Preparation of a recombinant cholesterol esterase immobilized enzyme preparation and use thereof

Abstract

This invention discloses the preparation and application of a recombinant cholesterol esterase immobilized enzyme preparation. This invention will... Ophiostoma piceae Cholesterol esterase mutant gene ope-D136P Linked to pPICZαA plasmid, via signal peptide replacement and rDNA Gene insertion yielded the pEESB-3 plasmid; the protein disulfide isomerase gene was inserted. pdi The pPIC9K plasmid was ligated to obtain the pEESB-4 plasmid. This plasmid was electroporated into Pichia pastoris GS115, and after resistance selection and methanol-induced expression selection, recombinant Pichia pastoris engineered strains were obtained. Recombinant cholesterol esterase was obtained from this strain after methanol-induced expression. An aminated immobilization vector was prepared using APTES and TEOS as silane precursors. After grafting with 2,4-dihydroxyacetophenone and nickel ion chelation, the recombinant cholesterol esterase was immobilized to obtain an immobilized enzyme preparation. This immobilized enzyme preparation can catalyze the synthesis of stigmasterol ferulic acid ester from stigmasterol in a non-aqueous phase system, with a conversion rate of 33% (based on stigmasterol) or 32% (based on ferulic acid).

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology, green chemical engineering, and biomanufacturing technology, and particularly relates to the preparation and application of a recombinant cholesterol esterase immobilized enzyme preparation. Background Technology

[0002] Highly efficient heterologous expression of Ophiostoma piceae cholesterol esterase (OPE) is of positive significance for improving its large-scale production and industrial application. Early on, Cedillo et al. (2012) used the pPIC9K expression vector with Pichia pastoris as the expression strain to achieve heterologous functional expression of the cholesterol esterase encoding gene (OPE), but the expression level remained low. By optimizing the culture medium (YEPS with methanol as an inducer), the fermentation level of recombinant Pichia pastoris for producing recombinant OPE was as follows: with p-nitrophenylbutyrate (pNPB) as the substrate, only 16 U / mL of esterase activity was detected in the fermentation broth. Cedillo et al. (2014) further optimized the expression host strain and fermentation medium, increasing the fermentation level in their 5L fermenter to 30 U / mL.

[0003] Molina-Gutiérrez et al. (2021) compared the effects of hydrophobic adsorption and covalent cross-linking with different supports on the immobilization of cholesterol esterase OPE. The results showed that two preparation methods—immobilizing OPE via hydrophobic interactions using a strongly hydrophobic octyl immobilization support and directly preparing OPE cross-linked enzyme aggregates using glutaraldehyde—exhibited better immobilization effects. In the further catalytic conversion of waste cooking oil into biodiesel, the OPE cross-linked enzyme aggregates performed best, with a fatty acid methyl ester yield of 92.3%, when the yield was used as the evaluation criterion.

[0004] Esterification of phytosterols can improve their lipid solubility (e.g., esterification with long-chain fatty acids) or endow them with new physiological functions (e.g., esterification with phenolic acids imparts antioxidant activity). Currently, biocatalysts used in the synthesis of phytosterol esters are mainly concentrated in a few types, such as Candida rugosalipase, Candida antarctica lipase B (CALB), and Burkholderia sp. lipase. The Martínez MJ research group in Spain (Molina-Gutiérrez et al., 2017) compared the catalytic effects of immobilized OPE, commercial enzyme preparation CALB, and commercial enzyme preparation CALA (Candidaantarctica lipase A) on the synthesis of β-sitosterol esters. When isooctane was used as a solvent, the catalytic effect of immobilized OPE was better than that of CALB and CALA; the conversion rate of β-sitosterol exceeded 90% within 3 hours. In a solvent-free system, using long-chain fatty acid methyl esters as acyl donors can further increase the concentration of β-sitosterol in the reaction system to 80 mmol / L, and β-sitosterol can be completely esterified within 2 hours.

[0005] Improving the expression level of recombinant proteins is of positive significance for reducing the production cost of industrial enzyme preparations and promoting their widespread application. When using Pichia pastoris as a high-efficiency expression host, the expression vector cassette contains upstream and downstream sequences of the AOX gene (methanol oxidase gene). Therefore, after the foreign gene is inserted into the multiple cloning site and introduced into Pichia pastoris, it can undergo homologous recombination with the methanol oxidase gene on the Pichia pastoris genome. Using this recombination technology, recombinant strains with 1-8 foreign genes can generally be obtained. Further increasing the copy number of the foreign gene is challenging. Considering the high copy number of rDNA genes in cells, the upstream and downstream sequences of the rDNA gene can be used as homologous arms to further increase the copy number of the foreign gene in Pichia pastoris. As the copy number of the foreign gene increases, the secretion capacity of the foreign protein may become the decisive factor limiting the high production of foreign protein in recombinant Pichia pastoris. Therefore, further screening and optimization of secretion signal peptides are of great significance for improving the secretion capacity of recombinant Pichia pastoris. Furthermore, when foreign proteins with disulfide bonds are heterologously expressed in Pichia pastoris, they require the assistance of chaperone proteins to fold correctly. Therefore, co-expressing the protein disulfide bond isomerase encoding gene together with the foreign protein encoding gene will help to further improve the level of foreign protein production by recombinant Pichia pastoris.

