Immobilized non-specific peroxygenase and application thereof in hydroxy fatty acid synthesis
By using the immobilized enzyme carrier resin LX-1000HAPA and glutaraldehyde cross-linking method to prepare immobilized nonspecific peroxygenase DcaUPO-A161C, the problem of poor stability of the free enzyme was solved, and efficient hydroxy fatty acid synthesis was achieved, which has potential for industrial application.
Patent Information
- Application Number
- CN202510642835.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-09-09
AI Technical Summary
Free nonspecific peroxygenase has poor stability and cannot meet the needs of efficient synthesis of hydroxy fatty acids on an industrial scale. Existing immobilization technology also has problems such as decreased enzyme activity and high cost.
Commercial immobilized enzyme carrier resins such as LX-1000HAPA were used to prepare immobilized nonspecific peroxygenase DcaUPO-A161C by glutaraldehyde cross-linking. The key factors of the immobilization process were optimized to improve the stability and activity of the enzyme.
The immobilized enzyme has achieved efficient catalysis of hydroxy fatty acid synthesis, improved the stability and reusability of the enzyme, and has important industrial application potential.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of enzyme engineering, in particular to an immobilized non-specific peroxygenase and application thereof in the synthesis of hydroxy fatty acids. Background Art
[0002] Hydroxy fatty acids are fatty acids containing one or more hydroxyl groups in their chemical structure. They are widely distributed in animals, plants, and microorganisms and are precursors in the synthesis of flavors and fragrances, cosmetic bases, and surfactants. Hydroxy fatty acids also have anti-diabetic, anti-inflammatory, and anti-cancer activities, so this type of substance has also attracted much attention in the study of the regulatory mechanisms of metabolic diseases. Compared with traditional chemical synthesis methods, the bioenzymatic synthesis of hydroxy fatty acids exhibits advantages such as mild reaction conditions, high substrate selectivity, and environmental friendliness. Among them, nonspecific peroxygenase (UPO), as a highly glycosylated sulfheme enzyme, catalyzes reactions including the hydroxylation of unactivated C-H bonds in n-alkanes, which can catalyze the synthesis of hydroxy fatty acids from fatty acids. Moreover, UPO only requires H2O2 to participate in the catalytic process, without the need for a complex electron transport chain. In contrast, other types of enzymes reported (currently mainly concentrated in hydratases and P450 monooxygenases), such as hydratases, have problems such as a single catalytic product, can only hydroxylate the double bonds of unsaturated fatty acids, and have a narrow catalytic substrate range; another type of enzyme, such as P450 monooxygenase, requires the addition of chemical doses of the coenzyme factor NAD(P)H during the reaction process, or needs to be used in conjunction with a cofactor regeneration system, resulting in complex reaction routes and high costs. Therefore, both hydratases and P450 monooxygenases are difficult to meet the huge industrial demand for hydroxy fatty acid products. In summary, UPO is currently one of the most promising oxidases for the synthesis of hydroxy fatty acids.
[0003] Nonspecific peroxygenases are highly tolerant to H₂O₂ and can catalyze the oxidative hydroxylation of fatty acids of varying chain lengths. However, free nonspecific peroxygenases are unstable, cannot be reused multiple times, and are unable to meet industrial-scale requirements, limiting their application in the efficient synthesis of hydroxylated fatty acids. Current research suggests that immobilization technology may be the optimal solution for improving enzyme performance and economic efficiency. Immobilization utilizes techniques such as physical adsorption and chemical cross-linking to combine the free enzyme with a carrier, confining the enzyme to a specific space and significantly improving its stability. Furthermore, immobilization can maintain enzyme activity and enable its recycling. Summary of the Invention
[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an immobilized nonspecific peroxygenase.
[0005] Another object of the present invention is to provide the use of the immobilized nonspecific peroxygenase in the synthesis of hydroxy fatty acids.
