where m represents the mass (mg) of the original free enzyme added; c is the lipase concentration (mg / mL) in the supernatant after encapsulation; V is the total volume (mL) of the supernatant after encapsulation.

[0107] The enzyme loading is defined as the percentage of the mass of the enzyme encapsulated in the carrier MOF to the total mass of the entire enzyme@MOF composite catalyst. The formula is as follows:

[0108]

[0109] Where M represents the total mass of the obtained enzyme@MOF composite catalyst.

[0110] III. Synthesis and detection of isoamyl acetate

[0111] Mix 1 mL of 0.5 mmol / L acetic acid solution with different molar concentrations of isoamyl alcohol solutions (the molar ratios of alcohol to acid are 1:1, 2:1, 4:1, 8:1, 12:1, 16:1) in a high-temperature and high-pressure glass tube, add 30 mg of the enzyme complex, react at 40 °C, 60 °C, and 80 °C for 8, 16, 24, 32, 40, and 48 h. Centrifuge the obtained mixture at 10000 rpm for 5 min, take 1 μL of the supernatant for gas chromatography to detect the content of isoamyl acetate. The formula for calculating the yield of isoamyl acetate is as follows:

[0112]

[0113] Where c represents the concentration of isoamyl acetate, mmol / L; c0 represents the initial concentration of acetic acid, mmol / L.

[0114] This invention explored the effects of different reaction conditions on the crystallinity of CuBDC ( Figure 1) It is concluded that the crystallinity of CuBDC first increases and then decreases with the increase of stirring speed, the prolongation of stirring time, the prolongation of crystallization time, and the increase of the metal-ligand molar ratio. Therefore, the optimal synthesis conditions for subsequent enzyme encapsulation are determined as follows: the rotation speed is 100 r / min, the stirring time is 1 h, the crystallization time is 12 h, and the metal-ligand molar ratio is 2:1.

[0115] This invention explores the activation effect of different amino acids on lipase ( Figure 2 (a) in it), and it is concluded that proline, histidine, and serine have a positive promoting effect on lipase. Then, the lipases activated by proline, histidine, and serine with positive promoting effects are encapsulated in situ. The results are shown in Figure 2 (b) in it. It can be seen from the figure that the lipase after proline encapsulation can retain 85% of its enzyme activity at 40 °C, which is twice that of the directly in-situ encapsulated CRL@CuBDC catalyst.

[0116] The preparation process of the series of proline-CRL@CuBDC enzyme complexes prepared in this invention is as Figure 3 shown.

[0117] From the mapping diagram of the enzyme complex ( Figure 4 (a) in it), it can be seen that C, N, O, Cu, and the characteristic element S of the enzyme are evenly distributed, indicating that the lipase is evenly distributed in the prepared carrier material. Figure 4 (b) in it is the transmission electron microscope diffraction imaging of the sample. From the confocal laser scanning microscopy (CLSM) imaging of the sample ( Figure 4 (c) in it), the fluorescence of the lipase in the corresponding position of the enzyme complex can be observed in the dark field, proving that the lipase has been successfully encapsulated into the carrier material. Figure 5 It also proves that the enzyme complex has good lipase loading capacity. When proline pre-activates the lipase and then encapsulates it, the encapsulation rate of the enzyme complex is 96%, and the enzyme loading can be as high as 139 mg / g.

[0118] Table 5 shows the enzyme activity test results of different samples of this invention at different temperatures. The highest enzyme activity retention rate of the proline 30 -CRL@CuBDC prepared by this method is 115%, which is 2.05 times that of 56% of the direct in-situ encapsulation method. In addition, compared with the free enzyme, the optimal reaction temperature of the experimentally prepared enzyme complex has increased from 40 °C to 60 °C, and at the optimal temperatures corresponding to both, the enzyme activity retention rate of the enzyme complex can reach 1.15 times that of the free enzyme, which is attributed to the effective activation of the high-activity conformation of lipase by proline. In addition, it can be seen from Table 6 that proline 10 -CRL@CuBDC, proline 20 -CRL@CuBDC, proline30 -CRL@CuBDC retained a high enzyme activity within a wider pH range (pH = 4 - 11), and the enzyme activity retention rate was generally above 75%, proline 30 -CRL@CuBDC was 102% under the condition of pH 9, which was 1.79 times that of directly in-situ encapsulated CRL@CuBDC. The above results together indicate that the prepared samples can retain high enzyme activity at higher temperatures and within a wider pH range, which is beneficial to broadening the application range of lipase in high-temperature and acid-base environments.

