Lipase immobilization method and application of lipase immobilization method in improvement of catalytic activity and / or stability of immobilized lipase

Abstract

The invention discloses a preparation method of immobilized lipase and application of the immobilized lipase in improvement of catalytic activity and / or stability of the immobilized lipase, the preparation method comprises the following steps: carrying out a coordination reaction on a lipase solution, a zinc salt and an organic ligand under a weakly alkaline condition to obtain the immobilized lipase, the lipase solution is a propanol solution containing lipase. According to the method, one-step synthesis of the lipase (at) MOF biological composite material is realized through a direct aggregation conformation locking strategy, and compared with a traditional solvothermal method, a spray layer-by-layer liquid phase epitaxy method and a phase change method, the method is simpler and more convenient to operate, crystallization is performed under mild conditions, lipase is immobilized, the influence on lipase is smaller, and the method is suitable for industrial production. The lipase has strong biological catalytic activity and excellent stability which are obviously superior to those of natural free lipase, can be recycled for multiple times, and has important popularization and application values in large-scale utilization of lipase in industrial production in biological catalytic reaction.

Description

Technical Field

[0001] The present invention relates to the field of protein engineering, and in particular to a method for preparing immobilized lipase and application of the method in improving the catalytic activity and / or stability of the immobilized lipase. Background Art

[0002] Lipase is a type of hydrolase that can catalyze the cleavage of ester bonds under environmentally friendly conditions. Due to its low cost and good thermal stability, lipase has great potential in important industrial fields such as food processing, environmental remediation, organic synthesis, biofuels and pharmaceutical production. The thermal stability of lipase depends on its unique "lid" conformation, that is, the active center (Ser-His-Asp catalytic triad) is buried in a hydrophobic pocket covered by a removable α-helical "lid". This "lid" can protect the active center from external losses, however, it inevitably limits the accessibility of the buried active center, affecting the catalytic activity of lipase. Therefore, how to solve the contradiction between the catalytic activity and stability of lipase is a difficult problem that technicians in this field focus on.

[0003] Immobilized lipase is a composite lipase catalyst that is immobilized and bound to a carrier by physical or chemical methods. Nanomaterials are commonly used carriers for immobilized lipase, which stabilize the overall conformation of lipase by setting up an "armor", and the interfacial interaction between lipase and nanomaterials provides the possibility of adjusting the "lid" to a conformation suitable for the purpose.

[0004] In the prior art, there are methods for obtaining highly active and highly stable immobilized lipases through nanocarriers. For example, lipases are fixed on the surface of graphene, and the conformation of lipases is regulated by molecular interactions between lipases and graphene. However, since lipases are fixed on the surface of graphene, most of the structural domains of lipases are still exposed to the external environment, which makes the surface-immobilized lipases easily leached out, and the catalytic stability is not ideal, making it difficult to recycle. In the prior art, there are also methods for designing nanopores based on mesh chemistry, which encapsulate lipases in nanopores and mediate the captured lipases into an open-lid conformation through specific host-guest interactions. Although this nanopore strategy provides a new idea for activating lipases, it still faces many challenges: (1) Due to the size matching problem between nanopores and lipases, the geometry and chemical structure of nanopores must be carefully designed to accommodate the bulky lipases; (2) The uncertainty problem of lipase interface activation: usually, lipases open the lid through the interface activation with nanopores to obtain highly active lipases. However, this interfacial activation requires certain specific pore modifications, and this pore microenvironment limits the study of high-activity lipases; (3) The synthesis steps are complicated, and since the orientation of the lipase encapsulated in the nanopore is difficult to predict, a lot of repeated debugging is required. Summary of the invention

[0005] In order to solve the problem in the prior art of lacking a simpler method for preparing immobilized lipase with high catalytic activity and high stability, the present invention provides a method for preparing immobilized lipase and its application in improving the catalytic activity and / or stability of immobilized lipase.

[0006] The first object of the present invention is to provide a method for preparing immobilized lipase.

[0007] The second object of the present invention is to provide immobilized lipase prepared according to the preparation method.

[0008] The third object of the present invention is to provide use of the immobilized lipase as a catalyst.

[0009] The fourth object of the present invention is to provide application of the preparation method in improving the stability of lipase.