[0006] The purpose of the present invention is achieved through the following technical solutions:
[0007] A method for preparing immobilized nonspecific peroxygenase comprises the following steps:
[0008] After the resin is washed, a nonspecific peroxygenase solution is added to carry out an immobilization reaction. After the reaction is completed, the resin is washed to obtain immobilized nonspecific peroxygenase.
[0009] The cleaning is performed using sodium phosphate buffer for 1 to 5 times.
[0010] The resin is a commercial immobilized enzyme carrier resin; preferably at least one of LX-1000EA, LX-1000HAPA, LX-1000HA, LKZ218, ECR8404M, ECR8215M, ECR8285M, ECR8525, ECR8806M, PCG900C, and PT0150; more preferably LX-1000HAPA.
[0011] When the connecting group of the resin is an amino group, the preparation method of the immobilized nonspecific peroxygenase comprises the following steps:
[0012] After cleaning the resin, the resin is activated with glutaraldehyde solution, rinsed after activation, and then added with nonspecific peroxygenase solution to carry out immobilization reaction. After the reaction is completed, the resin is washed to obtain immobilized nonspecific peroxygenase.
[0013] The concentration of the glutaraldehyde solution is 0.3-0.8%, preferably 0.3%.
[0014] The activation conditions are as follows: 20-30° C. and 200-400 rpm for 30-90 minutes.
[0015] The rinsing is performed using sodium phosphate buffer for 1 to 5 times.
[0016] The non-specific peroxygenase is an enzyme that can catalyze the reaction of fatty acids to produce hydroxy fatty acids; preferably, it is DcaUPO-A161C enzyme, and its amino acid sequence is shown in SEQ ID NO.1.
[0017] The concentration of the nonspecific peroxygenase solution is 1-2 mg / mL, and the specific enzyme activity is 250-350 U / mg.
[0018] The immobilization reaction conditions are 20-30° C., 200-400 rpm, reaction for 0.5-12 h; preferably 25° C., 300 rpm, reaction for 3 h.
[0019] The washing is performed using sodium phosphate buffer for 1 to 5 times.
[0020] An immobilized nonspecific peroxygenase is prepared by the above preparation method.
[0021] The application of the above-mentioned immobilized non-specific peroxygenase in catalyzing fatty acid reaction to produce hydroxy fatty acids.
[0022] The present invention has the following advantages and effects compared to the prior art:
[0023] The present invention utilizes heterologous expression in Escherichia coli to obtain pure enzyme DcaUPO-A161C. First, by screening 11 resin supports and optimizing key immobilization factors (cross-linker concentration, buffer pH, and immobilization time), the optimal immobilized enzyme, DcaUPO-A161C@HAPA, was obtained. Second, the enzymatic properties of DcaUPO-A161C@HAPA were investigated to determine optimal parameters for the hydroxy fatty acid synthesis process. The DcaUPO-A161C@HAPA produced by this invention exhibits significantly improved performance across all aspects, enabling efficient synthesis of hydroxy fatty acids and possessing significant potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is an SDS-PAGE image of the protein in Example 1 of the present invention; wherein, M represents marker, and lane 1 represents pure DcaUPO-A161C enzyme.
[0025] Figure 2 This is the protein quantitative curve of the supernatant collected during the filtration and washing process of the immobilized enzyme in Example 3 of the present invention.
[0026] Figure 3 This is the optimization result of the cross-linking agent concentration in Example 4 of the present invention.
[0027] Figure 4 This is the optimization result of the buffer pH in Example 4 of the present invention.
[0028] Figure 5 This is the optimization result of the immobilization time in Example 4 of the present invention.
[0029] Figure 6 Schematic diagram of the catalytic oxidation effect of C6-C18 fatty acids by DcaUPO-A161C (a) and DcaUPO-A161C@HAPA (b) in Example 5 of the present invention.
[0030] Figure 7 Optimum temperature response curves of DcaUPO-A161C and DcaUPO-A161C@HAPA in Example 6 of the present invention.
