Process for the enzymatic production of free bilirubin

Abstract

This invention discloses a production process for the enzyme-catalyzed preparation of free bilirubin, relating to the field of functional enzyme preparation and application technology. This enzyme-catalyzed preparation of free bilirubin utilizes GUS thermophilic enzymes, using porcine bile as a substrate, to obtain free bilirubin through enzymatic conversion and hydrolysis. The GUS thermophilic enzymes include one or more combinations of ACI185 GUS thermophilic enzymes, HOJ9 GUS thermophilic enzymes, MFN3 GUS thermophilic enzymes, and PVN8 GUS thermophilic enzymes. The enzyme-catalyzed conversion and hydrolysis temperature is 70–90°C, and the pH value is 4–5. This invention, by introducing GUS thermophilic enzymes, achieves high enzyme activity, high thermal stability, high specificity, and high catalytic efficiency under high-temperature conditions. It can increase the enzyme-catalyzed conversion temperature, significantly reduce bile viscosity, disrupt the colloidal encapsulation of the substrate, improve the substrate conversion rate, and effectively inhibit and kill endogenous contaminating enzymes and bacteria, improving product purity and inhibiting product decomposition.

Description

Technical Field

[0001] This invention relates to the field of functional enzyme preparation and application technology, and in particular to a production process for the enzyme-catalyzed preparation of free bilirubin. Background Technology

[0002] Bilirubin is a natural tetrapyrrole pigment found in animal bile. Based on its form, it can be divided into conjugated bilirubin and free bilirubin. Conjugated bilirubin generally refers to bilirubin bound to glucuronic acid, xylopyranose, taurine, and sulfate, while free bilirubin is unconjugated and has excellent lipid solubility, making it a key medicinal raw material for artificial bezoar. It is also widely used in other traditional Chinese medicine preparations for clearing heat and calming the nerves, and can also be used as a biochemical analysis standard.

[0003] Pig bile contains 0.04%–0.06% bilirubin, making it one of the main sources for the industrial extraction of high-purity bilirubin. However, most of the natural bilirubin in pig bile exists in a water-soluble bound form, which does not directly meet the requirements for use in pharmacopoeias. It must be converted into a free form before it can be used. Compared to bound bilirubin, free bilirubin is easier to separate and purify, has more stable pharmacological activity, and is less prone to hydrolysis and deterioration during storage, making it more valuable in practical applications.

[0004] Currently, the main preparation processes for extracting free bilirubin include chemical hydrolysis and room-temperature enzymatic conversion. Chemical hydrolysis relies primarily on strong acids or bases to hydrolyze bile substrates; however, the reaction conditions are harsh, and the process easily leads to the oxidative degradation of bilirubin, generating degradation products with no medicinal value. This results in a high level of impurities in the obtained bilirubin product, low extraction efficiency, and the large amount of acidic and alkaline waste generated during the process also poses a significant environmental burden.

[0005] The room-temperature enzymatic conversion method for producing free bilirubin mainly involves the hydrolysis of conjugated bilirubin under the catalysis of β-glucuronidase. However, at room temperature, bile has high viscosity, and the protein colloids in bile can encapsulate substrate molecules, hindering sufficient contact between the enzyme and substrate. Furthermore, at room temperature or low temperatures, it is highly susceptible to bacterial growth, and endogenous enzymes in bile can continuously decompose the target product. In addition, ordinary β-glucuronidase has poor thermal stability and limited catalytic activity, resulting in incomplete substrate conversion and very low utilization efficiency of raw materials.

[0006] Therefore, it is of great significance to develop a new production process for preparing free bilirubin in order to solve the problems of high product loss, low conversion efficiency and insufficient raw material utilization in traditional processes. Summary of the Invention

[0007] The purpose of this invention is to provide a production process for the enzyme-catalyzed preparation of free bilirubin, in order to solve the technical problems of large product loss, low conversion efficiency and insufficient raw material utilization in the traditional process for preparing free bilirubin from pig bile.

[0008] The technical problem to be solved by this invention can be achieved through the following technical solution: This invention provides a production process for the enzyme-catalyzed preparation of free bilirubin. The enzyme-catalyzed preparation of free bilirubin utilizes GUS thermophilic enzyme, with porcine bile as a substrate, to obtain free bilirubin through enzymatic conversion and hydrolysis. GUS thermophilic enzymes include one or more combinations of ACI185 GUS thermophilic enzyme, HOJ9 GUS thermophilic enzyme, MFN3 GUS thermophilic enzyme and PVN8 GUS thermophilic enzyme; The nucleotide sequences of ACI185 GUS thermophilic enzymes are shown in SEQ ID NO:1, HOJ9 GUS thermophilic enzymes are shown in SEQ ID NO:2, MFN3 GUS thermophilic enzymes are shown in SEQ ID NO:3, and PVN8 GUS thermophilic enzymes are shown in SEQ ID NO:4.