[0010] The fifth object of the present invention is to provide application of the preparation method in improving the catalytic activity of lipase.

[0011] The sixth object of the present invention is to provide the application of the preparation method in improving the catalytic activity and / or stability of immobilized lipase.

[0012] In order to achieve the above object, the present invention is implemented by the following scheme:

[0013] The present invention overcomes the limitation of complex pore design of previous nanopore methods, solves the problem of difficulty in confining large-size lipases, and avoids the uncertainty problem of lipase interface activation. By inducing lipase aggregation and self-activation into an open-capped conformation with an organic solvent, a highly active lipase is obtained in advance, and then nucleation is promoted on the surface of the lipase through an in-situ encapsulation method, and the open-capped conformation of the lipase is quickly locked in a two-dimensional metal organic framework (MOF). The long-range ordered pores of the two-dimensional MOF can build a bridge between the internal lipase aggregates and the external catalytic substrate, confining the lipase in the MOF pores, so that the locked lipase aggregation is highly accessible, and has significantly higher biocatalytic activity and stability than free lipase.

[0014] The present invention claims a method for preparing an immobilized lipase, comprising the following steps: subjecting a lipase solution, a zinc salt and an organic ligand to a coordination reaction under weakly alkaline conditions to obtain an immobilized lipase;

[0015] The lipase solution is a propanol solution containing lipase, wherein the mass volume ratio of the lipase to propanol is (5-20) mg:0.8 ml; the lipase, Zn 2+ The molar ratio of the organic ligand to the organic ligand is 3.375:(0.5-4):1.

[0016] The chemical formula of propanol is C HO, and there are two isomers: n-propanol and isopropanol. In a specific embodiment of the present invention, n-propanol and isopropanol can both induce lipase to aggregate and self-activate into an open-capped conformation. Specifically, the lipase solution is obtained by dissolving the lipase in a n-propanol solution or an isopropanol solution.

[0017] Preferably, in the propanol solution, the volume ratio of propanol to water is (0.025-4):1.

[0018] More preferably, in the propanol solution, the volume ratio of propanol to water is 4:1.

[0019] Preferably, the propanol is n-propanol.

[0020] Preferably, the mass volume ratio of the lipase to the propanol is 10 mg:0.8 ml.

[0021] Preferably, the lipase, Zn 2+ The molar ratio of the organic ligand to the organic ligand is 3.375:(0.5-4):1.

[0022] More preferably, the lipase, Zn 2+ The molar ratio of the organic ligand to the organic ligand is 3.375:2:1.

[0023] Zinc salt refers to salt compounds containing zinc, which provides the necessary Zn for the synthesis of MOF.2+ , including but not limited to zinc oxide, zinc acetate and zinc nitrate, can be used for the rapid encapsulation of the lipase in the open-capping conformation using a two-dimensional MOF framework through an in situ generation method of the present invention.

[0024] Preferably, the zinc salt is zinc nitrate.

[0025] Preferably, the weakly alkaline condition includes a pH of 10-11.

[0026] Preferably, the preparation method comprises the following steps: subjecting the lipase solution, the organic ligand solution and the weakly alkaline zinc salt solution to a coordination reaction to obtain immobilized lipase; the organic ligand solution is an ethanol solution containing the organic ligand; the zinc salt solution is an aqueous solution containing zinc salt.

[0027] More preferably, in the ethanol solution, the volume ratio of ethanol to water is (2-4):1.

[0028] Further preferably, in the ethanol solution, the volume ratio of ethanol to water is 3:1.

[0029] More preferably, the pH of the zinc salt solution is 10-11.

[0030] Preferably, the preparation method further comprises purifying the obtained immobilized lipase.

[0031] More preferably, the purification method comprises solid-liquid separation and washing.

[0032] Further preferably, the purification method comprises centrifugation, washing and drying.

[0033] More preferably, the washing method comprises the following steps: washing the precipitate obtained by centrifugation with water or ethanol.

[0034] More preferably, the drying method comprises vacuum drying.

[0035] Preferably, the lipase is Lipozyme TL 100L.

