A heme oxygenase mutant and use thereof
By modifying the structure of the heme oxygenase mutant HOT0 and fusing it with a soluble tag, the catalytic efficiency was improved, the problem of insufficient catalytic capacity of heme oxygenase was solved, and the efficient preparation of bilirubin was achieved, which is suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GENERAL HOSPITAL OF NUCLEAR IND
- Filing Date
- 2026-05-21
- Publication Date
- 2026-06-16
AI Technical Summary
In existing technologies, the catalytic capacity of heme oxygenase is limited, which makes the first step of porphyrin epoxidation and cleavage in the process of biliverdin to bilirubin a rate-limiting step, resulting in insufficient catalytic efficiency and difficulty in meeting the needs of industrial applications.
By remodeling the heme oxygenase mutant HOT0, introducing specific amino acid mutations (M29S, L33V, V142N, M146Y, H205T), and combining it with the soluble tag SUMO, the catalytic efficiency of the enzyme was improved.
The enzyme activity of the heme oxygenase mutant HOT5 was significantly increased, with the catalytic efficiency improved to 2.1 times the original level. Furthermore, the enzyme activity was further improved to 4.22 times through fusion with the SUMO tag, thus realizing the efficient and green synthesis of bilirubin, which is suitable for large-scale industrial production.
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Figure CN122214293A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biocatalysis technology, and in particular to a heme oxygenase mutant and its applications. Background Technology
[0002] Heme oxygenase (HO) is a key enzyme in the metabolism of heme, catalyzing the degradation of heme into biliverdin (BV), carbon monoxide (CO), and iron ions (Fe). 2+ Biliverdin, an intermediate product of heme metabolism, plays a vital role in regulating and maintaining cellular homeostasis. Numerous studies have demonstrated its beneficial effects, such as cell protection, anti-inflammatory effects, and antioxidant effects. It can be further converted into bilirubin by biliverdin reductase, ultimately participating in bile excretion and contributing to the body's antioxidant and anti-inflammatory physiological processes.
[0003] In recent years, the biological functions of bilirubin have expanded from its traditional understanding as a metabolic waste product to its status as an important endogenous bioactive molecule. Particularly in reproductive medicine, research has shown that bilirubin plays a crucial regulatory role in the reproductive system. Physiological concentrations of bilirubin can protect testicular Sertoli cells and spermatogenic cells from oxidative stress damage through their antioxidant properties, maintaining the stability of spermatogenesis. In the ovary, bilirubin participates in follicle development and corpus luteum function regulation, affecting female reproductive endocrine homeostasis. Abnormal expression of bilirubin metabolism-related enzymes in placental tissue is closely related to pregnancy complications such as preeclampsia and recurrent miscarriage. Therefore, the bilirubin metabolic pathway not only affects the body's overall redox state but also plays a vital role in maintaining reproductive system function and in the development of reproductive-related diseases.
[0004] Currently, biliverdin and bilirubin, as two compounds closely related in biological metabolism, are widely used in the fields of medicine, health products, cosmetics, and biochemical research, with a huge market demand for both. Among various production methods for biliverdin and bilirubin, the enzymatic method exhibits advantages such as mild reaction conditions, high optical purity of the product, simple operation, and economic applicability, showing promising prospects for industrial application. In the two-step biochemical reaction catalyzed by enzymes, heme undergoes oxidative cleavage of the α-methylene bridge on its porphyrin ring under the catalysis of heme oxygenase, opening the ring to generate biliverdin IXα with a linear tetrapyrrole structure. This step usually requires the participation of NADPH. Subsequently, biliverdin is hydrogenated and reduced by biliverdin reductase to generate bilirubin. The oxidative cleavage of the α-methylene bridge on the porphyrin ring in the first step is the rate-limiting step of the entire heme degradation reaction, while the catalytic ability of heme oxygenase is very limited under natural conditions.