[0031] Figure 8The temperature tolerance of DcaUPO-A161C (a) and DcaUPO-A161C@HAPA (b) in Example 6 of the present invention.
[0032] Figure 9 The effect of pH on the enzyme activity of DcaUPO-A161C and DcaUPO-A161C@HAPA in Example 6. (a) Optimal reaction pH curves of the free enzyme and the immobilized enzyme; (b) pH tolerance of the free enzyme and the immobilized enzyme.
[0033] Figure 10 This is the effect of H2O2 on the enzyme activity of DcaUPO-A161C and DcaUPO-A161C@HAPA in Example 6 of the present invention.
[0034] Figure 11 Figure 6 shows the effects of organic solvents on the enzyme activity of DcaUPO-A161C and DcaUPO-A161C@HAPA. (a) Effect of organic solvents on the activity of free and immobilized enzymes; (b) Optimal DMSO concentration curves for free and immobilized enzymes.
[0035] Figure 12 This is the effect of metal ions and EDTA on the enzyme activity of DcaUPO-A161C and DcaUPO-A161C@HAPA in Example 6 of the present invention.
[0036] Figure 13 The results of the hydroxy fatty acid synthesis using nonanoic acid, lauric acid, and pentadecanoic acid as substrates in Example 7 of the present invention are shown. (a) Time-conversion curves of the three types of substrates catalyzed by the immobilized enzyme. (b) Continuous synthesis of ω-1 hydroxy fatty acids by the immobilized enzyme.
[0037] Figure 14 This is a comparison of the total concentration of ω-1 hydroxy fatty acid products synthesized by free enzyme and immobilized enzyme in Example 7 of the present invention. DETAILED DESCRIPTION
[0038] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.
[0039] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.
[0040] Materials and reagents: The glycerol-preserved strain pET28a-DcaUPO-A161C / BL21(DE3) was a strain preserved by our research group; the empty plasmid pET28a was purchased from a commercial company; Escherichia coli BL21(DE3) competent cells were from Weidi Biotechnology Co., Ltd.; the plasmid extraction kit was from Sangon Biotech (Shanghai) Co., Ltd.; all chemicals were purchased from Sigma-Aldrich, MacLean, or Aladdin Reagent Company and were of the highest purity, requiring no further purification.
[0041] Example 1 Expression, purification and enzyme activity detection of target protein
[0042] The target protein DcaUPO-A161C was expressed and purified according to the method in the literature "Li Tiantian. Characterization of enzymatic properties and molecular modification of nonspecific peroxygenase UPO[D]. South China University of Technology, 2023." and SDS-PAGE detection ( Figure 1 ) showed that the protein DcaUPO-A161C was expressed and enriched. The desalted protein was then concentrated and assayed for enzyme activity using the NBD (3,4-methylenedioxynitrobenzene) method. Finally, the protein was aliquoted and stored frozen at -80°C.
[0043] The expression and purification of the target protein and the results of enzyme activity detection are shown in Table 1
[0044] Table 1 Purification conditions of DcaUPO-A161C
[0045]
[0046]
[0047] Example 2 Detection of fatty acids and hydroxy fatty acids and enzyme activity test using the fatty acid method
[0048] 2.1 Detection methods of fatty acids and hydroxy fatty acids
[0049] A Shimadzu TQ 8050 gas chromatograph-mass spectrometer (GC-MS) was used, equipped with an SH-I-5Sil MS capillary column (30 m × 0.25 mm × 0.25 μm). The key operating conditions are as follows:
[0050] (1) Carrier gas conditions: High-purity helium (purity ≥ 99.999%), constant flow mode, flow rate 1.69 mL / min;
[0051] (2) Injection parameters: Injection volume 1 μL, split injection (split ratio 50:1), injection port temperature 280°C;
[0052] (3) Ionization and transmission: Electron impact ion source (EI, 70 eV), ion source temperature 230°C, transmission line interface temperature 300°C, solvent delay time 5 min;
[0053] (4) Chromatographic procedure: The initial column temperature was maintained at 50°C for 2 min, then increased to 250°C at a rate of 20°C / min and maintained for 14 min. For C18 unsaturated fatty acids, the final temperature of 250°C was extended to 18 min.
