Method for improving ring opening rate of monascus milovastatin through enzymolysis treatment

Abstract

The invention relates to the technical field of biology, in particular to a method for improving the ring opening rate of monascus milovastatin through enzymolysis treatment, which comprises the following steps: preparing a pH response type functional carrier, activating carboxyl on the surface of the carrier, covalently immobilizing esterase, extracting and purifying lovastatin lactone, catalyzing ring opening by immobilized enzyme, recovering the immobilized enzyme and purifying lovastatin ring-opening acid. According to the scheme, the pH response type modified carrier is constructed to immobilize esterase, the reversible ionization characteristic of carboxyl functional groups of the carrier is utilized to realize accurate regulation and control of an enzymolysis microenvironment, the pH response carrier can present a dynamic swelling-shrinking behavior in different reaction stages, and the reaction process can be accelerated through a substrate enrichment effect in the initial reaction stage; accumulated products can be dredged through the swelling effect in the middle stage of the reaction; in the self-regulation stage, the stability of the microenvironment in which the enzyme is located can be maintained through contraction, so that the ring-opening conversion effect is greatly improved, the enzyme always keeps relatively high activity in a relatively long reaction period, and the ring-opening rate of the monascus milovastatin is greatly increased.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for improving the ring-opening rate of red yeast rice milovastatin through enzymatic hydrolysis. Background Technology

[0002] Lovastatin is an important lipid-lowering active ingredient widely used in the prevention and treatment of cardiovascular diseases. Its natural source is mainly red yeast rice, a traditional food and medicine product formed by the fermentation of rice by Monascus purpureus. Lovastatin in red yeast rice mainly exists in the lactone form. However, the lactone form of lovastatin needs to be converted into the open-ring acid form in vivo to fully exert its physiological activity. The open-ring acid form has stronger water solubility and bioavailability. Therefore, efficiently converting lovastatin lactone in red yeast rice into the open-ring acid form is a key step in enhancing its medicinal and health-promoting value. Currently, the ring-opening process of lovastatin lactone is mainly divided into two categories: chemical methods and enzymatic methods. Although the chemical method is simple to operate, it is prone to generating byproducts, destroying other active ingredients in red yeast rice, and leaving acid and alkali reagent residues that cause environmental pollution. The enzymatic method, using esterases as catalysts, has advantages such as mild reaction conditions, high specificity, and no byproduct pollution, and has become a current research hotspot. However, its application still relies on traditional free enzyme systems, and a highly efficient and stable process has not yet been developed.

[0003] Existing enzymatic methods for the ring-opening process of lovastatin have significant technical drawbacks. On the one hand, traditional free esterase systems are sensitive to the reaction environment; the hydrolysis of lovastatin lactone generates acidic products, leading to a continuous decrease in system pH and rapid inactivation of the free esterase. This results in poor ring-opening conversion and the inability to recover and reuse the free enzyme, leading to high enzyme preparation costs. On the other hand, some studies have attempted to improve enzyme stability using immobilized enzyme technology, but the pore size of the carrier remains unchanged during the reaction, resulting in poor enzyme activity maintenance and reusability. Therefore, we propose a method to improve the ring-opening rate of lovastatin through enzymatic hydrolysis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for improving the ring-opening rate of red yeast rice milovastatin through enzymatic hydrolysis, thereby solving the technical problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for improving the ring-opening rate of milovastatin from red yeast rice through enzymatic hydrolysis includes the following steps: S1: Prepare a pH-responsive functional carrier by using precipitation polymerization to prepare a polymer containing carboxyl functional groups. The carrier is in a swollen state when pH > 6.5 and in a shrinking state when pH < 5.0. S2: Activate the carboxyl groups on the surface of the carrier by using a carbodiimide chemical method to activate the carboxyl groups on the surface of the pH-responsive functional carrier; S3: Esterase covalent immobilization, where esterase is covalently immobilized onto an activated carrier to obtain a pH-responsive immobilized esterase; S4: Lovastatin lactone was extracted and purified from red yeast rice using a gradient extraction method. S5: Immobilized enzyme-catalyzed ring-opening: The pH-responsive immobilized esterase is mixed with lovastatin lactone substrate, and the enzyme-catalyzed ring-opening reaction is carried out at 37°C and pH 6.0-7.2. S6: Recover the immobilized enzyme by separating the immobilized enzyme from the reaction solution through ceramic membrane filtration and recovering the immobilized enzyme; S7: Purify lovastatin cyclophosphamide. The lovastatin cyclophosphamide in the reaction solution was purified by preparative high performance liquid chromatography to obtain lovastatin cyclophosphamide crystals.

[0006] In one possible implementation, step S1, the preparation of the pH-responsive functional carrier includes: mixing methacrylic acid, hydroxyethyl methacrylate, and N,N'-methylenebisacrylamide in a mass ratio of 5~6:2~3:1 to form a monomer system; dissolving the monomer system and 1% polyvinylpyrrolidone (by mass of the total monomers) in a mixed solvent composed of anhydrous ethanol and deionized water in a volume ratio of 3:2 to prepare a reaction solution; purging the reaction solution with nitrogen gas to remove oxygen, then adding 2% azobisisobutyronitrile (azobisisobutyronitrile) by mass of the total monomers, and reacting for 6 hours at 65±0.5℃ and 250 r / min with stirring; the reaction product is centrifuged, washed, and vacuum dried to obtain a white powdery pH-responsive functional carrier; the carboxyl density of the carrier is 1.3~1.6 mmol / g.