[0006] Currently, the immobilization of most industrial enzyme preparations requires extensive pretreatment of the fermented enzyme solution, including but not limited to bacterial separation, bacterial concentration or spray drying, and separation and purification of the target protein, which significantly increases the production cost of enzyme preparations. Developing and establishing an affinity chromatography-based immobilization strategy allows for the immobilization of recombinant target proteins simply by adding appropriate components to the fermentation broth and adjusting the pH, effectively simplifying the production process and reducing production costs.

[0007] The natural substrate of lipases is triglycerides, and the hydroxyl group pocket of their active site is compatible with glycerol. However, the hydroxyl group structure of phytosterols is much larger than that of glycerol molecules, thus the catalytic activity of lipases for sterols (esters) is generally low. Hakalin et al. (2018) and Molina-Gutiérrez et al. (2017) achieved good catalytic effects by using recombinant OPE to catalyze sterol esterification reactions. However, there are currently no reports on the use of this enzyme to catalyze phenolic acid sterol esters. On the other hand, in the enzymatic synthesis of phenolic acid sterol esters, the polarity of the two substrates is significantly different: sterols are weakly polar compounds, while phenolic acids are strongly polar compounds. Therefore, a suitable reaction medium is very important in the enzymatic synthesis of sterol esters. Considering the polarity difference between the two substrates, the catalytic effect of a single solvent is not good, and binary or multi-component mixed solvents are generally used as reaction media. He et al. (2024) synthesized phenolic acid sterol esters using toluene / ionic liquid as the reaction medium via chemical catalysis (ionic liquid can also be used as a catalyst), achieving a substrate concentration of 0.05 mol / L and a conversion rate of 99%. When using lipase to catalyze the reaction of sitosterol and vinyl caffeate, a binary solvent of n-hexane and methyl ethyl ketone (8:2, v / v) also yielded good catalytic results. Summary of the Invention

[0008] The purpose of this invention is to provide a preparation and application of a recombinant cholesterol esterase immobilized enzyme preparation.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for constructing a recombinant Pichia pastoris engineered strain expressing cholesterol esterase includes the following steps:

[0011] (1) The Ophiostoma piceae cholesterol esterase mutant gene ope-D136P was ligated between the EcoRI and NotRI restriction sites of the pPICZαA plasmid to obtain the pEESB-1 plasmid; the signal peptide coding sequence in the pEESB-1 plasmid was replaced to obtain the pEESB-2 plasmid; the Pichia pastoris rDNA gene was ligated to the BamHI restriction site of the pEESB-2 plasmid to obtain the pEESB-3 plasmid;

[0012] (2) The Pichia pastoris protein disulfide isomerase encoding gene pdi was ligated between the EcoRI and NotI restriction sites of pPIC9K plasmid to obtain pEESB-4 plasmid;

[0013] (3) Electroporate pEESB-3 plasmid into competent Pichia pastoris cells, and obtain recombinant Pichia pastoris engineered strains expressing cholesterol esterase by Zeocin resistance selection and methanol-induced expression selection; or, electroporate pEESB-3 plasmid into competent Pichia pastoris cells, and obtain first-generation recombinant strains by Zeocin resistance selection and methanol-induced expression selection, and then electroporate pEESB-4 plasmid into competent cells of the first-generation recombinant strains, and obtain recombinant Pichia pastoris engineered strains expressing cholesterol esterase by G418 resistance selection and methanol-induced expression selection.

[0014] Furthermore, the nucleotide sequence of the Ophiostoma piceae cholesterol esterase mutant gene ope-D136P is shown in SEQ ID NO.1; the signal peptide coding sequence is replaced by the sequence shown in SEQ ID NO.2 with the sequence shown in SEQ ID NO.3; the nucleotide sequence of the Pichia pastoris rDNA gene is shown in SEQ ID NO.4; and the nucleotide sequence of the pdi gene is shown in SEQ ID NO.5.

[0015] A recombinant Pichia pastoris engineered strain was constructed using the method described above.

[0016] A method for preparing recombinant cholesterol esterase involves inducing the above-mentioned recombinant Pichia pastoris engineered strain with methanol, collecting the fermentation supernatant, and obtaining a crude enzyme solution of recombinant cholesterol esterase.

[0017] A recombinant cholesterol esterase was prepared by the method described above.