[0054] For qualitative analysis, the target product was preliminarily identified based on the characteristic fragment ions of the standard, and the molecular structure was confirmed by matching the Wiley11th and NIST 20 mass spectral libraries (similarity > 85%). For quantitative analysis, the external standard method was used to calculate the target concentration based on the total ion current chromatogram (TIC) peak area.
[0055] 2.2 Fatty acid enzyme activity detection method
[0056] (1) To facilitate the activity detection of the immobilized enzyme, the activity detection method was adjusted as follows:
[0057] The amount of UPO required to catalyze lauric acid to produce 1 μmol ω-1 hydroxylauric acid within 1 min at 25°C and 300 rpm shaking was defined as 1 enzyme activity unit, recorded as 1 U.
[0058] (2) Reaction system: 1 mM lauric acid solution (20 mM, DMSO solvent), 1 mM H2O2 solution, 5 μM free enzyme or 15 mg immobilized enzyme, and sodium phosphate buffer (100 mM, pH 7.0, made up to a final volume of 1 mL) were added to a 5 mL glass reaction bottle in sequence. The reaction was then kept in a constant temperature oscillation at 25°C and 300 rpm for 6 h. H2O2 was added in equal amounts at 2 h and 4 h, respectively. After the reaction was terminated, 1 mL of methyl tert-butyl ether (MTBE) was added, and the reaction was vortexed (3 min, 2000 rpm), centrifuged (12000 rpm, 3 min), the organic phase was collected, and dried over anhydrous Na2SO4. Finally, 100 μL of the derivatization reagent N, O-bis(trimethylsilyl)trifluoroacetamide and 50 μL of pyridine were added and reacted at 70°C for 1 h to generate trimethylsilane derivatives. The derivatized product was filtered through a 0.22 μm organic filter membrane and analyzed by GC-MS.
[0059] Example 3 Immobilization and screening of enzyme protein
[0060] 3.1 Preparation of immobilized enzyme using amino carrier
[0061] Weigh 300 mg of amino resins of different types, refer to Table 2 for specific types, add sodium phosphate buffer (50 mM, pH 8.0), and repeat washing three times to remove impurities. The washed resin is mixed with 5 mL of glutaraldehyde solution (0.3%, v / v), placed in a 25°C constant temperature oscillator (300 rpm) for reaction for 1 hour to complete the activation of the aldehyde group on the carrier surface. After the reaction is completed, the activated resin is rinsed three times with sodium phosphate buffer to completely remove unbound glutaraldehyde. Then, 5 mL of DcaUPO-A161C enzyme solution (0.6 mg / mL, 305 U / mg, obtained by diluting the enzyme solution purified in Example 1) is added to the resin and immobilized for 3 hours at 25°C and 300 rpm. After completion, 150 μL of the supernatant is taken and the concentration of unbound free protein is quantitatively analyzed by the Bradford method (see the standard curve). Figure 2 ), calculate the enzyme loading and immobilization rate. Finally, wash the immobilized enzyme three times to remove physically adsorbed enzyme protein and store it at 4°C for future use.
[0062] 3.2 Preparation of immobilized enzymes using other types of carriers
[0063] Weigh 300 mg of different types of non-amino resins (see Table 2 for specific types), add sodium phosphate buffer (50 mM, pH 8.0), and repeat washing three times to remove impurities. Add 5 mL of DcaUPO-A161C enzyme solution (0.6 mg / mL, 305 U / mg) to the washed resin and immobilize for 3 hours at 25°C and 300 rpm. After completion, take 150 μL of the supernatant and quantify the concentration of unbound free protein using the Bradford method (see the standard curve). Figure 2 ), calculate the enzyme loading and immobilization rate. Finally, wash the immobilized enzyme three times to remove physically adsorbed / weakly physically adsorbed enzyme protein, and seal it and store it at 4°C until it is ready for catalytic reaction.