[0007] In one possible implementation, step S2, the activation of the carboxyl groups on the carrier surface, includes: dispersing the pH-responsive functional carrier prepared in step S1 in 0.1 mol / L MES buffer at pH=5.5; subsequently adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), wherein the molar ratio of EDC to the carrier carboxyl groups is 2.4:1, and the molar ratio of NHS to EDC is 1:1; reacting at 25°C and 180 r / min for 2 h, centrifuging to collect the activated carrier, and washing with 0.05 mol / L PBS buffer at pH=7.0.

[0008] In one possible implementation, in step S3, the esterase is derived from porcine pancreas and has an enzyme activity ≥300 U / mg; the immobilization reaction includes: dissolving the esterase in 0.05 mol / L Tris-HCl buffer at pH=7.5, and then... The enzyme was filtered through a filter membrane to obtain an esterase solution with an enzyme concentration of 1 mg / mL. The activated carrier obtained in step S2 was dispersed in the esterase solution at a carrier-to-enzyme mass ratio of 10:1. The reaction was carried out at 30℃ and 120 r / min for 10 h with shaking. During the reaction, the pH of the system was maintained at 7.5±0.1 by adding 0.1 mol / L NaOH solution. After the reaction was completed, the immobilized enzyme was collected by centrifugation and washed with Tris-HCl buffer at pH=7.5 until no free enzyme residue was found, thus obtaining the pH-responsive immobilized esterase.

[0009] In one possible implementation, step S4, the gradient extraction includes: pulverizing red yeast rice through an 80-mesh sieve; mixing the red yeast rice powder with a 75% (v / v) ethanol aqueous solution at a material-to-liquid ratio of 1:15 (g:mL); ultrasonically extracting at 45°C and 350W for 60 min; filtering the extract; repeating the extraction once with the residue; combining the filtrates and concentrating under reduced pressure to 1 / 5 of the original volume; defatting the concentrate with an equal volume of n-hexane; discarding the organic phase; repeating this process three times; adjusting the pH of the lower aqueous phase to 3.0 with 0.1 mol / L HCl; extracting three times with ethyl acetate; combining the organic phases and concentrating under reduced pressure to dryness; recrystallizing twice with a methanol-water mixed solvent at a volume ratio of 7:3; and vacuum drying to obtain the purified lovastatin lactone.

[0010] In one possible implementation, in step S5, the lovastatin lactone substrate is prepared by dissolving the lovastatin lactone concentrate obtained in step S4 in 0.02 mol / L PBS buffer at pH=7.0, with an initial concentration of 3.0 g / L; in the catalytic ring-opening reaction, the mass ratio of immobilized esterase to lovastatin lactone is 2.5~3:1, and the total reaction time is 15 h.

[0011] In one possible implementation, in step S6, the pore size of the ceramic membrane is 0.1 mm. The operating pressure was 0.2 MPa and the crossflow rate was 1.5 m / s. The membrane retentate was washed three times with Tris-HCl buffer at pH 7.5, and the immobilized enzyme was collected.

[0012] In one possible implementation, in step S7, the mobile phase of the preparative high-performance liquid chromatography is acetonitrile-0.1% phosphoric acid aqueous solution, the volume ratio of acetonitrile to 0.1% phosphoric acid aqueous solution is 55:45, and the detection wavelength is 238 nm; the target fraction is collected according to the retention time of lovastatin open-ring acid standard, and the fraction is freeze-dried to obtain lovastatin open-ring acid crystals.

[0013] Beneficial effects compared to existing technologies: 1. In this scheme, a dynamic catalytic system for immobilizing esterases on a pH-responsive modified carrier is constructed. The reversible ionization properties of the carrier's carboxyl functional groups enable precise control of the enzymatic microenvironment, significantly improving the ring-opening efficiency and enzyme activity stability of lovastatin. The pH-responsive carrier exhibits dynamic swelling-shrinkage behavior at different reaction stages. In the initial stage, it accelerates the reaction process through substrate enrichment, providing sufficient substrate for the enzymatic reaction. In the middle stage, swelling helps to channel accumulated products, preventing them from inhibiting enzyme activity. In the self-regulating stage, shrinkage maintains the stability of the enzyme's microenvironment, greatly improving the ring-opening conversion effect and allowing the enzyme to maintain high activity throughout a longer reaction period, significantly increasing the ring-opening rate of red yeast rice milvastatin. 2. In this scheme, the carboxyl groups on the carrier surface are activated, and then the esterase is stably immobilized on the carrier surface through covalent bonding, ensuring a tight binding between the enzyme and the carrier and effectively preserving the enzyme's activity. After the reaction, the immobilized enzyme can be efficiently recovered using membrane filtration technology. The recovered carrier structure is stable and will not be damaged during the separation process. The immobilized enzyme can maintain good catalytic performance even after multiple reuses. This high stability and reusability not only reduce the amount of enzyme preparation added at one time but also avoid the waste caused by the loss of free enzyme, significantly reducing the overall cost of the process. Attached Figure Description

[0014] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0015] Figure 1 This is a schematic diagram of the enzymatic hydrolysis and ring-opening method for red yeast rice milovastatin according to the present invention. Detailed Implementation

[0016] Preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention can also be implemented in various different forms, and therefore the present invention is not limited to the embodiments described below. In addition, for the purpose of more clearly describing the present invention, parts not connected to the invention will be omitted from the drawings. The technical solutions in this application are designed to address the problems described in the background, and are generally as follows: This invention introduces a method to improve the ring-opening rate of milovastatin from red yeast rice through enzymatic hydrolysis. By constructing a dynamic catalytic system of esterase immobilized on a pH-responsive modified carrier, the enzymatic hydrolysis process is regulated by utilizing the pH-responsive swelling-shrinkage characteristics of the carrier, thus solving the problems of product accumulation leading to enzyme inactivation and low ring-opening rate in traditional enzymatic hydrolysis.