[0018] A method for preparing a recombinant cholesterol esterase immobilized enzyme preparation includes the following steps:

[0019] (1) The silane precursor was dissolved in anhydrous ethanol, and PEG1000 solution and ammonia solution were added. After aging, washing and drying, an amino-modified immobilized support was obtained.

[0020] (2) The amino-modified immobilized support was reacted with 2,4-dihydroxyacetophenone solution in diethyl ether in the dark to obtain the immobilized support after grafting;

[0021] (3) The grafted immobilized carrier was reacted with nickel sulfate solution by shaking to obtain an immobilized carrier that chelates nickel ions;

[0022] (4) Immobilization: The recombinant cholesterol esterase described in claim 5 is contacted and adsorbed with the immobilization carrier that chelates nickel ions in a binding buffer to obtain a recombinant cholesterol esterase immobilized enzyme preparation;

[0023] Further, in step (1), the silane precursor comprises 3-aminopropyltriethoxysilane and tetraethyl silicate in a molar ratio of 1:3, and the molar ratio of the silane precursor to anhydrous ethanol is 1:8.17; in step (2), the amount of the amino-modified immobilization support is 0.02 g / mL, and the concentration of the 2,4-dihydroxyacetophenone solution is 242.4 mmol / L.

[0024] A recombinant cholesterol esterase immobilized enzyme preparation is prepared by the above-described preparation method.

[0025] Application of the above-mentioned recombinant cholesterol esterase immobilized enzyme preparation in the catalytic synthesis of stigmasterol ferulic acid ester;

[0026] Furthermore, using stigmasterol and ferulic acid as substrates, stigmasterol ferulic acid ester was synthesized by oscillating reaction in a non-aqueous reaction system composed of hexane and 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt under the catalysis of a recombinant cholesterol esterase immobilized enzyme preparation.

[0027] The significant advantages of this invention are:

[0028] (1) In this invention, the Ophiostoma piceae cholesterol esterase mutant gene ope-D136P was efficiently secreted and expressed in Pichia pastoris. The enzyme activity of the recombinant strain during shake-flask fermentation reached 31.15 U / mL (5.02 U / OD). 600 );

[0029] (2) The immobilized carrier prepared by the present invention can directly and specifically adsorb the target recombinant protein OPE-D136P from the concentrated fermentation broth, and can complete the immobilization without separation and purification steps, making the operation simple.

[0030] (3) The recombinant cholesterol esterase immobilized enzyme preparation prepared by the present invention can be used to catalyze the synthesis of stigmasterol ferulic acid ester with high conversion efficiency and good prospects for industrial application. Attached Figure Description

[0031] Figure 1 : Schematic diagram of the synthesis process of immobilized carrier.

[0032] Figure 2 Fourier transform infrared spectroscopy of the immobilized support. A: Amino-modified immobilized material; B: Grafted immobilized support.

[0033] Figure 3 SEM image of the recombinant cholesterol esterase immobilized enzyme preparation.

[0034] Figure 4 Qualitative analysis of the synthesis of stigmasterol ferulic acid ester catalyzed by recombinant cholesterol esterase immobilized enzyme preparation. Sample 1: stigmasterol standard; Sample 2: ferulic acid standard; Sample 3: hexane / tert-butanol extract; Sample 4: ion exchange chromatography. The corresponding Rf values ​​for each substance are: 0.65 (Sample 1); 0.30 (Sample 2); 0.29 / 0.65 / 0.98 (Sample 3, from bottom to top); and 0 (Sample 4).

[0035] Figure 5 HPLC analysis of the synthesis of stigmasterol ferulic acid ester catalyzed by recombinant cholesterol esterase immobilized enzyme preparation. Detailed Implementation

[0036] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0037] The YPD liquid culture medium formula involved in this invention is as follows: 10g yeast extract, 20g glucose, and 20g peptone are added to every 1L of distilled water.

[0038] The YPD solid culture medium formula involved in this invention is as follows: 10g yeast extract, 20g glucose, 20g peptone, and 20g agar are added to every 1L of distilled water.

[0039] The YEPS liquid culture medium of the present invention is formulated as follows: 20g sucrose, 20g peptone and 10g yeast extract are added to every 1L of distilled water.

[0040] Example 1:

[0041] 1. High-efficiency expression of the Ophiostoma picae cholesterol esterase mutant gene ope-D136P

[0042] 1.1 Construction of expression vector pPICZαA-ope-D136P

[0043] The nucleotide sequence (SEQ ID NO. 1) of the Ophiostoma picae cholesterol esterase mutant gene ope-D136P was ligated between the EcoRI and NotI restriction sites of the pPICZαA vector. The ligation product was transformed into E. coli DH5α competent cells, and plasmids were extracted from positive transformants. The recombinant plasmid, verified by double digestion with restriction endonucleases EcoRI and NotI and sequencing, was named pEESB-1.