[0009] Preferably, the temperature for enzyme-catalyzed conversion and hydrolysis is 70–90°C, and the pH value is 4–5.

[0010] More preferably, SEQ ID NO:1: SEQ ID NO:2: SEQ ID NO:3: SEQ ID NO:4:

[0011] By adopting the above technical solution, in the production process of enzyme-catalyzed preparation of free bilirubin of the present invention, the enzymatic hydrolysis temperature is controlled at 70-90℃ and the pH value is 4-5. Under these temperature conditions, the viscosity of bile can be significantly reduced, the fluidity can be greatly improved, and the proteins and other macromolecules that originally hindered the enzymatic hydrolysis of the substrate can undergo irreversible denaturation, thereby destroying the encapsulation of the substrate by proteins and other colloids. The substrate molecules can be fully exposed, clearing the way for subsequent enzymatic hydrolysis reactions, thereby increasing the contact probability between the enzyme and the substrate and the efficiency of the enzymatic hydrolysis reaction.

[0012] Meanwhile, the high-temperature environment can effectively inhibit or kill most of the miscellaneous bacteria. At room temperature or low temperature, bile is not only prone to the growth of miscellaneous bacteria, but the endogenous enzymes it contains can also decompose the target product, resulting in limited catalytic activity, incomplete substrate conversion, and low product yield. However, at the enzymatic hydrolysis temperature of this invention, the endogenous enzymes contained in bile can be denatured and inactivated, solving the problem of product decomposition from the source, avoiding the oxidative degradation of free bilirubin, thereby improving product yield and substrate conversion rate, and protecting the stability of free bilirubin.

[0013] However, the spatial structure of conventional β-glucuronidase is also destroyed under these high-temperature conditions, causing it to rapidly and irreversibly lose its activity, making substrate conversion impossible at this temperature. Therefore, this invention provides a GUS thermophilic enzyme. After thermophilic recombination, this GUS thermophilic enzyme can achieve high enzyme activity at the enzymatic hydrolysis temperature provided by this invention, exhibits good thermal stability, and is not easily inactivated under high-temperature conditions. Simultaneously, it can specifically hydrolyze the glucuronide bonds of the substrate, removing hydrophilic groups to generate lipid-soluble free bilirubin. This not only demonstrates high specificity but also significantly improves catalytic efficiency at high temperatures. It can greatly improve the conversion rate, reduce byproducts, shorten the reaction cycle, and effectively overcome the shortcomings of existing traditional processes.

[0014] Preferably, the GUS thermophilic enzyme is prepared according to the following method: S101. Using the GUS gene coding sequence as the target fragment and pET28a(+) as the recombinant expression vector, the target fragment and the recombinant expression vector were ligated using T4 phage DNase to obtain the recombinant plasmid. S102. The recombinant plasmid was transformed into Escherichia coli Rosetta(DE3) competent cells to obtain the corresponding engineered expression strain; S103. After the corresponding engineered expression strain is activated and cultured, it is inoculated into liquid LB medium containing Kan antibiotic and chloramphenicol, an inducer is added, and the culture is continued for 16-18 hours. Then, the precipitated bacterial cells are collected by centrifugation. S104. The precipitated bacterial cells are lysed, loaded onto a column, purified, and eluted. The eluent is then collected and dialyzed to obtain GUS thermophilic enzyme.

[0015] Preferably, the GUS gene coding sequence includes any one of ACI185, HOJ9, MFN3, and PVN8.

[0016] Preferably, the liquid LB medium containing Kan antibiotic and chloramphenicol has a Kan antibiotic content of 45-55 μg / mL and a chloramphenicol content of 45-55 μg / mL; the inoculum size is 1-1.1%.

[0017] Preferably, the inducing agent includes isopropyl-β-D-thiogalactoside at a concentration of 0.4–0.5 mM.

[0018] Preferably, the pyrolysis in step S104 mainly includes the following steps: Add the precipitated bacterial cells to the lysis buffer, vortex and resuspend until there are no obvious clumps of bacterial cells, then sonicate to disrupt the cells, and centrifuge at 4-5℃ and 8000-8500 rpm for 10-15 min, and collect the supernatant.

[0019] Preferably, the lysis buffer comprises 20-25 mM Tris-HCl buffer, 500-520 mM sodium chloride and 5-8 mM imidazole; the mass-to-volume ratio of the precipitated bacterial cells to the lysis buffer is 1 g: (8-10) mL.

[0020] More preferably, the pH of the Tris-HCl buffer is 8.0.

[0021] More preferably, step S104 further includes the following steps: Add 5 column volumes of column equilibration buffer to the nickel column, then add the supernatant obtained from lysis (i.e., crude enzyme solution) for column purification. Next, add 5 column volumes of washing buffer to remove contaminating proteins from the column material. Finally, add 5 column volumes of elution buffer according to the concentration gradient for elution. Collect the eluent and add it to a dialysis bag. Add dialysis solution A and stir and dialyze overnight. Replace with dialysis solution B and stir and dialyze for 6-8 hours to obtain GUS thermophilic enzyme.