[0017] Example 1 Please refer to Figure 1This embodiment describes a method for enzymatic hydrolysis and ring-opening of red yeast rice milovastatin based on a pH-responsive carrier. The specific method is as follows: S1: Preparation of pH-responsive functional carriers pH-responsive polymer microspheres containing carboxyl functional groups were prepared by precipitation polymerization. The specific process was as follows: 5.0 g of functional monomer methacrylic acid (MAA), 3.0 g of hydrophilic monomer hydroxyethyl methacrylate (HEMA), and 1.0 g of crosslinking agent N,N'-methylenebisacrylamide (MBA) were mixed at a mass ratio of 5:3:1 to form a monomer system with a total mass of 9.0 g. 0.09 g of stabilizer polyvinylpyrrolidone (PVP, molecular weight 58000) was added to the above monomer mixture, representing 1% of the total monomer mass. The above materials were dissolved together in a mixed solvent consisting of 300 mL of anhydrous ethanol and 200 mL of deionized water (volume ratio 3:2), and the mixture was magnetically stirred for 30 min to homogenize the system, resulting in a reaction solution with a total monomer concentration of 18 g / L.

[0018] The reaction solution was transferred to a 1000 mL three-necked flask, and a mechanical stirrer, nitrogen delivery tube, and reflux condenser were installed. High-purity nitrogen was purged for 30 min to purge the oxygen from the solution, with the purging rate controlled at 50 mL / min. The flask was then placed in a constant temperature water bath to maintain the reaction temperature at 65 ± 0.5 °C, and the stirring rate was set to 250 r / min. 0.18 g of the oil-soluble initiator azobisisobutyronitrile (AIBN) was added, which was 2% of the total monomer mass.

[0019] After the reaction continued for 6 hours, heating and stirring were stopped, and the product was transferred to a centrifuge tube. The tube was centrifuged at 8000 rpm for 20 minutes, and the lower precipitate was collected. The precipitate was washed five times with deionized water, centrifuged at 8000 rpm for 15 minutes after each wash, until the supernatant showed an absorbance of no more than 0.02 at 220 nm and the pH stabilized at 7.0 ± 0.1. Finally, the precipitate was placed in a vacuum drying oven and dried at 55°C and −0.095 MPa for 18 hours to obtain a white powdery pH-responsive carboxyl functional carrier, weighing 8.2 g, with a monomer conversion rate of 91.1%.

[0020] This step introduces MAA monomers, causing the carrier molecular chain to covalently bind a large number of carboxyl groups ( The functional group undergoes reversible ionization under different pH conditions: when the system pH > 6.5, Ionization Swelling occurs between chain segments due to increased electrostatic repulsion, increasing the carrier pore size from the initial 80-120 nm to 300-500 nm; when pH < 5.0, Protonization The electrostatic repulsion weakened, the carrier network shrank, and the pore size returned to its initial range. The carboxyl density of the carrier, determined by acid-base titration, was 1.3 mmol / g. The particle size distribution and polydispersity index (PDI), measured using dynamic light scattering (DLS), were 180–350 nm and 0.15, respectively.

[0021] S2: Active sites on the surface of the activator The carboxyl groups on the surface of the carrier were activated using a carbodiimide chemical method. The specific process was as follows: 0.5 g of the carboxyl functional carrier prepared by S1 was taken, and its total carboxyl group was 0.5 g × 1.3 mmol / g = 0.65 mmol. It was dispersed in 50 mL of 0.1 mol / L LMES buffer (pH = 5.5) and sonicated for 10 min (power 200 W) to ensure uniform dispersion of the carrier. Subsequently, 0.3 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.18 g of N-hydroxysuccinimide (NHS) were added. The molar ratio of EDC to the carboxyl groups of the carrier was approximately 2.4:1, and the molar ratio of NHS to EDC was 1:1.

[0022] The mixture was placed in a constant temperature shaker and reacted at 25℃ and 180 r / min for 2 h. After the reaction, it was centrifuged at 10000 r / min for 15 min to collect the activated carrier. It was washed three times with 0.05 mol / L PBS buffer (pH=7.0) and centrifuged after each wash to obtain the surface-activated pH-responsive carrier.

[0023] Fourier transform infrared spectroscopy (FTIR) was used to detect changes in functional groups before and after activation of the support. The results showed that after activation, the functional groups at 1735 cm⁻¹... -1 The presence of the characteristic absorption peak of the NHS ester bond at the point indicates that the carboxyl group was successfully activated.