[0005] Chinese patent "A Heme Oxygenase Mutant and Its Application" (publication number CN118126970A) screened out a highly resistant Corynebacterium (Corynebacterium sp. The heme oxygenase mutant derived from [source] can exhibit good catalytic effects in the free state, but it only represents a preliminary enzyme engineering modification of heme oxygenase, and its industrial application prospects are still not ideal, with catalytic ability needing further improvement. Summary of the Invention
[0006] Objective: In order to overcome the shortcomings of the existing technology, the present invention provides a heme oxygenase mutant and its application, which improves catalytic efficiency and enhances the efficiency of industrial applications.
[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0008] In a first aspect, the present invention provides a heme oxygenase mutant HO T5 The heme oxygenase mutant HO T5 The amino acid sequence of heme oxygenase, as shown in SEQ ID NO.1, is obtained by mutating methionine at position 29 to proline, leucine at position 33 to phenylalanine, valine at position 142 to glycine, methionine at position 146 to lysine, and histidine at position 205 to threonine. The heme oxygenase mutant HO T5 The amino acid sequence is shown in SEQ ID NO.3.
[0009] Based on the final experimental results of Chinese Patent "A Heme Oxygenase Mutant and Its Application" (Publication No. CN118126970A), this invention selected the heme oxygenase mutant M6, which exhibited the best catalytic effect, for further analysis and research. In this invention, the heme oxygenase mutant M6 was renamed heme oxygenase mutant HO. T0 Experiments were conducted. This invention constructed a structural remodeling mutant library locally in the substrate pocket through enzyme structure analysis and screened for the optimal mutant. Subsequently, by combining this with a strategy of adding soluble tags, a significant increase in enzyme activity was ultimately achieved.
[0010] In some embodiments, the heme oxygenase mutant HO T5 The N-terminus also includes a soluble tag selected from SUMO, TN11 and NUSA.
[0011] In some embodiments, the heme oxygenase mutant HO T5 The N-terminus also includes a soluble tag, which is SUMO, and the amino acid sequence of SUMO is shown in SEQ ID NO.8.
[0012] In some embodiments, the heme oxygenase mutant HOT5 The expression host is Escherichia coli BL21(DE3) competent cells.
[0013] Secondly, the present invention provides encoding the heme oxygenase mutant HO as described in the first aspect. T5 The gene, the DNA sequence of which is shown in SEQ ID NO.4.
[0014] Thirdly, the present invention provides a heme oxygenase mutant, wherein the heme oxygenase mutant is SUMO-HO. T5 Enzyme; the SUMO-HO T5 The amino acid sequence of the enzyme is shown in SEQ ID NO.5.
[0015] Fourthly, the present invention provides encoding as described in the third aspect, SUMO-HO. T5 The gene for the enzyme, the DNA sequence of which is shown in SEQ ID NO.6.
[0016] Fifthly, the present invention provides a heme oxygenase mutant HO as described in the first aspect. T5 Or as described in the third aspect, SUMO-HO T5 Application of enzymes in the preparation of bilirubin or products containing bilirubin.
[0017] In a sixth aspect, the present invention provides a method for preparing bilirubin, using the heme oxygenase mutant HO as described in the first aspect. T5 Or as described in the third aspect, SUMO-HO T5 The enzyme, the preparation method of which includes: Dissolve heme chloride in NaOH solution to obtain the reaction mother liquor; will HO T5 or SUMO-HO T5 After enzyme expression, the wet bacterial cells were mixed with PBS (pH=7.5) to lyse the cells and obtain the lysed enzyme solution. The supernatant of the lysed enzyme solution was collected by centrifugation. Add the supernatant of the cytolytic enzyme solution, crude biliverdin reductase powder, and NADP to the reaction system. + Coenzyme, glucose, crude GDH enzyme powder, and PBS buffer are added to the reaction system via feedstock, and the reaction proceeds to obtain the final product.
[0018] Biliverdin reductase catalyzes the conversion of biliverdin to bilirubin, and this catalysis requires the participation of NADPH. Since NADPH is relatively expensive, glucose dehydrogenase (GDH) is introduced, along with a small amount of NADP. + NADPH can then be recycled and regenerated.