[0044] 1.2 Replacement of the coding sequence of the signal peptide in the expression vector pPICZαA-ope-D136P

[0045] Using plasmid pEESB-1 as a template, whole-plasmid PCR amplification was performed using the first-round primer pairs Ost1-1R (5'-TCCAAGAGAACCAAACCTGCCTCATCGTTTCGAATAATTA-3') and Ost1-1F (5'-GGTTCTCTTGGAGTCTTCTGCTGCTCCAGTCAACACTACA-3'). The amplified product was digested with DpnI at 37℃ for 12 h to remove methylated template plasmid. The purified product was then transformed into E. coli DH5α competent cells. Positive transformants were picked, plasmids were extracted, and sequencing was performed to verify the correct insertion of the first-round primer sequences, yielding the intermediate plasmid. Using the intermediate plasmid as a template, a second round of whole-plasmid PCR amplification was performed using the second-round primer pair Ost1-2R (5'-ACATAGGAACAATCCCACAATCCAAGAGAACCAAACCT-3') / Ost1-2F (5'-TTGTTCCTATGTTTTTTCAACGTGTCTTCTGCTGCTCCAGT-3'). The aforementioned DpnI digestion, transformation, and plasmid extraction steps were repeated. Sequencing confirmed that both primer sequences were correctly inserted, and the signal peptide coding sequence in the resulting recombinant expression plasmid had been replaced by the target signal peptide coding sequence (SEQ ID NO.3) from the original plasmid's signal peptide coding sequence (SEQ ID NO.2). The final plasmid was named pEESB-2.

[0046] 1.3 Inserting the homologous recombination arm rDNA gene into the pEESB-2 plasmid

[0047] Using Pichia pastoris GS115 genomic DNA as a template, PCR amplification was performed using primer pairs rDNA-F (5'-CGCGGATCCTAGTTAGGTTACCGTTTTTCCTAATATT-3') and rDNA-R (5'-AAAGGATCCCTTCCACCAACAGTCAACCACC-3') to obtain the Pichia pastoris rDNA gene (SEQ ID NO. 4). The PCR amplification product was recovered and purified by agarose gel electrophoresis. The recovered PCR amplification product and pEESB-2 plasmid were digested with the restriction endonuclease BamHI. The digestion products were identified and purified by agarose gel electrophoresis, and then ligated using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells, and the plasmid was extracted from positive transformants. The recombinant plasmid that was verified by sequencing was named pEESB-3.

[0048] 1.4 Construction of co-expression plasmid pPIC9K-pdi

[0049] Genomic DNA was extracted from Pichia pastoris GS115 and used as a template. PCR amplification was performed using primer pairs pdi-F (5'-CCGGAATTCATGCAATTCAACTGGGATATTAAAACTGTGG-3') and pdi-R (5'-AAATATGCGGCCGCTTAAAGCTCGTCGTGAGCGTCTGCC-3') to obtain the Pichia pastoris protein disulfide isomerase encoding gene pdi (SEQ ID NO. 5). The PCR amplification product was recovered and purified by agarose gel electrophoresis. The recovered PCR amplification product and pPIC9K plasmid were double-digested with restriction endonucleases EcoRI and NotI, respectively. The digestion products were identified and purified by agarose gel electrophoresis, and then ligated using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells, and plasmids were extracted from positive transformants. The plasmid that was verified by sequencing was named pEESB-4.

[0050] 1.5 Construction and screening of recombinant Pichia pastoris strains that efficiently express the mutant gene ope-D136P

[0051] (1) Preparation of Pichia pastoris GS115 competent cells

[0052] Take the Pichia pastoris GS115 glycerol culture stored at -80℃, inoculate it into 3 mL of YPD liquid medium, and incubate at 28℃ and 120 rpm for 12 h; streak the culture on YPD solid medium plates to isolate single colonies, and incubate upside down at 30℃ for 2-3 days; pick a single colony and inoculate it into 3 mL of YPD liquid medium, and incubate overnight at 30℃ and 120 rpm; the next day, transfer it to 50 mL of YPD liquid medium at an inoculation rate of 1% (v / v), and incubate at 30℃ and 120 rpm until the bacterial growth rate reaches OD. 600 Reach an OD of 1.2-1.5 (approximately 4 hours); centrifuge the above bacterial culture at 4000 rpm for 5 minutes at 4°C, discard the supernatant, resuspend the bacterial precipitate in sterile ddH2O pre-cooled at 4°C, wash, and collect the bacterial cells by centrifugation under the same conditions, repeating the washing process twice; finally, resuspend the bacterial cells in 1 mol / L sorbitol solution pre-cooled at 4°C, and adjust the OD of the bacterial culture. 600 Reduce the temperature to 6.0~7.5 and store in separate containers.