[0024] S3: Covalent immobilization of esterase The esterase was covalently immobilized on an S2-activated carrier. The specific procedure was as follows: Prepare the esterase solution: Weigh 0.1 g of esterase (derived from porcine pancreas, enzyme activity ≥300 U / mg), dissolve it in 100 mL of 0.05 mol / L Tris-HCl buffer (pH=7.5), stir magnetically for 30 min to ensure complete dissolution, and then... Insoluble matter was removed by membrane filtration to obtain an esterase solution with an enzyme concentration of 1 mg / mL; The activated carrier obtained in S2 was dispersed in the above enzyme solution, so that the mass ratio of carrier to enzyme was 10:1; the mixture was placed in a constant temperature water bath at 30℃ and shaken at a rate of 120 r / min for 10 h, during which the pH of the system was maintained at 7.5±0.1 by adding 0.1 mol / L NaOH solution dropwise. After the reaction was complete, the mixture was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 15 minutes. The precipitate was collected and washed six times with Tris-HCl buffer (pH 7.5). After each wash, the supernatant was collected, and the concentration of free protein was determined by UV spectrophotometry (280 nm) and Coomassie Brilliant Blue G-250 method, respectively. Ultraviolet spectrophotometry: Use an ultraviolet-visible spectrophotometer to detect absorbance at 280 nm until the absorbance value is ≤0.01 (corresponding to a free protein concentration ≤0.01 mg / mL). Coomassie Brilliant Blue Method: Take 0.5 mL of supernatant, add 5 mL of Coomassie Brilliant Blue reagent, shake to mix, let stand for 5 min, and detect the absorbance at 595 nm. Calculate the free protein concentration by referring to the bovine serum albumin standard curve. The results show that the free protein concentration in the final supernatant is ≤0.005 mg / mL, confirming no free enzyme residue, and obtaining a pH-responsive immobilized esterase.

[0025] Total enzyme activity assay: The p-nitrophenylacetic acid method was used. At 37℃ and pH 7.5, using 5 mmol / L p-nitrophenylacetic acid (dissolved in a 1:1 volume ratio ethanol-water mixture) as the substrate, the formation rate of p-nitrophenol was monitored at a wavelength of 405 nm. One unit of enzyme activity (U) was defined as the amount of p-nitrophenol produced per minute. The amount of enzyme required for p-nitrophenol.

[0026] The specific activity of an immobilized enzyme is the ratio of its total activity to its total mass.

[0027] Enzyme activity recovery rate of immobilized enzymes According to the formula Calculate, where, Total activity of immobilized enzymes (unit: U). Total activity of the initial free enzyme (unit: U).

[0028] According to measurements, in this embodiment, =30000U, =25500U, therefore =85%, the total mass of the immobilized enzyme is about 0.6 g, so the specific activity of the immobilized enzyme is 425 U / g.

[0029] S4: Extraction and purification of lovastatin lactone from red yeast rice Lovastatin lactone was extracted from red yeast rice using a gradient extraction method. The specific process was as follows: Red yeast rice (lovastatin content ≥1.2%) was pulverized and passed through an 80-mesh sieve. 100g of red yeast rice powder was placed in a 2000mL Erlenmeyer flask, and 1500mL of 75% ethanol aqueous solution was added, with a material-to-liquid ratio of 1:15 (g:mL). The Erlenmeyer flask was placed in an ultrasonic extractor, with a power of 350W, a temperature of 45℃, and an extraction time of 60min, during which the mixture was stirred once every 15min.

[0030] After extraction, the residue was filtered through double-layered filter paper and the filtrate was collected. The residue was extracted once more with 500 mL of 75% ethanol, and the two filtrates were combined. The filtrate was then transferred to a rotary evaporator and concentrated under reduced pressure at 60 °C and −0.085 MPa to 1 / 5 of the original volume. Add an equal volume of n-hexane to the concentrate, stir magnetically for 30 min, allow to stand and separate into layers, discard the upper organic phase, and repeat the defatting process 3 times; adjust the pH of the lower aqueous phase to 3.0 with 0.1 mol / L HCl, extract with ethyl acetate 3 times (each time the volume of the extractant is 1 / 2 of the aqueous phase), and combine the ethyl acetate phases; The ethyl acetate phase was concentrated to dryness under reduced pressure, recrystallized twice with a methanol-water mixed solvent (volume ratio 7:3), and dried under vacuum to obtain the refined lovastatin lactone. High-performance liquid chromatography (HPLC) was used. Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution (55:45, v / v), the flow rate was 1 mL / min, the column temperature was 30℃, the detection wavelength was 238 nm, and the reference standard was lovastatin lactone (purity ≥99.5%). The results showed that the purity of the purified product was 97.8%. Gas chromatography (GC) was used to detect n-hexane residues. Chromatographic conditions: DB-5 capillary column (30m × 0.32mm × 0.25m). The column temperature program was 50℃ (hold for 3 min) → increased to 150℃ (hold for 2 min) at a rate of 10℃ / min. The FID detector showed that the residual hexane content was ≤0.005%. Dissolve the above-mentioned concentrate in 0.02 mol / L PBS buffer (pH=7.0) to prepare the initial concentration. The substrate solution is 3.0 g / L.

[0031] S5: pH-responsive immobilized enzyme catalyzes the ring-opening reaction of lovastatin. The immobilized enzyme prepared by S3 was used to catalyze the ring-opening of lovastatin lactone. The specific process was as follows: 500 mL of substrate solution prepared by S4 was placed in a 1000 mL reaction vessel. The initial lactone mass was 500 mL × 3.0 g / L = 1.5 g. 7.5 g of the immobilized esterase prepared by S3 was added (the mass ratio of enzyme to substrate was 2.5:1). The initial pH of the system was adjusted to 7.2 with 0.1 mol / L NaOH. The reaction vessel was placed in a constant temperature water bath, and the reaction temperature was maintained at 37 °C. The stirring rate was 200 r / min. The pH value of the system was recorded in real time using an online pH monitor. The total reaction time was 15 h.