[0036] Specifically, the preparation method further comprises the following steps:

[0037] S1. Dissolve lipase in n-propanol solution (water: n-propanol = 1: (0.025-4), v / v) or isopropanol solution (water: isopropanol = 1: (0.025-4), v / v) to obtain a lipase solution at a final concentration of (5-20) mg / mL, dissolve HHTP in ethanol solution (water: ethanol = 1: (2-4), v / v) to obtain an organic ligand solution at a final concentration of 10 mM, adjust Zn with NaOH 2+The pH value of the aqueous zinc nitrate solution with a concentration of (0.004-0.032) mmol) is adjusted to 10-11 to obtain a zinc salt solution;

[0038] S2. The lipase solution, the organic ligand solution and the zinc salt solution obtained in step S1 are mixed and then allowed to stand for a long time;

[0039] S3. The reaction product obtained in step S2 is centrifuged to collect the precipitate, and then the precipitate is washed with water and ethanol in sequence, and then dried sufficiently to obtain the product.

[0040] The immobilized lipase prepared according to any of the preparation methods should also be within the protection scope of the present invention.

[0041] The use of the immobilized lipase as a catalyst should also be within the scope of protection of the present invention.

[0042] Preferably, the catalyst is used to catalyze a hydrolysis reaction and / or an esterification reaction.

[0043] More preferably, the substrate for the hydrolysis reaction comprises p-nitrophenylbutyrate.

[0044] More preferably, the substrates for the esterification reaction include oleic acid and fatty alcohol.

[0045] Preferably, the catalyst is a heterogeneous catalyst.

[0046] A catalyst comprises the immobilized lipase.

[0047] The application of any of the preparation methods to improve the stability of lipase should also be within the protection scope of the present invention.

[0048] The application of any of the preparation methods in improving the catalytic activity of lipase should also be within the protection scope of the present invention.

[0049] The application of any of the preparation methods to improve the catalytic activity and / or stability of immobilized lipase should also be within the protection scope of the present invention.

[0050] Preferably, the catalytic activity includes the activity of catalyzing hydrolysis reaction and / or esterification reaction.

[0051] More preferably, the substrate for the hydrolysis reaction comprises p-nitrophenylbutyrate.

[0052] More preferably, the substrates for the esterification reaction include oleic acid and fatty alcohol.

[0053] More preferably, the fatty alcohol has 5 to 18 carbon atoms.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The present invention realizes the one-step synthesis of lipase@MOF biocomposite materials through a direct aggregation conformation locking strategy. Compared with the traditional solvothermal method, spray layer-by-layer liquid phase epitaxy method and phase change method, this method is simpler to operate and can be crystallized under mild conditions to immobilize the lipase, with less impact on the lipase. It has a powerful biocatalytic activity and excellent stability that is significantly superior to natural free lipase, can be repeatedly recycled, and has important promotion and application value in the large-scale utilization of lipase biocatalytic reactions in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 These are the PXRD test results of Zn-HHTP synthesized in mild aqueous phase in Example 1; a is Zn-HHTP synthesized under different pH conditions; b is Zn-HHTP synthesized under different ligand to metal ratios.

[0057] Figure 2 This is a schematic diagram of the process for preparing immobilized lipase in Example 2.

[0058] Figure 3 These are the characterization and identification results of LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP in Example 4; a is the PXRD detection result of LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP; b and c are the SEM detection results of LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP, respectively.

[0059] Figure 4 Statistical results of enzyme encapsulation efficiency of LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP in Example 4; a is the standard curve of lipase solution; b is the statistical results of enzyme encapsulation efficiency.

[0060] Figure 5 This is the equation for the lipase-catalyzed hydrolysis of p-nitrophenylbutyrate to produce p-nitrophenol and butyric acid in Example 5.

[0061] Figure 6 are the evaluation results of the catalytic performance of the hydrolysis reaction of different catalysts in Example 5; a is the relative activity of each catalyst in terms of the initial catalytic rate at the same dosage; b is the fitting curve of the catalytic rate of p-nitrophenylbutyrate concentration dependence of each catalyst; c is the standard curve of p-nitrophenol hydrolysis products; d is the stability test result.