[0053] (2) Construction of recombinant Pichia pastoris GS115 / pEESB-3 strain

[0054] The pEESB-3 plasmid was linearized by single-enzyme digestion with the restriction endonuclease ScaI. The digestion product was identified and purified by agarose gel electrophoresis. 60 μL of Pichia pastoris GS115 competent cells were added to 500–1000 ng of the linearized pEESB-3 plasmid. After thorough mixing, the mixture was incubated on ice for 5 min. The mixture was then transferred to a pre-chilled 0.1 cm electroporation cuvette. Electroporation was performed at 1500 V, 25 μF, and 200 Ω for 5 ms. Immediately after electroporation, 1 mL of pre-chilled 1 mol / L sorbitol solution (4 °C) was added to the cuvette. After mixing, the entire liquid was transferred to a 1.5 mL EP tube and incubated at 30 °C for 1–2 h. The cultured bacterial solution was concentrated to 1 / 3–1 / 2 of its original volume by centrifugation. 100–200 μL of the concentrated solution was spread onto a plate containing 100 μg / mL sorbitol. On Zeocin's YPD solid medium plates, incubate upside down at 28°C for 2-3 days until single colonies grow; pick a single colony and streak it onto a new YPD solid medium plate, numbering it 1#, 2#, 3#, etc. in sequence.

[0055] (3) Screening of recombinant Pichia pastoris GS115 / pEESB-3 strain

[0056] Initial screening: Different numbered transformants obtained in step (2) were selected and inoculated into 2 mL EP tubes containing 300 μL of YEPS liquid medium, and cultured at 28℃ for 24 h. Methanol was added to each tube to a final concentration of 0.5% (w / v) for induction expression. The tubes were then cultured at 28℃, with methanol added to a final concentration of 0.5% (w / v) every 24 h for a total of 96 h. After induction, 200 μL of fermentation broth was used to determine its OD. 600 The remaining fermentation broth was centrifuged at 8000 rpm for 2 min, and the supernatant was collected to determine the cholesterol esterase activity. The enzyme activity level per unit cell density of each strain (U / OD) was calculated. 600 ).

[0057] Take 10 μL of appropriately diluted fermentation supernatant and add it to a reaction system containing 180 μL of potassium phosphate buffer (pH 7.4, 20 mmol / L K₂HPO₄-KH₂PO₄) and 10 μL of substrate p-nitrophenol laurate (pNPL, 10 mmol / L). Record the change in absorbance at 410 nm over 5 min at 40 °C. Enzyme activity units (U) are defined as the amount of enzyme required to catalyze the release of 1 μmol of p-nitrophenol (pNP) per minute under the above conditions.

[0058] Secondary screening: Based on the initial screening results, the top 5 transformants with the highest enzyme activity per unit cell density were selected (from 0.25 U / OD). 600 Up to 0.48U / OD600 Secondary screening was performed. Single colonies were picked from the plates and inoculated into 3 mL of YPES liquid medium, incubated overnight at 28°C. The next day, 1% (v / v) inoculum was transferred to 250 mL Erlenmeyer flasks containing 50 mL of YPES liquid medium and incubated at 28°C for 24 h. Then, methanol was added to a final concentration of 1% (v / v) to induce expression. Methanol was added every 24 h to a final concentration of 1% (v / v), for a total induction culture of 96 h. The OD of the fermentation broth was measured using the same method as the initial screening. 600 And supernatant enzyme activity, calculate U / OD 600 Finally, a recombinant strain with the highest cholesterol esterase expression level was obtained through screening and named GS115 / pEESB-3. Its enzyme activity during shake-flask fermentation reached 3.2 U / mL (0.57 U / OD). 600 ).

[0059] (4) Construction and screening of recombinant Pichia pastoris strains GS115 / pEESB-3 / pEESB-4

[0060] The recombinant strain GS115 / pEESB-3, which had the highest ope-D136P expression level obtained in step (3), was selected, and its competent cells were prepared according to step (1). The pEESB-4 plasmid was linearized with ScaI and then electroporated into GS115 / pEESB-3 competent cells according to step (2). The transformed bacterial culture was plated on YPD plates containing G418 (0.5 mg / mL) for screening; the remaining transformation operations were the same as in step (2). The primary and secondary screening methods for transformants were performed according to step (3). Finally, a recombinant strain with the highest cholesterol esterase expression level was obtained and named GS115 / pEESB-3 / pEESB-4. Its enzyme activity during shake-flask fermentation after secondary screening was further increased to 31.15 U / mL (5.02 U / OD). 600 ).