[0032] The reaction exhibits three characteristic stages: the initial stage (0-3h), where the system pH stabilizes at 7.2±0.05, the carrier is in a contracted state with an average pore size of approximately 100nm, and the local concentration of substrate inside the carrier, determined by microdialysis, is 3.96g / L, which is 32% higher than the average concentration of the system, significantly accelerating the enzymatic reaction. At 3h, the ring-opening conversion rate reaches 45.2%; the reaction progress stage (3-9h), with the formation of acidic products and lovastatin ring-opening acid, the system pH gradually decreases to 6.0±0.1, the carrier undergoes a swelling phase transition, and the average pore size increases to 400nm. At this time, the relative enzyme activity still maintains 92% of the initial activity; the self-regulation stage (9-15h), the carrier swelling promotes product diffusion, the system pH rises back to 6.8±0.2, and the carrier shrinks again to a pore size of 150nm, forming a dynamic equilibrium.

[0033] The ring-opening conversion rate was determined by sampling at the corresponding time points and then subjected to a 0.22... After filtration through a filter membrane, the remaining lovastatin lactone concentration in the reaction system was analyzed by high-performance liquid chromatography (HPLC) (under the same purity detection conditions as S4). The concentration was quantified using a lactone standard curve. ,according to The ring-opening conversion rate was calculated to be 96.1% at 15 h. The ring-opening acid concentration was simultaneously detected (using lovastatin ring-opening acid standard). The results showed that the ring-opening acid concentration was 1.36 g / L at 3 h, 2.15 g / L at 6 h, 2.58 g / L at 9 h, 2.72 g / L at 12 h, and 2.88 g / L at 15 h.

[0034] The relative enzyme activity was determined by taking a small amount of immobilized enzyme from the reaction system at the corresponding time point, washing it gently with buffer, and measuring its residual enzyme activity under the same standard enzyme activity assay conditions as S3. The residual enzyme activity was calculated by comparing it with the initial enzyme activity (defined as 100%) of the immobilized enzyme at the start of the reaction (0h).

[0035] Samples were taken at 3h, 6h, 9h, 12h, and 15h, and purified by 0.22... The enzyme was tested after filtration through a membrane. The results showed that at 6 h, the pH of the system stabilized at 6.3±0.2, and the relative enzyme activity remained at 90% of the initial activity; at 12 h, the pH fluctuated at 6.0±0.3, and the relative enzyme activity still reached 78%.

[0036] S6: Separation and recovery of immobilized enzymes and reaction solution Immobilized enzymes and products are separated via membrane separation. The specific process is as follows: After the reaction is complete, the reaction mixture of S5 is passed through a ceramic membrane filter (pore size 0.1). Cross-flow filtration was performed at an operating pressure of 0.2 MPa and a cross-flow rate of 1.5 m / s. The retentate was a pH-responsive immobilized enzyme, and the permeate was a product solution containing lovastatin cyclohexanoate. The membrane was washed three times with 50 mL of Tris-HCl buffer (pH 7.5), and the immobilized enzyme was collected and stored at 4°C. The permeate was then used for further purification. The collected immobilized enzyme weighed 7.32 g, the recovery rate was 97.6%, and the relative activity of the recovered enzyme was 82% of the initial activity. The particle size distribution of the recovered carrier was detected by DLS, and the results showed that it was 175~340nm with PDI=0.16. This was not significantly different from that of the fresh carrier (180~350nm with PDI=0.15), proving that the carrier was not broken during the filtration process. S7: Purification and efficacy evaluation of lovastatin secoidic acid The product was purified using preparative HPLC. The specific process was as follows: the permeate obtained from S6 was injected into a preparative HPLC system (equipped with a C18 column, 50 mm × 20 mm, 5... The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution (volume ratio 55:45), the flow rate was 12 mL / min, the column temperature was 30℃, and the detection wavelength was 238 nm. The target fraction was collected according to the retention time of the standard (retention time of open-ring acid was 8.5 min, and retention time of lactone was 12.3 min). The collected fraction was freeze-dried at −50℃ and 10 Pa for 24 h to obtain lovastatin open-ring acid crystals, and the purity was 99.4% as determined by HPLC.

[0037] Performance evaluation indicators include: Open-loop conversion rate : ,in, The initial lactone concentration (g / L) This represents the concentration of the remaining lactone (g / L) after the reaction. In this example... =96.1%.

[0038] Open-ring acid yield : ,in, The actual mass (g) of the open-ring acid obtained. The mass (g) of the theoretically fully converted open-ring acid is given. In this embodiment, the mass of the open-ring acid crystals is obtained by weighing. The yield is 1.40g. The theoretical mass of the open-ring acid = initial lactone mass × (locastatin open-ring acid molecular weight / lovastatin lactone molecular weight) = 1.5g × (422.5 / 404.5) ≈ 1.56g. Therefore, the yield of the open-ring acid is... =93.2%.

[0039] Stability of immobilized enzyme after repeated use: The immobilized enzyme recovered from S6 was reused 8 times under the conditions of S5. The stability of the 8th reaction was... It still reached 84.3%.

[0040] Example 2 This embodiment only changes the monomer mass ratio in step S1, while the remaining steps are the same as in Example 1, aiming to verify the influence of monomer ratio on carrier performance and ring-opening effect.