[0064] 3.3 Immobilized carrier screening experiment
[0065] According to 3.1 and 3.2, 11 immobilized enzymes were prepared and used to screen the immobilized carriers. First, the immobilization rate was calculated, followed by the conversion rate. The reaction system required for the conversion rate calculation is as follows: 1mM tetradecanoic acid solution (20mM, DMSO solvent), 1.75mM H2O2 solution, 30mg immobilized enzyme and sodium phosphate buffer (100mM, pH 7.0, filled to a final volume of 1mL) were added to a 5mL glass reaction bottle in sequence. Then, the reaction was kept at 30℃ and 300rpm for 12h. At the 6th hour, an equal amount of H2O2 was added.
[0066] The calculation formulas for immobilization rate, enzyme loading capacity and conversion rate are as follows:
[0067]
[0068]
[0069] After the reaction, the product was detected by GC-MS. The specific steps are as shown in Example 2. For more accurate screening, the immobilized enzyme was reused for three rounds.
[0070] The results of vector screening are shown in Table 2. It can be seen that the best vector is LX-1000HAPA, which was used in subsequent experiments.
[0071] Table 2 Types of DcaUPO-A161C immobilization carriers and screening results
[0072]
[0073] *LX-1000HAPA was purchased from Xi’an Lanxiao Technology New Materials Co., Ltd.
[0074] Example 4 Optimization of key factors in the immobilization process
[0075] After screening and obtaining the optimal immobilization carrier LX-1000HAPA, the immobilization process was further optimized. The cross-linker concentration, buffer pH, and immobilization time in the preparation method of 3.1 in Example 3 were adjusted to obtain a more effective immobilized enzyme. The specific adjustments were as follows:
[0076] (1) Cross-linking agent concentration
[0077] Referring to the method described in Example 3 and Example 2, the effects of LX-1000 HAPA pretreated with different glutaraldehyde concentrations (0.3% to 0.8%, v / v) on the enzyme loading and activity after immobilization were measured, wherein the amount of immobilized enzyme added was changed to 30 mg.
[0078] (2) Buffer pH
[0079] The buffer added in 3.1 of Example 3 was replaced with citrate buffer (pH 5.0), sodium phosphate buffer (pH 6.0-8.0) and Tris-HCl (pH 9.0-10.0) in sequence, and the optimal pH for enzyme immobilization was measured. The amount of immobilized enzyme added was changed to 30 mg.
[0080] (3) Fixation time
[0081] The optimal immobilization time was selected based on the enzyme loading and activity after immobilization at different times (0.5-24 h), and the amount of immobilized enzyme added was changed to 30 mg.
[0082] The experimental results are as follows Figures 3 to 5As shown, it can be seen that the optimal concentration of the carrier pre-activated cross-linker is 0.6% (v / v), the optimal pH of the buffer is 8, and the optimal immobilization time at 25°C is 3 h. Based on the optimal parameters obtained by optimization, DcaUPO-A161C@HAPA was prepared for subsequent experiments.
[0083] Example 5 Catalytic Ability of Free Enzyme and Immobilized Enzyme on Fatty Acid Substrates
[0084] In order to test the catalytic ability of free enzyme and immobilized enzyme on different substrates, fatty acids of different lengths were used for verification. The substrates included 13 kinds of substrates, including hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, lauric acid, tetradecanoic acid, pentadecanoic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. The specific steps are as follows:
[0085] 5.1 Detection of free enzyme
[0086] To a 5 mL glass reaction vial, 1 mM fatty acid solution (20 mM, DMSO), 2 mM H₂O₂ solution, 5 μM free enzyme, and sodium phosphate buffer (100 mM, pH 7.0, made up to a final volume of 1 mL) were added sequentially. The reaction was then incubated at 30°C, 300 rpm, and shaken for 24 h. An equal amount of H₂O₂ was added at 12 h. Following completion of the reaction, the reaction was assayed according to the method in Example 2, and the conversion rate was calculated according to the method in Example 3.