[0062] Figure 7The catalytic performance evaluation results of the esterification reaction of different catalysts in Example 5; a is the esterification reaction principle; b is the esterification reaction of oleic acid and n-decanol under the action of each catalyst at the same dosage over time 1 HNMR spectrum; c is the catalytic kinetics of the esterification reaction of each catalyst at the same dosage; d is the recyclability of LA-n-prop@Zn-HHTP for catalytic esterification conversion; e is the esterification conversion rate of lipase (Lipozyme TL 100L) and LA-n-prop@Zn-HHTP for n-pentanol and oleyl alcohol.

[0063] Figure 8 These are the characterization and identification results of LA-n-prop@Zn-HHTP after completing 5 cycles of esterification reaction in Example 5; a is the PXRD detection result; b is the SEM detection result. DETAILED DESCRIPTION

[0064] The present invention is further elaborated in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified. The lipase (Lipozyme TL 100L) involved in the following examples was purchased from Beijing Gao Ruisen Technology Co., Ltd. The lipase purified by dialysis was collected by freeze drying; p-nitrophenyl butyrate (p-NPB, AR), zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H 2 O, 99%), oleic acid and various chain length fatty alcohols were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China; 2,3,6,7,10,11-hexahydroxytriphenyl (HHTP) and deuterated chloroform (CDCl 3 ) was purchased from Beijing Bailingwei Technology Co., Ltd., China.

[0065] Example 1 A method for mild aqueous phase synthesis of Zn-HHTP

[0066] This embodiment provides a method for preparing a Zn-HHTP two-dimensional MOF material by mild aqueous phase synthesis. By adjusting the pH and the ratio between the ligand and the metal, a 2D Zn-HHTP material with high crystallinity is obtained. The specific steps are as follows:

[0067] (1) pH adjustment

[0068] HHTP was dissolved in ethanol solution (water: ethanol = 1:3, v / v) at a final concentration of 10 mM to prepare HHTP solution. Take a 2 mL glass vial and add 0.8 mL Zn(NO 3 )2 Aqueous solution (Zn 2+ 0 μL, 0.8 μL, 8 μL or 80 μL of NaOH was added to a flask with a concentration of 0.016 mmol), mixed evenly, and the pH was adjusted to 8, 9, 10 or 11 accordingly; then 0.8 mL of HHTP solution was added, and the mixture was allowed to stand at room temperature (25° C.) for 12 hours to obtain the product.

[0069] (2) The ratio between ligand and metal (HHTP:Zn 2+ )

[0070] HHTP was dissolved in ethanol solution (water: ethanol = 1:3, v / v) at a final concentration of 10 mM to prepare HHTP solution. Take a 2 mL glass vial and add 0.8 mL Zn(NO 3 ) 2 Aqueous solution (Zn 2+ The concentration is 0.008mmol, 0.016mmol or 0.0032mmol, that is, HHTP:Zn 2+ =10mM:5mM, 10mM:20mM or 10mM:40mM) was added with 8μL NaOH (0.008mmol), mixed evenly, and the pH was adjusted to 10; then 0.8mL HHTP solution was added, and the mixture was allowed to stand at room temperature (25°C) for 12 hours to obtain a product, which was recorded as Zn-HHTP.

[0071] (3) Test results

[0072] The Zn-HHTP synthesized at different pH values ​​was characterized and identified by powder X-ray diffraction (PXRD), and the PXRD card of the standard Zn-HHTP (Simulated Zn-HHTP) derived from the CCDC crystallographic database was used as a control. The results are shown in Figure 1 As shown in Figure a, at pH = 8 and pH = 9, the synthesized Zn-HHTP does not have XRD diffraction peaks and is an amorphous structure; at pH = 10 and pH = 11, it has diffraction peaks similar to those of Simulated Zn-HHTP and has a certain degree of crystallinity. To prevent the influence of high pH conditions on enzyme activity, pH = 10 was selected as the optimal pH condition for synthesizing Zn-HHTP.

[0073] The Zn-HHTP synthesized with different ligand to metal ratios was characterized by powder X-ray diffraction (PXRD). Figure 1 As shown in Figure b, after changing the ratio of ligand to metal ion, in the three ratios of 10mM:5mM, 10mM:20mM and 10mM:40mM, when HHTP:Zn 2+=10mM:20mM and 10mM:40mM Zn-HHTP synthesized has stronger XRD diffraction peaks and no other impurity peaks. In order to prevent the influence of excessive metal ion concentration on enzyme activity, the ratio of 10mM:20mM was selected as the optimal ligand to metal ratio for synthesizing Zn-HHTP.