[0041] S1: Preparation of pH-responsive functional carriers The difference from Example 1 is that the mass ratio of the functional monomer methacrylic acid (MAA), the hydrophilic monomer hydroxyethyl methacrylate (HEMA), and the crosslinking agent N,N'-methylenebisacrylamide (MBA) was adjusted to 6:2:1 to increase the proportion of MAA and improve the carboxyl density. All other operations (precipitation polymerization, PVP dosage 1%, AIBN dosage 2%, reaction temperature 65±0.5℃, centrifugation and drying conditions, etc.) were the same as S1 in Example 1. Testing showed that the carboxyl density of the carrier in this example was 1.6 mmol / g (higher than 1.3 mmol / g in Example 1), the particle size distribution was 170~330 nm, the polydispersity index (PDI) was 0.13, the pore size was 75~110 nm at pH < 5.0, and the pore size was 320~520 nm at pH > 6.5, with a greater swelling range than in Example 1.

[0042] Steps S2-S7 The operation parameters for carrier activation, esterase immobilization, lovastatin extraction, catalytic ring opening, enzyme separation and recovery, and product purification are exactly the same as those for S2 to S7 in Example 1.

[0043] Test results: Open-loop conversion rate =97.5% (higher than 96.1% in Example 1); Open-ring acid yield =94.8% (higher than 93.2% in Example 1); Stability of immobilized enzyme after repeated use: After 8 repetitions under S5 conditions, the stability of the 8th reaction was... =86.5% (higher than 84.3% in Example 1).

[0044] The improvement is due to the higher carboxyl group density, which enhances the pH response range and buffering capacity of the carrier, allowing for more efficient consumption of the H2 produced in the reaction. + This allows for the creation of a more stable microenvironment for immobilized enzymes, extending the duration of enzyme activity.

[0045] Example 3 This embodiment improves catalytic efficiency by optimizing the mass ratio of enzyme to substrate in step S5, while the remaining steps remain the same as in Example 1.

[0046] S5: pH-responsive immobilized enzyme catalyzes the ring-opening reaction of lovastatin. The difference from Example 1 is that the mass ratio of enzyme to substrate is adjusted to 3:1 (2.5:1 in Example 1). That is, 500 mL of substrate solution prepared by S4 (initial concentration 3.0 g / L) is taken and 9.0 g of immobilized esterase prepared in S3 is added. The remaining operations (initial pH 7.2, reaction temperature 37℃, stirring rate 200 r / min, reaction time 15 h, etc.) are the same as S5 in Example 1.

[0047] Steps S1~S4 and S6~S7 are exactly the same as S1~S4 and S6~S7 in Example 1, that is, the operating parameters for carrier preparation, activation, enzyme immobilization, lovastatin extraction, enzyme separation and recovery and product purification are all the same.

[0048] Test results: Open-loop conversion rate =96.8% (higher than 96.1% in Example 1); Open-ring acid yield =93.9% (higher than 93.2% in Example 1); Reaction kinetics: The ring-opening conversion rate reached 48.5% after 3 hours (45.2% in Example 1), and the reaction rate was significantly improved in the early stage. Stability of immobilized enzyme after repeated use: After 8 repetitions under S5 conditions, the stability of the 8th reaction was... =85.1% (higher than 84.3% in Example 1).

[0049] The improvement is due to the fact that moderately increasing the amount of enzyme can accelerate the binding of the substrate to the enzyme's active site, and the pH-responsive carrier can still effectively regulate the microenvironment, avoiding the waste of activity caused by excessive enzyme.

[0050] Comparative Example 1: This comparative example uses a non-pH-responsive carrier without carboxyl groups to compare and verify the key role of pH response characteristics in the ring-opening rate. The variable is the carrier monomer composition in step S1, and the remaining steps are consistent with those in Example 1.

[0051] S1: Preparation of non-pH-responsive carriers The difference from Example 1 is that methacrylic acid (MAA) was replaced with the non-functional monomer styrene. The monomer system consisted of a mixture of styrene, HEMA, and MBA in a mass ratio of 5:3:1 (without carboxyl functional groups). All other operations (precipitation polymerization, 1% PVP, 2% AIBN, reaction temperature 65±0.5℃, centrifugation and drying conditions, etc.) were the same as in S1 of Example 1. DLS analysis showed that within the pH range of 4.0–7.0, the pore size remained stable at 180–220 nm with no significant swelling-shrinkage changes, and the carboxyl density was 0.02 mmol / g.

[0052] Steps S2 to S7 are exactly the same as S2 to S7 in Example 1, that is, the operating parameters for carrier activation, esterase immobilization, lovastatin extraction, catalytic ring opening, enzyme separation and recovery, and product purification are all the same.

[0053] Test results: Open-loop conversion rate =73.5% (lower than 96.1% in Example 1); Open-ring acid yield =68.2% (lower than 93.2% in Example 1); Enzyme activity stability: After 6 hours of reaction, the pH of the system dropped to 4.5, and the relative enzyme activity was only 38% (90% in Example 1), which could not maintain catalytic activity; Stability of immobilized enzyme after repeated use: After 8 repetitions under S5 conditions, the stability of the 8th reaction was... =45.1% (far lower than 84.3% in Example 1).

[0054] The results showed that carriers without pH-responsive properties could not regulate the enzymatic hydrolysis microenvironment, and the H+ generated in the reaction... + Continuous accumulation leads to a sharp drop in the pH of the system, rapid inactivation of esterases (optimal pH 7.0~7.5), and a significant decrease in the ring-opening rate.

[0055] Comparative Example 2: This comparative example uses free esterase instead of immobilized enzyme to compare and verify the effect of immobilized carrier (especially pH response characteristics) on ring-opening rate and enzyme reusability. The core variable is the enzyme morphology in steps S3 to S5, and the remaining steps are consistent with those in Example 1.