[0022] More preferably, the column equilibration buffer comprises 18–24 mM Tris-HCl buffer, 490–510 mM sodium chloride, and 4–6 mM imidazole; the washing buffer comprises 20–24 mM Tris-HCl buffer, 500–520 mM sodium chloride, and 100–120 mM imidazole; the concentration gradient elution buffer comprises 20–25 mM Tris-HCl buffer, 500–520 mM sodium chloride, and 150 / 200 / 500 mM imidazole; dialysate A comprises 20–24 mM Tris-HCl buffer, 500–520 mM sodium chloride, and 1–2 mM dithiothreitol; and dialysate B comprises 20–24 mM Tris-HCl buffer, 150–160 mM sodium chloride, and 1–2 mM dithiothreitol.

[0023] More preferably, step S103 specifically includes: The corresponding engineered expression strains were activated on LB plates containing Kan antibiotic and chloramphenicol resistance, cultured overnight at 36-37°C, and single clones were picked and inoculated into liquid LB medium containing Kan antibiotic and chloramphenicol for further overnight culture. Transfer the overnight cultured monoclonal strain to liquid LB medium containing Kansin antibiotics and chloramphenicol, and incubate at 35–37°C and 200–220 rpm until OD500. 600 Adjust the concentration to 0.6–0.8, then add an inducer and continue culturing at 15–16°C and 200–220 rpm for 16–18 h. Finally, centrifuge at 8000–8500 rpm for 10–15 min, discard the supernatant, collect the precipitated cells, and store at -80–-70°C.

[0024] By adopting the above technical solution, the present invention first introduces the corresponding restriction site through primer amplification, then uses the GUS gene coding sequence as the target fragment and performs double digestion with the linearized pET28a(+) vector, and uses T4 phage DNase to ligate the vector and the target fragment. Then, positive clone recombinant plasmids are extracted from the obtained recombinant plasmids and verified by restriction endonuclease double digestion electrophoresis. The obtained recombinant plasmids with correct enzyme digestion are sequenced to confirm that there is no mutation in the inserted sequence of the target fragment. Subsequently, they are transformed into Escherichia coli Rosetta(DE3) competent cells to obtain four corresponding engineered expression strains.

[0025] Secondly, the obtained engineered expression strains are cultured. The addition of an inducer can induce the transcription process of the lactose operon, thereby inducing the expression of the protein corresponding to the downstream gene of the lactose operon, and thus obtaining bacterial cells enriched with the target enzyme.

[0026] Finally, the obtained bacterial cells were subjected to lysis, column loading, impurity removal, elution, and dialysis. Tris-HCl buffer effectively stabilized the pH value, sodium chloride eliminated the adsorption of electrostatic impurities during the process, and imidazole competed for nickel column sites. Different concentration differences enabled the elution, purification, and collection of GUS thermophilic enzymes.

[0027] Preferably, the enzyme-catalyzed production process of free bilirubin includes the following steps: S201. Take pig bile and filter it. Adjust the pH of the filtrate to 5-5.5 using acetic acid. Add an antioxidant and sonicate at 30-32℃ for 20-30 minutes to obtain pretreated pig bile. S202. Add GUS thermophilic enzyme to pretreated porcine bile to adjust OD. 600 The concentration was 10–10.2, and the mixture was stirred to carry out enzyme-catalyzed conversion and hydrolysis to obtain a hydrolyzed pig bile suspension; S203. Adjust the pH of the hydrolyzed pig bile suspension to 6.5-7, add chloroform for extraction, and then rotary evaporate to obtain crude bilirubin; add crude bilirubin to boiling anhydrous ethanol and dissolve it, filter and dry to obtain free bilirubin.

[0028] Preferably, the mass-to-volume ratio of GUS thermophilic enzyme to pretreated porcine bile is (1-1.2) g: 10 mL.

[0029] By adopting the above technical solution, the present invention applies the obtained GUS thermophilic enzyme to the process of extracting free bilirubin from pig bile. Due to the characteristics of GUS thermophilic enzyme, pig bile can be converted and hydrolyzed at high temperature, thereby effectively avoiding the problems of high bile viscosity, low substrate conversion rate and low product yield caused by colloidal encapsulation of substrate in conventional room temperature environment. In addition, high temperature can effectively inhibit endogenous enzymes and bacteria in bile, avoid product decomposition, and obtain high purity and high yield of free bilirubin.