[0074] Example 2 Preparation method of immobilized lipase LA-n-prop@Zn-HHTP

[0075] This embodiment provides a method for preparing immobilized lipase, the process is as follows Figure 2 As shown, it mainly includes the synthesis of lipase self-activated aggregates (LA), conformation locking and organic solvent removal. The specific steps are as follows:

[0076] (1) LA synthesis

[0077] Lipase (Lipozyme TL 100L) was dissolved in n-propanol solution (water: n-propanol=1:4, v / v) at a final concentration of 10 mg / mL to obtain a lipase solution.

[0078] (2) Conformational locking

[0079] The method for synthesizing Zn-HHTP in Example 1 was used to encapsulate lipase in situ in Zn-HHTP. The specific steps are as follows:

[0080] HHTP was dissolved in an ethanol solution (water:ethanol=1:3, v / v) at a final concentration of 10 mM to prepare an HHTP solution.

[0081] Take a 2 mL glass vial and add 0.8 mL Zn(NO 3 ) 2 Aqueous solution (Zn 2+ The concentration is 0.016mmol, that is, HHTP:Zn 2+ =10mM:20mM) was added with 8μL NaOH (0.008mmol), mixed evenly, and the pH was adjusted to 10; then 0.08mL lipase solution and 0.8mL HHTP solution were added, and the mixture was allowed to stand at room temperature (25°C) for 12 hours to obtain a reaction product.

[0082] (3) Removal of organic solvents

[0083] The reaction product was centrifuged at 9000 rpm for 5 min at 25 °C, and the precipitate was collected and washed twice with deionized water and once with ethanol to fully remove n-propanol. It was then vacuum dried overnight (12 h) to obtain immobilized lipase, which was recorded as LA-n-prop@Zn-HHTP.

[0084] Example 3 Preparation method of immobilized lipase LA-isoprop@Zn-HHTP

[0085] This example provides a method for preparing immobilized lipase, the process is similar to that of Example 2, and the specific steps are as follows:

[0086] (1) LA synthesis

[0087] Lipase (Lipozyme TL 100L) was dissolved in isopropanol (water:isopropanol=1:4, v / v) at a final concentration of 10 mg / mL to obtain a lipase solution.

[0088] (2) Conformational locking

[0089] The method for synthesizing Zn-HHTP in Example 1 was used to encapsulate lipase in situ in Zn-HHTP. The specific steps are as follows:

[0090] HHTP was dissolved in an ethanol solution (water:ethanol=1:3, v / v) at a final concentration of 10 mM to prepare an HHTP solution.

[0091] Take a 2 mL glass vial and add 0.8 mL Zn(NO 3 ) 2 Aqueous solution (Zn 2+ The concentration is 0.016mmol, that is, HHTP:Zn 2+ =10mM:20mM) was added with 8μL NaOH (0.008mmol), mixed evenly, and the pH was adjusted to 10; then 0.08mL lipase solution and 0.8mL HHTP solution were added, and the mixture was allowed to stand at room temperature (25°C) for 12 hours to obtain a reaction product.

[0092] (3) Removal of organic solvents

[0093] The reaction product was centrifuged at 9000 rpm for 5 min at 25 °C, and the precipitate was collected and washed twice with deionized water and once with ethanol to fully remove isopropanol. It was then vacuum dried overnight (12 h) to obtain immobilized lipase, which was recorded as LA-isoprop@Zn-HHTP.

[0094] Example 4 Characterization and Identification of Immobilized Lipase and Encapsulation Efficiency of Enzyme

[0095] 1. Characterization and identification

[0096] (1) Identification method

[0097] The LA-n-prop@Zn-HHTP prepared in Example 2 and the LA-isoprop@Zn-HHTP prepared in Example 3 were characterized and identified by powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM), using the PXRD card (Simulated Zn-HHTP) of the standard Zn-HHTP derived from the CCDC crystallographic database as a control.