[0119] Table 5 Enzyme activities of different samples at different temperatures

[0120]

[0121] Table 6 Enzyme activities of different samples at different pH values

[0122]

[0123]

[0124] To evaluate the catalytic performance of this catalyst, the immobilized proline 30 -CRL@CuBDC was used to catalyze the synthesis reaction of isoamyl acetate, and the substrates were acetic acid and isoamyl alcohol. The effects of different solvents and different molar ratios of alcohol to acid on the synthesis reaction of isoamyl acetate were investigated. As Figure 6 shown in (a, b), the optimal solvent system for the synthesis reaction was isooctane, and the optimal molar ratio of alcohol to acid was 8:1. Since the experimentally prepared enzyme complex had excellent performance at higher temperatures, the synthesis yield of the product under higher temperature conditions (40 - 80 °C) was investigated ( Figure 6 in (c)). The results showed that at 40 °C, the yield of isoamyl acetate synthesized by proline 30 -CRL@CuBDC was up to 1.2 times that of the free lipase and 2.4 times that of directly in-situ encapsulated CRL@CuBDC, and the catalytic performance improvement effect of the immobilized enzyme became more obvious with the increase of temperature. At the high temperature of 80 °C, the yield of isoamyl acetate catalyzed by the free lipase was only 15%, and the yield of directly in-situ encapsulated CRL@CuBDC was 49%, while the synthesis yield of isoamyl acetate by proline 30 -CRL@CuBDC was up to 94%, which was 6.3 times that of the free enzyme and 1.9 times that of CRL@CuBDC. It can be seen that the prepared enzyme complex has excellent catalytic performance under high temperature conditions, demonstrating the excellent protective effect of the MOF framework on the free lipase.

[0125] The stability of the enzyme complex is an important parameter for evaluating its performance. The protective effect of the MOF support on lipase is specifically reflected in aspects such as organic solvent tolerance, storage stability, recyclability, and high-temperature stability. The specific test results are shown in Figure 7 . In terms of organic solvent tolerance, after the enzyme complex was exposed to various organic solvents such as methanol (MeOH), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone (Acetone), and urea (Urea) at 40 °C for 30 min, the remaining enzyme activity was detected. It can be concluded that proline 30 -CRL@CuBDC has good tolerance to the above organic solvents and generally can maintain 70 - 90% of the enzyme activity, while the free enzyme showed a huge loss of enzyme activity, with only 20 - 40% of the enzyme activity retention rate. After the enzyme complex was placed at room temperature for 45 days, the remaining enzyme activity was detected, and it was found that the enzyme complex could retain 86% of the enzyme activity, showing excellent storage stability. The enzyme complex was subjected to the synthesis reaction of isoamyl acetate. After the reaction was completed, the catalyst was recovered by centrifugation, and the enzyme activity retention rate of the sample was tested. Then it was washed with deionized water for the next cycle reaction. The change in enzyme activity within 10 consecutive cycles was tested to explore the recycling performance of the enzyme complex. Finally, it was detected that it still had a relatively high enzyme activity retention rate of 87%. In addition, the stability of the enzyme complex under high-temperature conditions was explored. The remaining enzyme activity of the enzyme complex and free lipase after incubation in Tris-HCl buffer solution with pH 8 at 60 °C, 70 °C, 80 °C, and 90 °C for 2 h was measured, and the results are shown in Figure 7 (d). It can be seen that at 90 °C, the enzyme activity retention rate of proline 30 -CRL@CuBDC can reach 7.7 times that of the free enzyme and 2 times that of directly encapsulated CRL@CuBDC, indicating that the ability of lipase to resist high temperature is significantly enhanced after being encapsulated. This is because the stable framework structure formed by MOF restricts the breakage of the relatively weak non-covalent interactions (such as van der Waals forces or hydrogen bonds) around the active center of lipase under high-temperature conditions, thereby greatly reducing the conformational change of the enzyme molecule in the high-temperature environment, stabilizing the tertiary structure of lipase, reducing the inactivation of lipase under high-temperature conditions, and macroscopically showing a significant improvement in the high-temperature stability of the enzyme complex.