[0030] The beneficial effects of this invention are: 1. This invention provides a GUS thermophilic enzyme, including ACI185 GUS thermophilic enzyme, HOJ9 GUS thermophilic enzyme, MFN3 GUS thermophilic enzyme, and PVN8 GUS thermophilic enzyme, which can be used for enzymatic hydrolysis to prepare free bilirubin. This GUS thermophilic enzyme can achieve the highest enzyme activity at high temperatures, has good thermal stability, and can specifically hydrolyze the glucuronide bonds of the substrate, removing hydrophilic groups to generate lipid-soluble free bilirubin. It not only exhibits high specificity but also shows a significant improvement in catalytic efficiency at high temperatures. This significantly increases the conversion rate, reduces byproducts, and shortens the reaction cycle, effectively overcoming the shortcomings of existing traditional processes.

[0031] 2. The present invention provides a production process for the enzyme-catalyzed preparation of free bilirubin, wherein the enzyme-catalyzed conversion hydrolysis temperature is controlled at 70-90℃ and the pH value is 4-5. Under these high-temperature conditions, the viscosity of bile can be significantly reduced, the fluidity can be greatly improved, the colloid encapsulation of the substrate can be destroyed, and the substrate molecules can be fully exposed, thereby improving the conversion rate of the substrate and the yield of the product. At the same time, under these high temperatures, endogenous enzymes and bacteria can be effectively inhibited and killed, the purity of the product can be improved, and the decomposition of the product can be inhibited, thereby protecting the stability of free bilirubin and greatly reducing the impact of the external environment on the production process. Attached Figure Description

[0032] Figures 1-4 These are plasmid maps of the ACI185 GUS thermophilic enzyme, HOJ9 GUS thermophilic enzyme, MFN3 GUS thermophilic enzyme, and PVN8 GUS thermophilic enzyme obtained in Example 1 of this invention. Figures 5-8 These are schematic diagrams showing the SDS-PAGE analysis results of the ACI185 GUS thermophilic enzyme, HOJ9 GUS thermophilic enzyme, MFN3 GUS thermophilic enzyme and PVN8 GUS thermophilic enzyme obtained in Example 2 of the present invention. Figure 9 and Figure 10 These are schematic diagrams showing the results of the pH gradient test and pH stability test of GUS thermophilic enzyme in Example 3 of the present invention. Figure 11 and Figure 12 These are schematic diagrams showing the results of the temperature gradient test and thermal stability test of the GUS thermophilic enzyme in Example 3 of the present invention. Figure 13 This is a schematic diagram of the test results of the effect of metal ions on the enzyme activity of GUS thermophilic enzyme in Example 4 of the present invention; Figure 14 This is a high-performance liquid chromatogram of free bilirubin obtained in Example 5 of the present invention; Figure 15 This is a high-performance liquid chromatogram of free bilirubin obtained in Example 6 of the present invention; Figure 16 This is a high-performance liquid chromatogram of free bilirubin obtained in Example 7 of the present invention; Figure 17 This is a high-performance liquid chromatogram of free bilirubin obtained in Example 8 of the present invention; Figure 18 This is a high-performance liquid chromatogram of free bilirubin obtained in Comparative Example 1 of this invention; Figure 19 This is a high-performance liquid chromatogram of a bilirubin standard sample. Detailed Implementation