[0098] (2) Identification results

[0099] like Figure 3 As shown in a, compared with Simulated Zn-HHTP, LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP both have strong diffraction peaks at 2θ=4.70°, indicating that LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP have high crystallinity. Figure 3 As shown in Figures b to c, LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP both exhibit rod-like morphology, and the lipase is in situ encapsulated in the ordered pores of the quasi-mesoporous two-dimensional MOF (about 1.90 nm wide). The above results indicate that the immobilized lipase was successfully synthesized.

[0100] 2. Enzyme encapsulation efficiency

[0101] (1) Measurement method

[0102] In the step "LA synthesis" of Example 2 and Example 3, the amount of lipase (Lipozyme TL 100L) was adjusted, that is, the final concentration of lipase (Lipozyme TL 100L) in the lipase solution was 5 mg / mL, 10 mg / mL or 20 mg / mL.

[0103] At the same time, in the step "conformation locking" of Example 2 and Example 3, solutions that were left standing at room temperature (25°C) for 0 hours and 12 hours were taken respectively; after centrifugation, a standard curve of the lipase solution was constructed using the Bradford protein method (the concentration gradient was: 0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL and 500 μg / mL), and the concentration of lipase in the supernatant obtained by centrifugation was detected respectively. The main steps included: adding 20 μL of sample to a 96-well plate, adding 200 μL of Coomassie Brilliant Blue G-250 reagent, incubating at room temperature (25°C) for 5 minutes, and finally detecting the absorbance at 595 nm using a microplate reader (BioTek cytting 3). The concentration of lipase is proportional to the absorbance at 595 nm.

[0104] The enzyme encapsulation efficiency of LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP was calculated based on the concentration difference of lipase, and the calculation formula was: Enzyme encapsulation efficiency (%) = Loaded enzyme ÷ Added enzyme × 100%; wherein, Loaded enzyme = the concentration of lipase in the supernatant after standing for 0 hours - the concentration of lipase in the supernatant after standing for 12 hours, Added enzyme = the concentration of lipase in the supernatant after standing for 0 hours.

[0105] (2) Measurement results

[0106] The standard curve of lipase solution is as follows Figure 4 As shown in a in the figure, the regression linear equation is y = 0.00135x + 0.54306, R 2 =0.998. The statistical results of enzyme encapsulation efficiency of LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP are shown in Figure 4 As shown in b, under the condition of different lipase (Lipozyme TL 100L) dosage, the immobilized lipase synthesized by the two preparation methods can efficiently encapsulate the lipase, and the enzyme encapsulation efficiency is as high as 100%.

[0107] Example 5 Performance evaluation of immobilized lipase

[0108] 1. Evaluation of catalytic activity of hydrolysis reaction

[0109] (1) Detection method

[0110] Lipase is an ester hydrolase that catalyzes Figure 5 The hydrolysis reaction shown catalyzes the hydrolysis of p-nitrophenylbutyrate (p-NPB, CAS No.: 2635-84-9) to produce p-nitrophenol and butyric acid.

[0111] The hydrolysis reaction was carried out at room temperature (25° C.) using lipase (Lipozyme TL 100L), LA-n-prop@Zn-HHTP prepared in Example 2 or LA-isoprop@Zn-HHTP prepared in Example 3 as catalysts (working concentration of 5 μg / mL) and p-nitrophenylbutyrate as substrate (initial concentration of 1 mM).

[0112] The maximum absorbance wavelength of p-nitrophenol is 405 nm. The output of p-nitrophenol is detected by UV-visible spectrophotometer, and the catalytic rate is calculated.

[0113] The Michaelis-Menten model was used to construct a fitting curve of the catalytic rate and substrate concentration.

[0114] A standard curve of p-nitrophenol hydrolysis products was constructed by UV-visible spectrophotometer.

[0115] Each catalyst was stored in aqueous solution for 1 month, and the hydrolysis activity was detected by UV-visible spectrophotometer.

[0116] The catalytic kinetic parameters (maximum catalytic rate (V m ), Michaelis constant (K m ), peak enzyme reaction rate (k cat ).

[0117] (2) Test results

[0118] like Figure 6 As shown in a, the catalytic rates of LA-n-prop@Zn-HHTP prepared in Example 2 and LA-isoprop@Zn-HHTP prepared in Example 3 are significantly higher than that of the free lipase (Lipozyme TL 100L), which are 4.7 times and 3.9 times of that of the free lipase (Lipozyme TL 100L), respectively.