[0061] 2. Construction of immobilization carrier and preparation of immobilized recombinant OPE-D136P enzyme preparation

[0062] 2.1 Construction of the immobilization carrier

[0063] In this patent, the immobilized carrier preparation process is as follows: Figure 1 As shown, the specific experimental procedure and characterization results are as follows:

[0064] 2.1.1 Synthesis of immobilized supports for surface-modified amino groups

[0065] In centrifuge tubes, a certain volume of anhydrous ethanol, 3 mmol of silane precursor (composed of APTES and TEOS in a molar ratio of 1:3), 56 μL of 10 mmol / L PEG 1000 solution were added sequentially, and finally 12 μL of 25% ammonia solution was added (Table 1). The molar ratio (R value) of anhydrous ethanol to silane precursor in the system was set to 8.17, 19.58, and 31, respectively. The detailed proportions of each reaction system are shown in Table 1. After the reaction system was prepared, the following aging procedure was followed: aging at 4℃ with the cap closed for 12 h; aging at room temperature with the cap open for 12 h; centrifuging at 8000 rpm for 5 min to collect the precipitate, and continuing aging at room temperature with the cap open for another 12 h. After aging, the precipitate was washed twice with 1 mL ddH2O and once with 1 mL anhydrous ethanol, and the precipitate was collected after each washing by centrifugation at 8000 rpm for 5 min. The resulting precipitate was dried in a fume hood at room temperature for two days to obtain an amino-modified immobilized support, which was then transferred to a desiccator for long-term storage at room temperature. Before use, the support was weighed, and the amino content on the support surface was quantitatively determined. Experimental results (Table 1) showed that when the molar ratio (R value) of anhydrous ethanol to the silane precursor was 8.17, the immobilized support had the highest mass (162.1 mg) and the highest amino content on the support surface (4506.37 μmol / g). Fourier transform infrared spectroscopy (FTIR) further confirmed the presence of characteristic amino absorption peaks on the surface of the immobilized support prepared under these conditions. Figure 2 ).

[0066] Table 1. Synthesis reaction system of immobilized carrier and amino content on carrier surface

[0067]

[0068] * P<0.05, ** P<0.01

[0069] 2.1.2 Immobilization support for surface-modified amino groups grafted with 2,4-dihydroxyacetophenone

[0070] 2,4-Dihydroxyacetophenone solutions of different concentrations were prepared using diethyl ether as a solvent. 1 mL of a specific concentration of 2,4-dihydroxyacetophenone solution was added, along with a certain mass of the immobilized support with surface-modified amino groups prepared under the optimal conditions described in Section 2.1.1. The reaction was carried out at 25°C and 220 rpm in the dark for 48 hours with shaking. After the reaction, the mixture was centrifuged at 8000 rpm for 1 min, and the precipitate and supernatant were collected separately. The precipitate was washed twice with 500 μL of diethyl ether by centrifugation and then air-dried in a fume hood at room temperature for two days to obtain the grafted immobilized support for later use. The effect of changing the initial concentration of 2,4-dihydroxyacetophenone and the solid-liquid ratio (the ratio of the mass of the immobilized support with surface-modified amino groups to the volume of the 2,4-dihydroxyacetophenone solution) on the grafting effect was investigated. Experimental results showed that the grafting effect was optimal when the concentration of 2,4-dihydroxyacetophenone solution was 242.4 mmol / L and the amount of immobilized support with surface-modified amino groups was 0.02 g / mL, with 3675.62 ± 12.5 μmol of 2,4-dihydroxyacetophenone grafted per gram of support. The immobilized support after grafting was characterized by FTIR. The results showed that at 1630 cm⁻¹… -1 A characteristic absorption peak belonging to an imine bond (C=N) appeared at this point. Figure 2 This indicates that 2,4-dihydroxyacetophenone has been successfully grafted onto the vector.

[0071] 2.1.3 Chelation of nickel ions with the grafted immobilized support

[0072] Take 0.02 g of the grafted immobilized support prepared under the optimal conditions described in Section 2.1.2, immerse it in 1 mL of 0.1 mol / L NiSO4 aqueous solution, and react at 25 °C and 220 rpm for 3 h with shaking. After the reaction is complete, collect the precipitate by centrifugation at 8000 rpm for 1 min. The obtained precipitate is first washed once with deionized water, and then washed 2-3 times with 20 mmol / L pH 7.4 Na2HPO4-NaH2PO4 buffer to obtain the immobilized support chelating nickel ions.

[0073] 2.2 Preparation of recombinant cholesterol esterase immobilized enzyme preparation

[0074] Single colonies of recombinant strains GS115 / pEESB-3 / pEESB-4 were inoculated into 3 mL of YPES liquid medium and cultured overnight at 28°C. The next day, the inoculum was transferred at a rate of 1% (v / v) to 250 mL Erlenmeyer flasks containing 50 mL of YPES liquid medium and cultured at 28°C for 24 h. Then, methanol was added to a final concentration of 1% (v / v) to induce expression. Methanol was added every 24 h to a final concentration of 1% (v / v) for a total induction culture of 96 h. After induction, the fermentation broth was centrifuged at 12000 rpm for 30 min, and the supernatant was collected and concentrated to 1 / 3 of its original volume using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, which was used as the crude enzyme solution of recombinant cholesterol esterase.