[0033] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0034] Example Example 1: A GUS thermophilic enzyme was prepared according to the following method: S101. Using the sequences ACI185, HOJ9, MFN3, and PVN8 as target fragments, and pET28a(+) as the recombinant expression vector, recombinant plasmids were obtained by ligating the target fragments and the recombinant expression vector using T4 phage DNase. The recombinant plasmids of positive clones were selected using Kan resistance screening, and then verified by restriction endonuclease double digestion electrophoresis. Sequencing confirmed the absence of inserted sequence mutations. The sequences of ACI185 GUS thermophilic enzyme, HOJ9 GUS thermophilic enzyme, MFN3 GUS thermophilic enzyme, and PVN8 GUS thermophilic enzyme corresponded to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively. The maps of their recombinant plasmids are shown below. Figures 1 to 4 As shown; S102. The recombinant plasmid was transformed into Escherichia coli Rosetta(DE3) competent cells to obtain the corresponding engineered expression strain; S103. The corresponding engineered expression strain was activated and cultured. Specifically, the corresponding engineered expression strain was activated on LB agar plates containing Kansas antibiotic and chloramphenicol resistance, and cultured overnight at 37°C. Single clones were picked and inoculated into liquid LB medium containing Kansas antibiotic and chloramphenicol for further overnight culture. Then, the single clones cultured overnight were transferred to liquid LB medium containing Kansas antibiotic and chloramphenicol at a 1% inoculation rate and cultured at 37°C and 220 rpm until OD500. 600 The concentration was increased to 0.8, and then 0.5 mM of the inducer isopropyl-β-D-thiogalactoside was added. The mixture was cultured at 16℃ and 220 rpm for 16 h. Finally, the mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and four types of precipitated bacterial cells were collected and stored at -80℃. The liquid LB medium containing Kan antibiotic and chloramphenicol had a Kan antibiotic content of 50 μg / mL and a chloramphenicol content of 50 μg / mL. S104. The precipitated bacterial cells were lysed. Specifically, the four types of precipitated bacterial cells obtained above were added to lysis buffer, which consisted of 20 mM Tris-HCl buffer (pH 8.0), 500 mM sodium chloride, and 5 mM imidazole. The mass-to-volume ratio of precipitated bacterial cells to lysis buffer was 1 g: 10 mL. After resuspending the cells by shaking until no obvious clumps remained, the cells were sonicated and then centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was collected as the crude enzyme solution. The solution was then loaded onto a column for impurity removal and elution. Finally, the eluent was collected and dialyzed to obtain the GUS thermophilic enzyme. Specifically: Add 5 column volumes of column equilibration buffer to the nickel column. The equilibration buffer consists of 20 mM Tris-HCl buffer (pH 8.0), 500 mM sodium chloride, and 5 mM imidazole. Filter the crude enzyme solution through a 0.22 μM filter membrane. Purify the supernatant (crude enzyme solution) by loading it onto the column. Then, add 5 column volumes of washing buffer (20 mM Tris-HCl buffer, 500 mM sodium chloride, and 100 mM imidazole) to remove contaminating proteins from the column. Finally, elute with 5 column volumes of elution buffer following a concentration gradient. The gradient elution buffer consisted of 20 mM Tris-HCl buffer, 500 mM sodium chloride, and 150 / 200 / 500 mM imidazole. The eluent was collected and added to a dialysis bag. Dialysis solution A, consisting of 20 mM Tris-HCl buffer, 500 mM sodium chloride, and 1 mM dithiothreitol, was added. Dialysis was performed overnight with stirring. The solution was then replaced with dialysis solution B, consisting of 20 mM Tris-HCl buffer, 150 mM sodium chloride, and 1 mM dithiothreitol. After dialysis for 7 hours with stirring, GUS thermophiles were obtained, namely ACI185 GUS thermophile, HOJ9 GUS thermophile, MFN3 GUS thermophile, and PVN8 GUS thermophile.

[0035] Example 2, SDS-PAGE electrophoresis verification assay of a GUS thermophilic enzyme: Samples of ACI185 GUS thermophilic enzyme, HOJ9 GUS thermophilic enzyme, MFN3 GUS thermophilic enzyme, and PVN8 GUS thermophilic enzyme were respectively subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) electrophoresis for verification. Coomassie brilliant blue staining was used for staining. The detection results are as follows: Figures 5 to 8 As shown in the figure. Furthermore, by comparing the protein electrophoresis bands, the molecular weight of the GUS thermophilic enzyme is 6.5–7.5 kDa.

[0036] Well M contains the protein marker, Well 1 contains the total GUS thermophilic enzyme lysate, Well 2 contains the supernatant of the GUS thermophilic enzyme lysate, Well 3 contains the precipitate of the GUS thermophilic enzyme lysate, Well 4 contains the flow-through buffer, and Wells 5 to 9 contain 50 mM imidazole elution buffer, 100 mM imidazole elution buffer, 150 mM imidazole elution buffer, 200 mM imidazole elution buffer, and 500 mM imidazole elution buffer, respectively; the other components of the elution buffer are 20 mM Tris-HCl buffer and 500 mM sodium chloride.

[0037] Example 3, determination of the enzymatic hydrolysis temperature and pH value of a GUS thermophilic enzyme: 1. pH value measurement: Using pNPG (p-nitrobenzene-β-D-galactopyranoside) as a substrate, the pH value was measured at an enzymatic hydrolysis temperature of 85℃. Specifically, 10 μL of GUS thermophilic enzyme was accurately pipetted into 40 μL of 50 mM acetate-sodium acetate buffer at pH values ​​of 4.0, 4.5, 5.0, 5.5, and 6.0, and 50 mM Tris-HCl buffer at pH values ​​of 6.5, 7.0, 7.5, and 8.0, respectively. 1.25 mM of pNPG substrate was added to the buffer, and the reaction was stopped after 10 min by adding 200 μL of 0.4 M sodium carbonate solution. The enzyme activity of GUS thermophilic enzyme was then measured. The test results are shown below. Figure 9 As shown; Using the same method, buffer solutions containing different pH gradients of GUS thermophilic enzyme were incubated on ice for 30 min. Then, 1.25 mM pNPG substrate was added, and the reaction was continued for 10 min. Finally, 200 μL of 0.4 M sodium carbonate solution was added to terminate the reaction. The residual activity of the GUS thermophilic enzyme was measured to verify its pH stability. The test results are as follows: Figure 10 As shown.

[0038] The methods for determining enzyme activity mainly include: using different concentrations of p-nitrophenol dissolved in 50mM acetate-sodium acetate buffer as a standard curve, hydrolyzing pNPG substrate, and quantitatively testing p-nitrophenol at 405nm using a spectrophotometer to determine the enzyme activity of GUS thermophilic enzyme.