[0119] The kinetic curves of the catalytic rate and substrate concentration of LA-n-prop@Zn-HHTP prepared in Example 2 and LA-isoprop@Zn-HHTP prepared in Example 3 were simulated by Miichaelis-Menten, and the following results were obtained: Figure 6 The fitting curves shown in b and the catalytic kinetic parameters shown in Table 1 show that, compared with the free lipase (Lipozyme TL 100L), the V max Both were significantly improved, and K m Both decreased, K m The lower the value, the stronger the substrate binding ability, which further indicates that the lipase locked inside the Zn-HHTP still aggregates in an open-lid conformation; in addition, through k cat / K m The results of catalytic efficiency estimation showed that the catalytic efficiency of LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP was significantly higher than that of free lipase (Lipozyme TL 100L), which were 5.30 times and 3.63 times that of free lipase (Lipozyme TL 100L), respectively. Among them, LA-n-prop@Zn-HHTP had the highest catalytic efficiency.

[0120] Table 1 Summary of catalytic kinetic parameters

[0121]

[0122] like Figure 6 As shown in c, under the same test conditions, the catalytic hydrolysis of p-nitrophenyl butyrate (p-NPB, CAS No.: 2635-84-9) to generate p-nitrophenol showed color at 405nm and conformed to the linear relationship. The regression linear equation was y=0.01538x+0.0188, R 2 =0.996, which can be used to quantify the hydrolysis rate of p-nitrophenylbutyrate.

[0123] like Figure 6 As shown in (d), after one month of storage, the catalytic activity of the hydrolysis reaction of the free lipase (Lipozyme TL 100L) dropped to 72%, while LA-n-prop@Zn-HHTP and LA-isoprop@Zn-HHTP still maintained efficient catalytic activity of the hydrolysis reaction, which were 219% and 255% of the free lipase (Lipozyme TL 100L), respectively.

[0124] The above results show that the immobilized lipases prepared in Examples 2 and 3 of the present invention can efficiently catalyze the hydrolysis reaction, among which LA-n-prop@Zn-HHTP prepared in Example 2 has a higher catalytic hydrolysis activity, which is expected to play the highest catalytic role in the esterification reaction of lipase.

[0125] 2. Evaluation of catalytic activity of esterification reaction

[0126] (1) Detection method

[0127] Enzymatic esterification is another important method for large-scale synthesis of esters in the chemical industry.

[0128] Lipase (Lipozyme TL 100L) and LA-n-prop@Zn-HHTP prepared in Example 2 were used as catalysts (working concentration was 0.1% w / v), and oleic acid and fatty alcohols of different chain lengths (short-chain n-pentanol (n=5), n-decanol (n=10) or long-chain oleyl alcohol (n=18, n represents the number of carbon atoms in the fatty chain) were used as substrates (initial concentration was 0.2 mM). Figure 7 The esterification reaction shown in a in the figure has the following specific steps:

[0129] In a flask containing a catalyst, 100 μL of oleic acid and an equal amount of fatty alcohol were added to 1.6 mL of n-hexane, and the resulting suspension was placed at 37°C and stirred at 200 rpm for reaction; 100 μL of the reaction solution was taken at different reaction time points (2h, 5h, 8h, 10h and 24h), centrifuged, and 20 μL of the supernatant was mixed with 500 μL of deuterated chloroform and analyzed by nuclear magnetic resonance spectroscopy ( 1H NMR) was used to detect the conversion amount of oleic acid, and the esterification conversion rate was calculated as follows: esterification conversion rate (%) = integrated area of ​​product oleic acid ester at 2.31 ppm / integrated area of ​​reactant oleic acid at 2.37 ppm × 100%. The esterification process was repeated three times, and the esterification conversion rate data were expressed as an average value.

[0130] After the esterification reaction was completed, LA-n-prop@Zn-HHTP was recovered by centrifugation, washed with n-hexane three times, and then redispersed in n-hexane. The esterification reaction was repeated 5 times under the same conditions, that is, 5 cycles of esterification were completed, and lipase (Lipozyme TL 100L) was used as a control; then the conversion amount of oleic acid and the esterification conversion rate were detected according to the above method. In addition, the LA-n-prop@Zn-HHTP recovered after completing 5 cycles of esterification reaction was characterized and identified by powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM), and the PXRD card (Simulated Zn-HHTP) of the standard Zn-HHTP derived from the CCDC crystallography database was used as a control.