[0087] 5.2 Detection of immobilized enzyme
[0088] To a 5 mL glass reaction vial, add 1 mM fatty acid solution (20 mM, DMSO), 1 mM H₂O₂ solution, 15 mg of immobilized enzyme, and sodium phosphate buffer (100 mM, pH 7.0, to a final volume of 1 mL) in this order. The reaction was then incubated at 30°C, 300 rpm, and shaken for 6 h. Equal amounts of H₂O₂ were added at 2 and 4 h, respectively. Following completion of the reaction, assays were performed according to the method in Example 2, and conversion was calculated according to the method in Example 3.
[0089] 5.3 Experimental Results
[0090] The experimental results are shown in Table 3 and Figure 6 As shown, the enzyme catalyzes the conversion rate of each substrate to ω-1 hydroxy fatty acids as shown in Table 3, and it can be seen that by-products such as keto acids are produced; the specific situation of catalyzing the oxidation of fatty acids to synthesize various hydroxy fatty acids is shown in Figure 6 As shown, it can be seen that the substrate selectivity and hydroxylation position selectivity of the free enzyme (a) and the immobilized enzyme (b) are basically consistent.
[0091] Table 3. Reaction products of C6-C18 fatty acids catalyzed by DcaUPO-A161C and DcaUPO-A161C@HAPA
[0092]
[0093] Example 6 Effect of different catalytic conditions on enzyme activity
[0094] 6.1 Effect of different temperatures
[0095] Referring to the method in 2.2 of Example 2, the enzyme activity at different temperatures (25-45° C.) was measured to evaluate the optimal temperature of the enzyme.
[0096] At the same time, the temperature stability of the enzyme was investigated by incubating the enzyme with buffer at a 1:1 ratio at different temperatures (25-55°C).
[0097] The experimental results are as follows Figure 7 As shown in the figure, the optimum temperature for both free and immobilized enzymes is 35°C, and both maintain >80% relative activity within the range of 30-35°C. Figure 8 ), the temperature tolerance of the immobilized enzyme was improved. For example, after incubation at 45°C for 3 h, the residual enzyme activity of the immobilized enzyme (43%) was twice that of the free enzyme (23%).
[0098] 6.2 Effect of different pH values
[0099] Similarly, referring to the method of 2.2 in Example 2, lauric acid was used as a substrate, and the optimal pH of the enzyme was measured in the following buffers, including citrate buffer (pH 5.0), sodium phosphate buffer (6.0-8.0) and Tris-HCl (pH 9.0-10.0).
[0100] In addition, the residual activity of the enzyme was measured after incubation with different pH buffers at 4 °C for 12 h to determine pH stability.
[0101] The experimental results are as follows Figure 9 As shown by Figure 9 As shown in Figure a, there is not much difference between the free enzyme and the immobilized enzyme in the optimal reaction pH curve. When the pH rises from 5.0 to 7.0, the relative enzyme activities of the two gradually increase, reaching 97% and 90% respectively at pH 6.0, and reaching a peak at pH 7.0. It is worth noting that at pH 8.0, the immobilized enzyme still maintains 79% relative activity, showing a slightly better alkaline buffering capacity than the free enzyme (75%). Figure 9 As shown in Figure b, the activity fluctuation of the immobilized enzyme in the pH range of 5.0-9.0 was less than 11%, especially at pH 9.0, it still maintained 104% activity, showing better pH tolerance.