[0039] 2. Determination of enzymatic hydrolysis temperature: Using pNPG (p-nitrobenzene-β-D-galactopyranoside) as a substrate, the enzymatic hydrolysis temperature was determined at pH 5.0. Specifically, 10 μL of GUS thermophilic enzyme was accurately pipetted into 40 μL of 50 mM acetate-sodium acetate buffer at pH 5.0, and 1.25 mM of pNPG substrate was added to the buffer. The reaction temperatures were adjusted to 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, and 90℃, respectively. After reacting for 10 min at each temperature, 200 μL of 0.4 M sodium carbonate solution was added to terminate the reaction. The enzyme activity of GUS thermophilic enzyme was then measured. The test results are shown below. Figure 11 As shown; Using the same method, 50 mM acetate-sodium acetate buffer (pH 5.0) containing GUS thermophilic enzyme was incubated for 30 min at different temperature gradients. After incubation, the enzyme was cooled on ice, then 1.25 mM pNPG substrate was added, and the reaction was allowed to proceed for 10 min. Finally, 200 μL of 0.4 M sodium carbonate solution was added to terminate the reaction. The residual activity of the GUS thermophilic enzyme was measured to verify its thermostability. The test results are shown below. Figure 12 As shown.

[0040] according to Figure 9 and Figure 10 It can be seen that the activity of GUS thermophilic enzyme shows an increasing trend at pH 4 to 5, and the activity of GUS thermophilic enzyme reaches the highest value of 98.4 U / μL at pH 5.0. Furthermore, under pH 4 to 5 conditions, GUS thermophilic enzyme can maintain relatively stable enzyme activity for a long time.

[0041] according to Figure 11 and Figure 12 It can be seen that the enzyme activity of GUS thermophilic enzyme remains high at temperatures of 70-90℃ and can reach the maximum enzyme activity. At lower temperatures, the enzyme activity decreases. At the same time, it can exhibit good thermal stability and can maintain high enzyme activity for a long time under high temperature conditions.

[0042] Example 4, Test on the effect of metal ions on the enzyme activity of GUS thermophilic enzyme: Prepare Fe separately 2+ Mg 2+ Zn 2+ K + Fe 3+ Mn 2+ Ca 2+ Cu 2+ and Na +An ionic solution was prepared by adding GUS thermophilic enzyme solution to the solution and mixing until the ionic concentration reached 2 mM. The mixture was then incubated on ice for 30 min. The residual enzyme activity was measured at pH 5.0 and 85°C. A fire-extinguished GUS thermophilic enzyme solution was used as a control group (CK). Each group was tested three times. The test results are as follows: Figure 13 As shown.

[0043] according to Figure 13 It can be seen that the catalytic activity of GUS thermophilic enzymes is affected to some extent by metal ions, among which Fe... 2+ and Fe 3+ It has a significant inhibitory effect on ACI185 GUS thermophilic enzyme, while Zn 2+ Ca 2+ Cu 2+ Na + and K + It has a significant promoting effect on ACI185 GUS thermophilic enzyme.

[0044] Example 5: A process for producing free bilirubin via enzyme catalysis, comprising the following steps: S201. Take 1L of pig bile (total bilirubin concentration of 0.56mg / mL) and filter it. Adjust the pH of the filtrate to 5.0 using acetic acid. Add an antioxidant, namely sodium bisulfite with a concentration of 0.3%. Sonicate at 30℃ for 20min with an ultrasonic power of 3200W and 70Hz to obtain pretreated pig bile. S202. The ACI185 GUS thermophilic enzyme prepared in Example 1 was added to the pretreated porcine bile at a mass-to-volume ratio of 1g:10mL, and the OD was adjusted. 600 The concentration was 10. Under the enzymatic hydrolysis catalytic conditions of pH 5.0 and temperature 85℃, the enzyme catalytic conversion hydrolysis was carried out by stirring at 200 rpm for 0.5 h to obtain a hydrolyzed pig bile suspension. S203. Adjust the pH of the hydrolyzed pig bile suspension to 6.5, add 50% chloroform for extraction. Specifically, stir for 3 min, let stand for 10 min, stir again for 3 min, let stand for 1 h, and use a separatory funnel to separate the layers. Repeat twice, and then evaporate the lower layer liquid by rotary evaporation at 80℃ to obtain crude bilirubin. Add the crude bilirubin to boiling anhydrous ethanol and dissolve it. After filtration and drying, free bilirubin is obtained.