[0075] The crude recombinant cholesterol esterase solution was diluted with binding buffer (50 mmol / L Na2HPO4-NaH2PO4, pH 8.0, containing 500 mmol / L sodium chloride) to a protein concentration of 0.45 mg / mL. Different concentrations of imidazole were pre-added to the binding buffer, with final concentrations set at 0, 40, 80, and 160 mmol / L, respectively. 1 mL of the diluted crude enzyme solution was mixed with 0.02 g of the immobilization carrier for chelating nickel ions prepared according to section 2.1.3, and the mixture was incubated at 30 °C and 220 rpm for 6 h with shaking. After adsorption, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant and precipitate were collected separately. The precipitate was washed three times with 0.1 mL of imidazole-free binding buffer to obtain the immobilized recombinant cholesterol esterase enzyme preparation. The supernatant was used to determine residual enzyme activity and protein content. The enzyme load and enzyme activity recovery rate were calculated using the following formulas: Enzyme load (mg / g) = (Total added protein - Total residual protein in supernatant) / Carrier mass; Enzyme activity recovery rate (%) = [(Total added enzyme activity - Total residual enzyme activity in supernatant) / Total added enzyme activity] × 100%. SDS-PAGE was used for qualitative analysis. In addition to imidazole concentration, the effect of maximum enzyme load on immobilization efficiency was further investigated. After optimization, the final immobilization conditions were as follows: The crude recombinant cholesterol esterase enzyme solution was diluted with binding buffer containing 20 mmol / L imidazole to a protein concentration of 0.5 mg / mL. Then, 4 mL of this solution was mixed with 0.02 g of the immobilization carrier chelated with nickel ions prepared according to section 2.1.3, and the mixture was shaken at 30℃ and 220 rpm for 6 h for adsorption. After adsorption, the mixture was centrifuged at 8000 rpm for 10 min, and the precipitate was collected. The precipitate was washed three times with 0.1 mL of imidazole-free binding buffer to obtain the recombinant cholesterol esterase immobilized enzyme preparation. Under these optimal immobilization conditions, the maximum enzyme loading of the obtained recombinant cholesterol esterase immobilized enzyme preparation was 55.5 mg / g, and the enzyme activity recovery rate was 66.71%. Scanning electron microscopy showed that the particle size of the obtained recombinant cholesterol esterase immobilized enzyme preparation was in the micrometer range.

[0076] 3. The synthesis of stigmasterol ferulic acid ester catalyzed by immobilized recombinant OPE-D136P enzyme preparation.

[0077] The esterification reaction was carried out in 10 mL screw-cap vials. The reaction system consisted of the following components: 5 mmol / L stigmasterol (2.1 mg), 5 mmol / L ferulic acid (250 µL of 20 mmol / L stock solution), 315 µL n-hexane, 440 µL 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt ([EMIM]Tf2N), 40 mg recombinant cholesterol esterase immobilized enzyme preparation, and 0.3 g 4 Å molecular sieve. No recombinant cholesterol esterase immobilized enzyme preparation was added to the control group. The reaction flasks were incubated at 65 °C and 220 rpm with shaking for 5 days. After the reaction, the samples were processed as follows: First, all liquid phase fractions were removed from the reaction flasks; the solid residue was washed once with 500 µL of a 1:1, v / v mixture of n-hexane and tert-butanol, and the liquid phase fractions were combined. The combined liquids were centrifuged at 12000 rpm for 5 min, and the lower ionic liquid phase ([EMIM]Tf2N phase) and the upper organic phase (n-hexane / tert-butanol layer) were collected separately. Twice the volume of distilled water was added to the upper organic phase, mixed well, and incubated in a water bath at 65°C for 15 min. Then, the mixture was centrifuged at 12000 rpm for 5 min, and the lower aqueous phase and the upper organic phase (n-hexane / tert-butanol layer) were collected again separately. 10 µL of each of the final upper organic phase (n-hexane / tert-butanol extract) and the lower ionic liquid phase were analyzed by thin-layer chromatography using petroleum ether / ethyl acetate (4:1, v / v) as the developing solvent, and colorimetric analysis was performed using 10% sulfuric acid ethanol solution (v / v). Figure 4 Simultaneously, the final upper organic phase (n-hexane / tert-butanol extract) was quantitatively analyzed by HPLC: after evaporating the solvent from the organic phase, it was reconstituted with 1 mL of isopropanol, centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm organic phase filter membrane before HPLC analysis. The chromatographic conditions were as follows: PAD detector, 210 nm detection; Shim-pack GIST C18 column (5 μm, 250 mm × 4.6 mm), column temperature 30 ℃; mobile phase was a mixture of methanol and isopropanol (8:2, v / v), flow rate 1.0 mL / min, injection volume 5 μL. The HPLC results are as follows. Figure 5 As shown, the chromatographic peak with a retention time of 7.972 min corresponds to the substrate stigmasterol, and the chromatographic peak with a retention time of 12.341 min corresponds to the product stigmasterol ferulic acid ester. Stigmasterol ferulic acid ester was not detected in the control group sample (without the addition of recombinant cholesterol ester immobilized enzyme preparation). Calculations show that the conversion rate of stigmasterol ferulic acid ester catalyzed by the recombinant cholesterol ester immobilized enzyme preparation is approximately 33% based on stigmasterol and approximately 32% based on ferulic acid (ferulic acid content was determined spectrophotometrically).