[0126] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of MOF in-situ encapsulating proline-activated lipase, characterized in that, It includes the following steps: Mix the proline solution with the lipase solution to activate the lipase, then add the soluble copper salt solution and mix evenly, then add the terephthalic acid compound solution, mix and stir for a period of time and then keep it static at a constant temperature, separate the solid and liquid, wash the solid phase and dry it to obtain the proline-CRL@CuBDC enzyme complex.

2. The preparation method of a MOF in-situ encapsulating proline-activated lipase according to claim 1, characterized in that The mass ratio of proline in the proline solution to lipase in the lipase solution is 1.15 - 9.2:1; And / or, the mixing and activation time of the proline solution and the lipase solution is 1.5 ± 0.5 h.

3. The preparation method of a MOF in-situ encapsulating proline-activated lipase according to claim 1 or 2, characterized in that, The molar ratio of the soluble copper salt in the soluble copper salt solution to the terephthalic acid compound in the terephthalic acid compound solution is 1:1 - 4:1; And / or, the ratio of the soluble copper salt in the soluble copper salt solution to the lipase in the lipase solution is 2 - 3 mmol:50 mg.

4. The preparation method of a MOF in-situ encapsulating proline-activated lipase according to claim 1 or 2, characterized in that, The mixing and stirring time after adding the terephthalic acid compound solution is 0.5 - 2 h; the stirring speed is 50 - 200 r / min.

5. The preparation method of an MOF in-situ encapsulating proline-activated lipase according to claim 1 or 2, characterized in that, The constant temperature static means the temperature condition is 30 ± 2 °C; And / or, the static time is 6 - 14 h.

6. The preparation method of a MOF in-situ encapsulating proline-activated lipase according to claim 1 or 2, characterized in that, The concentration of the proline solution is 50 - 400 mmol / L, and the solvent is water; And / or, the concentration of the soluble copper salt solution is 60 ± 5 mmol / L, and the solvent is water; And / or, the concentration of the terephthalic acid compound is 30 ± 5 mmol / L, and the solvent is water; And / or, the concentration of the lipase solution is 25 ± 5 mg / mL, and the solvent is Tris-HCl buffer solution with pH = 7 - 8.

7. The preparation method of a MOF in-situ encapsulating proline-activated lipase according to claim 1 or 2, characterized in that, The soluble copper salt in the soluble copper salt solution is at least one of copper nitrate and copper acetate; And / or, the terephthalic acid compound in the terephthalic acid compound solution is at least one of terephthalic acid and disodium terephthalate.

8. A MOF in-situ encapsulating proline-activated lipase prepared by the preparation method according to any one of claims 1 - 7.

9. The application of the MOF in-situ encapsulating proline-activated lipase according to claim 8 as a catalyst in the catalytic synthesis of esters.

10. The application according to claim 9, wherein The application of the MOF in-situ encapsulating proline-activated lipase as a catalyst in the catalytic synthesis of isoamyl acetate; The specific steps are: mix the substrate with proline-CRL@CuBDC, and react at 40 - 80 °C for 8 - 48 h to achieve the catalytic synthesis of isoamyl acetate; And / or, the substrate is a mixture of glacial acetic acid and isoamyl alcohol; And / or, the molar ratio of alcohol to acid in the substrate is 1:1 - 16:1; And / or, the enzyme concentration is 0.1 - 1.0 mg / mL, and the concentration of the substrate is 0.5 - 10 mmol / L.

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