[0045] Example 6: A process for producing free bilirubin via enzyme catalysis, comprising the following steps: S201. Take 1L of pig bile (total bilirubin concentration of 0.56mg / mL) and filter it. Adjust the pH of the filtrate to 5.0 using acetic acid. Add an antioxidant, namely sodium bisulfite with a concentration of 0.3%. Sonicate at 30℃ for 20min with an ultrasonic power of 3200W and 70Hz to obtain pretreated pig bile. S202. The HOJ9 GUS thermophilic enzyme prepared in Example 1 was added to the pretreated porcine bile at a mass-to-volume ratio of 1g:10mL, and the OD was adjusted. 600 The concentration was 10. Under the enzymatic hydrolysis catalytic conditions of pH 5.0 and temperature 75℃, the enzyme catalytic conversion hydrolysis was carried out by stirring at 200 rpm. The hydrolysis reaction was carried out for 1 hour to obtain a hydrolyzed pig bile suspension. S203. Adjust the pH of the hydrolyzed pig bile suspension to 6.5, add 50% chloroform for extraction. Specifically, stir for 3 min, let stand for 10 min, stir again for 3 min, let stand for 1 h, and use a separatory funnel to separate the layers. Repeat twice, and then evaporate the lower layer liquid by rotary evaporation at 80℃ to obtain crude bilirubin. Add the crude bilirubin to boiling anhydrous ethanol and dissolve it. After filtration and drying, free bilirubin is obtained.

[0046] Example 7, an enzyme-catalyzed production process for free bilirubin, comprising the following process steps: S201. Take 1L of pig bile (total bilirubin concentration of 0.56mg / mL) and filter it. Adjust the pH of the filtrate to 5.0 using acetic acid. Add an antioxidant, namely sodium bisulfite with a concentration of 0.3%. Sonicate at 30℃ for 20min with an ultrasonic power of 3200W and 70Hz to obtain pretreated pig bile. S202. The MFN3 GUS thermophilic enzyme prepared in Example 1 was added to the pretreated porcine bile at a mass-to-volume ratio of 1g:10mL, and the OD was adjusted. 600 The concentration was 10. Under the enzymatic hydrolysis catalytic conditions of pH 5.0 and temperature 75℃, the enzyme catalytic conversion hydrolysis was carried out by stirring at 200 rpm. The hydrolysis reaction was carried out for 1 hour to obtain a hydrolyzed pig bile suspension. S203. Adjust the pH of the hydrolyzed pig bile suspension to 6.5, add 50% chloroform for extraction. Specifically, stir for 3 min, let stand for 10 min, stir again for 3 min, let stand for 1 h, and use a separatory funnel to separate the layers. Repeat twice, and then evaporate the lower layer liquid by rotary evaporation at 80℃ to obtain crude bilirubin. Add the crude bilirubin to boiling anhydrous ethanol and dissolve it. After filtration and drying, free bilirubin is obtained.

[0047] Example 8: A process for producing free bilirubin via enzyme catalysis, comprising the following steps: S201. Take 1L of pig bile (total bilirubin concentration of 0.56mg / mL) and filter it. Adjust the pH of the filtrate to 5.0 using acetic acid. Add an antioxidant, namely sodium bisulfite with a concentration of 0.3%. Sonicate at 30℃ for 20min with an ultrasonic power of 3200W and 70Hz to obtain pretreated pig bile. S202. The PVN8 GUS thermophilic enzyme prepared in Example 1 was added to the pretreated porcine bile at a mass-to-volume ratio of 1g:10mL, and the OD was adjusted. 600 The concentration was 10. Under the enzymatic hydrolysis catalytic conditions of pH 5.0 and temperature 75℃, the enzyme catalytic conversion hydrolysis was carried out by stirring at 200 rpm. The hydrolysis reaction was carried out for 1 hour to obtain a hydrolyzed pig bile suspension. S203. Adjust the pH of the hydrolyzed pig bile suspension to 6.5, add 50% chloroform for extraction. Specifically, stir for 3 min, let stand for 10 min, stir again for 3 min, let stand for 1 h, and use a separatory funnel to separate the layers. Repeat twice, and then evaporate the lower layer liquid by rotary evaporation at 80℃ to obtain crude bilirubin. Add the crude bilirubin to boiling anhydrous ethanol and dissolve it. After filtration and drying, free bilirubin is obtained.

[0048] Comparative Example Comparative Example 1: A production process for free bilirubin using chemical saponification, comprising the following steps: 1 L of pig bile (total bilirubin concentration of 0.56 mg / mL) was filtered, defatted with a mixture of petroleum ether and double-layered gauze, and then saponified after antioxidant treatment with pyrogallol. Specifically, sodium hydroxide solid was added to adjust the pH of the bile to 11, and the reaction was stirred at 100°C for 10 min to achieve full saponification. The temperature was then lowered to 50°C and acidified with sulfuric acid to a pH of 5.0. Finally, free bilirubin was obtained by extraction, distillation, washing and drying.

[0049] Performance testing The mass of free bilirubin obtained in Examples 5 to 8 and Comparative Example 1 was measured, and the yield of free bilirubin was calculated. High-performance liquid chromatography (HPLC) was used to test the bilirubin standard samples and the obtained free bilirubin to compare purity. The experimental results are shown in Table 1. Figures 14-19 As shown.