[0078] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for constructing a recombinant Pichia pastoris engineered strain expressing cholesterol esterase, characterized in that: Includes the following steps: (1) The Ophiostoma piceae cholesterol esterase mutant gene ope-D136P was ligated between the EcoRI and NotRI restriction sites of the pPICZαA plasmid to obtain the pEESB-1 plasmid; the signal peptide coding sequence in the pEESB-1 plasmid was replaced to obtain the pEESB-2 plasmid; the Pichia pastoris rDNA gene was ligated to the BamHI restriction site of the pEESB-2 plasmid to obtain the pEESB-3 plasmid; (2) The Pichia pastoris protein disulfide isomerase encoding gene pdi was ligated between the EcoRI and NotI restriction sites of pPIC9K plasmid to obtain pEESB-4 plasmid; (3) Electroporate the pEESB-3 plasmid into competent Pichia pastoris cells, and obtain recombinant Pichia pastoris engineered bacteria expressing cholesterol esterase by Zeocin resistance selection and methanol-induced expression selection; or, electroporate the pEESB-3 plasmid into competent Pichia pastoris cells, and obtain the first generation of recombinant bacteria by Zeocin resistance selection and methanol-induced expression selection, and then electroporate the pEESB-4 plasmid into competent cells of the first generation of recombinant bacteria, and obtain recombinant Pichia pastoris engineered bacteria expressing cholesterol esterase by G418 resistance selection and methanol-induced expression selection.

2. The construction method according to claim 1, characterized in that: The nucleotide sequence of the Ophiostoma piceae cholesterol esterase mutant gene ope-D136P is shown in SEQ ID NO.1; the signal peptide coding sequence is replaced by the sequence shown in SEQ ID NO.2 with the sequence shown in SEQ ID NO.3; the nucleotide sequence of the Pichia pastoris rDNA gene is shown in SEQ ID NO.4; and the nucleotide sequence of the pdi gene is shown in SEQ ID NO.

5.

3. A recombinant Pichia pastoris engineered strain, characterized in that: It is constructed by the construction method described in any one of claims 1 to 2.

4. A method for preparing recombinant cholesterol esterase, characterized in that: The recombinant Pichia pastoris engineered strain described in claim 3 was induced and cultured with methanol, and the fermentation supernatant was collected to obtain a crude enzyme solution of recombinant cholesterol esterase.

5. A recombinant cholesterol esterase, characterized in that: It is prepared by the preparation method described in claim 4.

6. A method for preparing a recombinant cholesterol esterase immobilized enzyme preparation, characterized in that: Includes the following steps: (1) The silane precursor was dissolved in anhydrous ethanol, and PEG1000 solution and ammonia solution were added. After aging, washing and drying, an amino-modified immobilized support was obtained. (2) The amino-modified immobilized support was reacted with 2,4-dihydroxyacetophenone solution in diethyl ether in the dark to obtain the grafted immobilized support; (3) The grafted immobilized carrier was reacted with nickel sulfate solution by shaking to obtain an immobilized carrier that chelates nickel ions; (4) Immobilization: The recombinant cholesterol esterase described in claim 5 is contacted and adsorbed with the immobilization carrier that chelates nickel ions in a binding buffer to obtain a recombinant cholesterol esterase immobilized enzyme preparation.

7. The preparation method according to claim 6, characterized in that: In step (1), the silane precursor comprises 3-aminopropyltriethoxysilane and tetraethyl silicate in a molar ratio of 1:3, and the molar ratio of the silane precursor to anhydrous ethanol is 1:8.17; in step (2), the amount of the amino-modified immobilization support is 0.02 g / mL, and the concentration of the 2,4-dihydroxyacetophenone solution is 242.4 mmol / L.

8. A recombinant cholesterol esterase immobilized enzyme preparation, characterized in that: It is prepared by the preparation method according to any one of claims 6 to 7.

9. The application of the recombinant cholesterol esterase immobilized enzyme preparation as described in claim 8 in the catalytic synthesis of stigmasterol ferulic acid ester.

10. The application according to claim 9, characterized in that: Using stigmasterol and ferulic acid as substrates, stigmasterol ferulic acid ester was synthesized by oscillating reaction in a non-aqueous reaction system composed of n-hexane and 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt under the catalysis of a recombinant cholesterol esterase immobilized enzyme preparation.

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