[0102] 6.3 Tolerance to H2O2 Interference
[0103] The enzyme was incubated in different H2O2 concentrations (2 mM, 5 mM, 10 mM and 20 mM) for 1 hour to evaluate H2O2 tolerance. After incubation, the residual activity was measured according to the method in 2.2 of Example 2.
[0104] The experimental results are as follows Figure 10 As shown in the results, the H2O2 tolerance of the immobilized enzyme was improved. For example, after incubation at 20 mM for 1 h, the residual activity was 2.5 times that of the free enzyme.
[0105] 6.4 Tolerance to interference from organic solvents, metal ions, and EDTA
[0106] The enzyme was dissolved in 10% (v / v) organic solvent (methanol, ethanol, isopropanol, ethyl acetate, acetonitrile, acetone, DMSO), 10 mM metal ions (Na + 、Fe 2+ 、Fe 3+ 、Ni 2+ Mg 2+ , K + ) and 10 mM EDTA for 4 h, and after completion of the incubation, the residual activity was measured according to the method in 2.2 of Example 2. In addition, the optimal DMSO concentration (1%-15%, v / v) for enzyme catalysis was also studied.
[0107] The experimental results are as follows Figure 11 and Figure 12 As shown, Figure 11 a shows that the immobilized enzyme exhibits significantly enhanced solvent stability compared to the free enzyme. For the free enzyme, DMSO has the weakest inhibitory effect (residual enzyme activity 93.0%), while methanol, ethanol and isopropanol cause the residual enzyme activity to drop to 55.6%, 44.7% and 45.2%, respectively. In comparison, the immobilized enzyme showed excellent tolerance in seven solvents (residual enzyme activity was >70%), among which the residual enzyme activity after treatment with isopropanol, ethyl acetate and DMSO was 84.0%, 86.5% and 85.4%, respectively.
[0108] Figure 11 Figure b shows that both enzymes maintained their maximum activity at 5% DMSO. However, at 15% DMSO, the relative activity of the free enzyme decreased to 55.1%, while the immobilized enzyme maintained 84.3%, showing a more stable performance.
[0109] Figure 12 It shows that all kinds of metal ions can damage free enzymes, but after the immobilized enzymes are incubated with metal ions and EDTA, the enzyme activity is improved to varying degrees, such as Fe 3+ and EDTA increased it by 46% and 36% respectively. This also shows that the Fe 3+ With Mg2+ They form a tight and stable interaction with the enzyme cofactor of UPO, so it is difficult for the chelating agent EDTA to remove them through chelation.
[0110] Example 7 Continuous catalytic performance of enzymes determined by cyclic catalysis
[0111] The optimized temperature and pH value obtained in Example 6 were used as the catalytic reaction conditions of the immobilized enzyme DcaUPO-A161C@HAPA, and the substrates were replaced with nonanoic acid / lauric acid / pentadecanoic acid. The immobilized enzyme was used to repeatedly catalyze each substrate, and the cycle was set to 5 rounds, with each round lasting 6 hours ( Figure 13 (a), at which point the reactions of the three substrates have reached adsorption-reaction equilibrium. After each round of catalytic reaction, the reaction products were extracted and separated using methyl tert-butyl ether (MTBE). After the extraction was complete and all liquid was removed from the reaction system, the next round of catalytic reaction could be initiated. The conversion rate of the extracted products was calculated using the method described in Example 3.
[0112] At the same time, in order to intuitively compare the catalytic effects of the immobilized enzyme and the free enzyme, free enzyme catalytic experiments were carried out on these three substrates under the same reaction conditions as the immobilized enzyme, and the amount of free enzyme added was controlled to 5 μM.