[0131] (2) Test results

[0132] The esterification reaction of oleic acid and n-decanol (n=10) catalyzed by LA-n-prop@Zn-HHTP is shown in Figure 2. Figure 7 As shown in b and c, the triple peak at δ = 2.37 ppm is attributed to the protons near the carboxyl group of oleic acid, and the proton peak moves to the high field region at δ = 2.31 ppm, which is attributed to the formation of oleic acid decyl ester; by integrating the change of peak area over time, it can be seen that within 24 hours, the esterification conversion rate under the heterogeneous catalyst of LA-n-prop@Zn-HHTP is as high as 85.47%, while the esterification conversion rate under the catalysis of free lipase (Lipozyme TL 100L) is only 27.91%. The above results show that LA-n-prop@Zn-HHTP has efficient catalytic activity for esterification reaction.

[0133] After completing 5 cycles of esterification, the catalytic conversion rates of oleic acid catalyzed by lipase (Lipozyme TL 100L) and LA-n-prop@Zn-HHTP were as follows: Figure 7 As shown in d, the esterification conversion rate of LA-n-prop@Zn-HHTP remained at 98.44%, and its catalytic activity was significantly higher than that of lipase (Lipozyme TL 100L). The characterization and identification of LA-n-prop@Zn-HHTP after completing 5 cycles of esterification reaction is shown in Figure 8As shown in a and b, it can be seen that LA-n-prop@Zn-HHTP still has good XRD diffraction peaks and complete morphology after recycling. The above results show that LA-n-prop@Zn-HHTP has excellent stability and recyclability and is a heterogeneous biocatalyst that can be recycled.

[0134] In order to further identify the extensiveness of LA-n-prop@Zn-HHTP as a heterogeneous catalyst to promote esterification reactions, the fatty alcohol substrates were also expanded from n-decanol to short-chain n-pentanol (n=5) and long-chain oleyl alcohol (n=18), such as Figure 7 As shown in Figure e, under the action of the heterogeneous catalyst LA-n-prop@Zn-HHTP for 24 hours, the esterification conversion rates of n-pentanol and oleyl alcohol were both over 80%, while the conversion rates of n-pentanol and oleyl alcohol by free lipase (Lipozyme TL 100L) were only 27.91% and 24.1%, respectively.

[0135] The above results show that the immobilized lipase LA-n-prop@Zn-HHTP prepared in the present invention can efficiently catalyze the esterification reaction, has strong stability, can be recycled and reused multiple times, and catalyzes a wider range of substrates.

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing immobilized lipase, characterized in that: The following steps are involved: The lipase solution, zinc salt and organic ligand are subjected to coordination reaction under weak alkaline conditions to obtain immobilized lipase; The lipase solution is a propanol solution containing lipase, wherein the mass volume ratio of the lipase to propanol is (5-20) mg:0.8 ml; The lipase, Zn 2+ The molar ratio of the organic ligand to the organic ligand is 3.375:(0.5-4):

1.

2. The preparation method according to claim 1, characterized in that: The weakly alkaline condition includes a pH of 10-11.

3. The preparation method according to claim 1, characterized in that: The zinc salt is zinc nitrate.

4. The preparation method according to claim 1, characterized in that: The organic ligand is 2,3,6,7,10,11-hexahydroxytriphenyl.

5. The immobilized lipase obtained according to the preparation method according to any one of claims 1 to 4.

6. Use of the immobilized lipase according to claim 5 as a catalyst.

7. Use of the preparation method according to any one of claims 1 to 4 in improving the stability of lipase.

8. Use of the preparation method according to any one of claims 1 to 4 in improving the catalytic activity of lipase.

9. Use of the preparation method according to any one of claims 1 to 4 in improving the catalytic activity and / or stability of immobilized lipase.

10. The use according to claim 8 or 9, characterized in that: The catalytic activity includes catalytic activity of hydrolysis reaction and / or esterification reaction.

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