[0056] S3: Preparation of free esterase solution The difference from Example 1 is that only the esterase solution was prepared (without immobilization). Specifically, 0.1 g of esterase (with the same enzyme activity as in Example 1, 300 U / mg) was weighed, dissolved in 100 mL of 0.05 mol / L Tris-HCl buffer (pH=7.5), and magnetically stirred for 30 min before being diluted with 0.22 mL of HCl. The solution was filtered through a membrane to obtain a free esterase solution (enzyme concentration 1 mg / mL), without a carrier fixation step.

[0057] S5: Free esterase-catalyzed ring-opening reaction of lovastatin The difference from Example 1 is that: to the substrate solution prepared with 500 mL of S4, an equal amount of free esterase solution (about 7.5 mL) was added, which was the same as the amount of enzyme protein in Example 1. The rest of the operation, such as initial pH 7.2, reaction temperature 37°C, stirring rate 200 r / min, and reaction time 15 h, was the same as S5 in Example 1.

[0058] Steps S1~S2, S4, S6~S7: S1~S2 (carrier preparation and activation) are omitted; S4 (lovastatin extraction and purification) is the same as in Example 1; S6 (enzyme separation and recovery) is performed only by filtering the product solution because there is no immobilized carrier; S7 (product purification evaluation) is the same as in Example 1.

[0059] Test results: Open-loop conversion rate =60.2% (lower than 96.1% in Example 1); Open-ring acid yield =54.5% (lower than 93.2% in Example 1); Enzyme activity stability: The pH of the system dropped to 4.0 after 4 hours of reaction, and the esterase was completely inactivated (the relative enzyme activity was still 78% after 12 hours in Example 1). Enzyme reusability: Free enzymes cannot be recovered and have no reusability.

[0060] The results showed that free esterases without carrier protection are easily inactivated by product acidification and cannot be recovered. Their ring-opening rate and economic efficiency are far lower than those of pH-responsive immobilized enzyme systems.

[0061] Data Comparison Table

[0062] Experimental data show that the final ring-opening conversion rates of Examples 1-3 all exceeded 96%, and the relative enzyme activity remained above 90% after 6 hours of reaction. This excellent performance is closely related to the unique pH-responsive characteristics of the carrier. Data shows that in the initial stage of the reaction (0-3h), the carrier was in a swollen state (pore size 300-500 nm), with a local substrate concentration reaching 3.96 g / L, 32% higher than the average concentration of the system. This substrate enrichment effect directly translated into an increased reaction rate, enabling Example 1 to achieve a conversion rate of 45.2% within 3 hours. As the reaction proceeded, the decrease in system pH caused the carrier to shrink (pore size decreased to approximately 100 nm). This change effectively regulated the product distribution in the microenvironment and alleviated the product inhibition effect, which is the structural basis for the long-term maintenance of enzyme activity.

[0063] Example 2, by optimizing the monomer ratio (MAA:HEMA:MBA = 6:2:1), increased the carrier carboxyl density to 1.6 mmol / g, achieving optimal overall performance: a ring-opening conversion rate of 97.5%, and a conversion rate still reaching 86.5% after 8 reuses. The underlying mechanism of this improvement lies in the fact that the higher carboxyl density not only enhances the carrier's pH response sensitivity but, more importantly, improves its inherent buffering capacity. The carrier can more effectively consume the H⁺ generated in the reaction through the ionization / protonation of its own carboxyl groups, thereby constructing a more stable microenvironment for the immobilized enzyme at the molecular level. This is the fundamental reason why its long-term operational stability is significantly superior to other examples.

[0064] The data from Comparative Example 1 (non-pH-responsive carrier) and Comparative Example 2 (free enzyme) conversely demonstrate the innovative value of this invention. Comparative Example 1 showed a ring-opening conversion rate of only 73.5%, and its reusability decreased sharply (45.1% after the 8th conversion), proving that a carrier lacking pH responsiveness cannot achieve effective microenvironment regulation. The results of Comparative Example 2 (conversion rate 60.2%, rapid enzyme inactivation) indicate that the free enzyme system is completely powerless in the face of reaction self-acidification. In contrast, the immobilized system constructed in this invention, through dynamic structural changes in the carrier, achieves a leap from passive acceptance to active regulation, simultaneously solving the two major challenges of reaction efficiency and enzyme stability.

[0065] Experimental data fully demonstrate that this invention achieves intelligent regulation of the enzymatic hydrolysis microenvironment by constructing a pH-responsive immobilized enzyme system. The carrier not only serves as a solid-phase support for the enzyme but also functions as a substrate enrichment device, product channeling device, and microenvironment buffer. This dynamic and adaptive catalytic system successfully overcomes the technical bottlenecks of traditional lovastatin enzymatic hydrolysis processes, such as easy enzyme inactivation, low conversion rate, and difficulty in reusing the enzyme.

[0066] Finally, it should be noted that the above embodiments are merely examples for clearly illustrating the present invention and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for improving the ring-opening rate of milovastatin from red yeast rice through enzymatic hydrolysis, characterized in that, Includes the following steps: S1: Prepare a pH-responsive functional carrier by using precipitation polymerization to prepare a polymer containing carboxyl functional groups. The carrier is in a swollen state when pH > 6.5 and in a shrinking state when pH < 5.

0. S2: Activate the carboxyl groups on the surface of the carrier by using a carbodiimide chemical method to activate the carboxyl groups on the surface of the pH-responsive functional carrier; S3: Esterase covalent immobilization, where esterase is covalently immobilized onto an activated carrier to obtain a pH-responsive immobilized esterase; S4: Lovastatin lactone was extracted and purified from red yeast rice using a gradient extraction method. S5: Immobilized enzyme-catalyzed ring-opening: The pH-responsive immobilized esterase is mixed with lovastatin lactone substrate, and the enzyme-catalyzed ring-opening reaction is carried out at 37°C and pH 6.0-7.