[0050] Table 1. Yield and production rate of free bilirubin

[0051] The above-disclosed embodiments are merely a few specific examples of the present invention. However, the embodiments of the present invention are not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A production process for the enzyme-catalyzed preparation of free bilirubin, characterized in that, The enzyme-catalyzed preparation of free bilirubin utilizes GUS thermophilic enzyme, with porcine bile as a substrate, to obtain free bilirubin through enzymatic conversion and hydrolysis. The GUS thermophilic enzymes include one or more combinations of ACI185 GUS thermophilic enzyme, HOJ9 GUS thermophilic enzyme, MFN3 GUS thermophilic enzyme and PVN8 GUS thermophilic enzyme; The nucleotide sequences of the ACI185 GUS thermophilic enzyme are shown in SEQ ID NO:1, the HOJ9 GUS thermophilic enzyme are shown in SEQ ID NO:2, the MFN3 GUS thermophilic enzyme are shown in SEQ ID NO:3, and the PVN8 GUS thermophilic enzyme are shown in SEQ ID NO:

4.

2. The production process for enzyme-catalyzed preparation of free bilirubin according to claim 1, characterized in that, The enzyme-catalyzed transformation and hydrolysis is carried out at a temperature of 70–90°C and a pH value of 4–5.

3. The production process for enzyme-catalyzed preparation of free bilirubin according to claim 1, characterized in that, The GUS thermophilic enzyme was prepared according to the following method: S101. Using the GUS gene coding sequence as the target fragment and pET28a(+) as the recombinant expression vector, the target fragment and the recombinant expression vector were ligated using T4 phage DNase to obtain the recombinant plasmid. S102. The recombinant plasmid was transformed into Escherichia coli Rosetta(DE3) competent cells to obtain the corresponding engineered expression strain; S103. After the corresponding engineered expression strain is activated and cultured, it is inoculated into liquid LB medium containing Kan antibiotic and chloramphenicol, an inducer is added, and the culture is continued for 16-18 hours. Then, the precipitated bacterial cells are collected by centrifugation. S104. The precipitated bacterial cells are lysed, loaded onto a column, purified, and eluted. The eluent is then collected and dialyzed to obtain GUS thermophilic enzyme.

4. The production process for enzyme-catalyzed preparation of free bilirubin according to claim 3, characterized in that, The GUS gene coding sequence includes any one of ACI185, HOJ9, MFN3, and PVN8.

5. The production process for enzyme-catalyzed preparation of free bilirubin according to claim 3, characterized in that, The liquid LB medium containing Kan antibiotic and chloramphenicol had a Kan antibiotic content of 45–55 μg / mL and a chloramphenicol content of 45–55 μg / mL; the inoculum size was 1–1.1%.

6. The production process for enzyme-catalyzed preparation of free bilirubin according to claim 3, characterized in that, The inducing agent includes isopropyl-β-D-thiogalactoside at a concentration of 0.4–0.5 mM.

7. The production process for enzyme-catalyzed preparation of free bilirubin according to claim 3, characterized in that, The pyrolysis in step S104 mainly includes the following steps: Add the precipitated bacterial cells to the lysis buffer, vortex and resuspend until there are no obvious clumps of bacterial cells, then sonicate to disrupt the cells, and centrifuge at 4-5℃ and 8000-8500 rpm for 10-15 min, and collect the supernatant.

8. The production process for enzyme-catalyzed preparation of free bilirubin according to claim 7, characterized in that, The lysis buffer comprises 20–25 mM Tris-HCl buffer, 500–520 mM sodium chloride, and 5–8 mM imidazole; the mass-to-volume ratio of the precipitated bacterial cells to the lysis buffer is 1 g: (8–10) mL.

9. The enzyme-catalyzed production process of free bilirubin according to claim 1, characterized in that, The process includes the following steps: S201. Take pig bile and filter it. Adjust the pH of the filtrate to 5-5.5 using acetic acid. Add an antioxidant and sonicate at 30-32℃ for 20-30 minutes to obtain pretreated pig bile. S202. Add GUS thermophilic enzyme to pretreated porcine bile to adjust OD. 600 The concentration was 10–10.2, and the mixture was stirred to carry out enzyme-catalyzed conversion and hydrolysis to obtain a hydrolyzed pig bile suspension; S203. Adjust the pH of the hydrolyzed pig bile suspension to 6.5-7, add chloroform for extraction, and then rotary evaporate to obtain crude bilirubin; add crude bilirubin to boiling anhydrous ethanol and dissolve it, filter and dry to obtain free bilirubin.

10. The enzyme-catalyzed production process of free bilirubin according to claim 9, characterized in that, The mass-to-volume ratio of the GUS thermophilic enzyme to the pretreated porcine bile is (1-1.2) g: 10 mL.

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