[0113] The experimental results are as follows Figure 13 As shown, Figure 13 Figure a shows that lauric acid (C12) is the most suitable substrate, and the conversion rate of its product ω-1 hydroxylauric acid reaches 66.9% within 6 hours, while the conversion rates of ω-1 hydroxylation products of nonanoic acid (C9) and pentadecanoic acid (C15) are only 4.7% and 10.3%, respectively. Figure 13 b) shows that the catalytic efficiency of the immobilized enzyme exhibited a gradual decline over five consecutive reaction cycles (cumulatively 30 hours). With nonanoic acid and pentadecanoic acid as substrates, the enzyme was completely inactivated after the third and fourth cycles, respectively. However, when catalyzing the oxidation of lauric acid, the immobilized enzyme retained 12.4% residual activity in the fifth cycle.
[0114] Comparison of free enzyme and immobilized system shows (Table 4, Figure 14 ), although the catalytic efficiency of DcaUPO-A161C was slightly higher than that of DcaUPO-A161C@HAPA, after 5 cycles, the total production of ω-1 hydroxynonanoic acid, ω-1 hydroxylauric acid and ω-1 hydroxypentadecanoic acid reached 20.0 mg / L, 298.1 mg / L and 85.0 mg / L, respectively, which were 3.2 times, 2.3 times and 1.7 times that of the free enzyme, respectively. This confirms that immobilization can significantly increase the total amount of product accumulation by reducing enzyme loss and realizing continuous operation.
[0115] Table 4 Comparison of conversion rates of hydroxy fatty acids synthesized by DcaUPO-A161C and DcaUPO-A161C@HAPA
[0116]
[0117]
[0118] In summary, the various properties of DcaUPO-A161C@HAPA have been improved to varying degrees, and it exhibits superior properties to free enzymes in the synthesis of hydroxyl fatty acids, thus having the potential for industrial application.
[0119] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A method for preparing immobilized nonspecific peroxygenase, characterized in that The steps include: After the resin is washed, a nonspecific peroxygenase solution is added to carry out an immobilization reaction. After the reaction is completed, the resin is washed to obtain immobilized nonspecific peroxygenase.
2. The method for preparing immobilized nonspecific peroxygenase according to claim 1, wherein: The cleaning is performed using sodium phosphate buffer for 1 to 5 times.
3. The method for preparing immobilized nonspecific peroxygenase according to claim 1, wherein: The resin is at least one of LX-1000EA, LX-1000HAPA, LX-1000HA, LKZ218, ECR8404M, ECR8215M, ECR8285M, ECR8525, ECR8806M, PCG900C, and PT0150.
4. The method for preparing immobilized nonspecific peroxygenase according to claim 1, wherein: When the connecting group of the resin is an amino group, the preparation method of the immobilized nonspecific peroxygenase comprises the following steps: After cleaning the resin, the resin is activated with glutaraldehyde solution, rinsed after activation, and then added with nonspecific peroxygenase solution to carry out immobilization reaction. After the reaction is completed, the resin is washed to obtain immobilized nonspecific peroxygenase.
5. The method for preparing immobilized nonspecific peroxygenase according to claim 4, wherein: The concentration of the glutaraldehyde solution is 0.3-0.8%; The activation conditions are as follows: 20-30°C, 200-400 rpm, 30-90 min; The rinsing is performed using sodium phosphate buffer for 1 to 5 times.
6. The method for preparing immobilized nonspecific peroxygenase according to claim 1, wherein: The non-specific peroxygenase is DcaUPO-A161C enzyme, and its amino acid sequence is shown in SEQ ID NO.
1.
7. The method for preparing immobilized nonspecific peroxygenase according to claim 1, wherein: The concentration of the nonspecific peroxygenase solution is 1-2 mg / mL, and the specific enzyme activity is 250-350 U / mg.
8. The method for preparing immobilized nonspecific peroxygenase according to claim 1, wherein: The immobilization reaction conditions are 20-30°C, 200-400 rpm, and 0.5-12h; The washing is performed using sodium phosphate buffer for 1 to 5 times.
9. An immobilized nonspecific peroxygenase prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the immobilized nonspecific peroxygenase according to claim 9 in catalyzing fatty acid reactions to produce hydroxy fatty acids.