2. S6: Recover the immobilized enzyme by separating the immobilized enzyme from the reaction solution through ceramic membrane filtration and recovering the immobilized enzyme; S7: Purify lovastatin cyclophosphamide. The lovastatin cyclophosphamide in the reaction solution was purified by preparative high performance liquid chromatography to obtain lovastatin cyclophosphamide crystals.

2. The method for improving the ring-opening rate of milovastatin by enzymatic hydrolysis as described in claim 1, characterized in that, In step S1, the preparation of the pH-responsive functional carrier includes: mixing methacrylic acid, hydroxyethyl methacrylate, and N,N'-methylenebisacrylamide in a mass ratio of 5~6:2~3:1 to form a monomer system; dissolving the monomer system and 1% polyvinylpyrrolidone (VPS) in a mixed solvent of anhydrous ethanol and deionized water in a volume ratio of 3:2 to prepare a reaction solution; purging the reaction solution with nitrogen gas to remove oxygen, then adding 2% azobisisobutyronitrile (AIBN) in VPS, and reacting at 65±0.5℃ and 250 r / min for 6 h with stirring; centrifuging, washing, and vacuum drying of the reaction product to obtain a white powdery pH-responsive functional carrier; the carboxyl density of the carrier is 1.3~1.6 mmol / g.

3. The method for improving the ring-opening rate of milovastatin by enzymatic hydrolysis as described in claim 1, characterized in that, In step S2, the activation of the carboxyl groups on the carrier surface includes: dispersing the pH-responsive functional carrier prepared in step S1 in 0.1 mol / L MES buffer at pH=5.5; then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS, wherein the molar ratio of EDC to the carrier carboxyl groups is 2.4:1 and the molar ratio of NHS to EDC is 1:1; after reacting at 25℃ and 180 r / min for 2 h, the activated carrier is collected by centrifugation and washed with 0.05 mol / L PBS buffer at pH=7.

0.

4. The method for improving the ring-opening rate of milovastatin by enzymatic hydrolysis as described in claim 1, characterized in that, In step S3, the esterase is derived from porcine pancreas and has an enzyme activity ≥300 U / mg; the immobilization reaction includes: dissolving the esterase in 0.05 mol / L Tris-HCl buffer at pH=7.5, and then... The enzyme was filtered through a filter membrane to obtain an esterase solution with an enzyme concentration of 1 mg / mL. The activated carrier obtained in step S2 was dispersed in the esterase solution at a carrier-to-enzyme mass ratio of 10:

1. The reaction was carried out at 30℃ and 120 r / min for 10 h with shaking. During the reaction, the pH of the system was maintained at 7.5±0.1 by adding 0.1 mol / L NaOH solution. After the reaction was completed, the immobilized enzyme was collected by centrifugation and washed with Tris-HCl buffer at pH 7.5 until no free enzyme residue was found, thus obtaining the pH-responsive immobilized esterase.

5. The method for improving the ring-opening rate of milovastatin by enzymatic hydrolysis as described in claim 1, characterized in that, In step S4, the gradient extraction includes: pulverizing red yeast rice through an 80-mesh sieve; mixing the red yeast rice powder with a 75% ethanol aqueous solution at a material-to-liquid ratio of 1:15 (g:mL); ultrasonically extracting at 45℃ and 350W for 60 min; filtering the extract; repeating the extraction once with the residue; combining the filtrates and concentrating under reduced pressure to 1 / 5 of the original volume; defatting the concentrate with an equal volume of n-hexane; discarding the organic phase; repeating this process three times; adjusting the pH of the lower aqueous phase to 3.0 with 0.1 mol / L HCl; extracting three times with ethyl acetate; combining the organic phases and concentrating under reduced pressure to dryness; recrystallizing twice with a methanol-water mixed solvent at a volume ratio of 7:3; and vacuum drying to obtain the purified lovastatin lactone.

6. The method for improving the ring-opening rate of milovastatin by enzymatic hydrolysis as described in claim 1, characterized in that, In step S5, the lovastatin lactone substrate is prepared by dissolving the lovastatin lactone concentrate obtained in step S4 in 0.02 mol / L PBS buffer at pH=7.0, with an initial concentration of 3.0 g / L; in the catalytic ring-opening reaction, the mass ratio of immobilized esterase to lovastatin lactone is 2.5~3:1, and the total reaction time is 15 h.

7. The method for improving the ring-opening rate of milovastatin by enzymatic hydrolysis as described in claim 1, characterized in that, In step S6, the pore size of the ceramic membrane is 0.1 mm. The operating pressure was 0.2 MPa and the crossflow rate was 1.5 m / s. The membrane retentate was washed three times with Tris-HCl buffer at pH 7.5, and the immobilized enzyme was collected.

8. The method for improving the ring-opening rate of milovastatin by enzymatic hydrolysis as described in claim 1, characterized in that, In step S7, the mobile phase of the preparative high-performance liquid chromatography is acetonitrile-0.1% phosphoric acid aqueous solution, the volume ratio of acetonitrile to 0.1% phosphoric acid aqueous solution is 55:45, and the detection wavelength is 238 nm. The target fraction is collected according to the retention time of the lovastatin open-ring acid standard, and the fraction is freeze-dried to obtain lovastatin open-ring acid crystals.

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