[0019] In some embodiments, the concentration of the mother liquor is 8-12 g / L, and the concentration of the NaOH solution is 0.15-0.25 M; The mass ratio of the wet bacterial cells to PBS is 1:1.5-2; The volume ratio of the cytolytic enzyme supernatant to the PBS buffer is 1:1-1.2; The crude biliverdin reductase dry powder, NADP + The mass ratio of coenzyme, glucose, and crude GDH enzyme powder is (4.5-5.5):(1-1.2):(4.5-5.5):(4.5-5.5). The flow rate is 0.15-0.3 mL / h, and the flow duration is 18-22 h; The final concentration of the mother liquor in the reaction system is 0.8-1.2 g / L; The reaction conditions are 25-35℃, stirring at 280-320 rpm, and a total reaction time of 24-25 h.
[0020] Beneficial effects: The heme oxygenase mutant SUMO-HO provided by this invention T5 With high enzyme activity and a simple process for preparing bilirubin based on biliverdin, it achieves efficient and green synthesis of bilirubin, which can be promoted to large-scale industrial production. Attached Figure Description
[0021] Figure 1 In the embodiments of the present invention, heme oxygenase HO T0 Schematic diagram of structural modeling and analysis.
[0022] Figure 2 SUMO-HO is an embodiment of the present invention. T5 A map of gene expression vectors.
[0023] Figure 3 This is a gel image comparing the expression effects of heme oxygenase fused with the SUMO tag in an embodiment of the present invention.
[0024] Figure 4 The images show the HPLC detection chromatograms of biliverdin and bilirubin in this embodiment of the invention. Detailed Implementation
[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use.
[0026] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may include different values. It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.
[0027] The present invention will be further described below with reference to the embodiments.
[0028] In the following examples, the materials and solvents used were sourced from sources including: *Escherichia coli* (…). Escherichia coli BL21(DE3) competent cells, culture dishes, and 96-well plates were all purchased from Sangon Biotech (Shanghai) Co., Ltd. LB medium components: 1% tryptone; 1% NaCl; 0.5% yeast extract; 1.5% agar. The medium was added to solid medium and sterilized at 121℃ for 20 min before use.
[0029] Solution reagents: 0.1M PBS buffer (pH 7.5); kanamycin sulfate, isopropyl-β-D-thiogalactoside (IPTG), heme chloride, biliverdin, bilirubin, NADP + Both glucose and glucose were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0030] Gene synthesis, primer synthesis, and mutant sequencing were completed by Genewiz (Suzhou) Biotechnology Co., Ltd.
[0031] Example 1: Heme oxygenase mutant HO T5 Construction
[0032] This embodiment provides a heme oxygenase mutant HO T5 .
[0033] I. Heme oxygenase mutant HO T0 The construction.
[0034] Heme oxygenase mutant HO T0The amino acid sequence is shown in SEQ ID NO.1. The amino acid sequence shown in SEQ ID NO.1 was optimized into the gene sequence SEQ ID NO.2 according to the codons of the E. coli expression system. This gene was then synthesized and loaded between the Nde I and Xhol I restriction sites of the pET-24a expression vector to obtain pET24a-HO. T0 Plasmid.
[0035] The synthesized pET24a-HO T0 The plasmid was transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing a final concentration of 50 mg / L kanamycin resistance, and incubated at 37°C for 12 h. The cells were then transferred to fresh LB liquid shake flasks and cultured at 220 rpm and 37°C until OD (outcome limit). 600 IPTG was added to the culture medium to a final concentration of 0.1 mM when the concentration reached 0.6-1.0. Expression was induced at 25℃ and 220 rpm for 16 h. Wet cells were collected by centrifugation at 5000 r / min and used as the HO (Heterotrophic Oxygenation) assay. T0 Crude enzyme sample.
[0036] II. Construction of a Heme Oxygenase Mutant Library
[0037] Input the SEQ ID NO.1 sequence into the HO using the SWISS-MODEL tool. T0 Homology modeling of the protein structure was performed (template PDB ID: 1iw1.3.A, GMQE=0.97, Identity=96.74), and the results were visualized using PyMOL software. Figure 1 As shown, the model already includes heme ligand molecules that coordinate with H2O. According to sequence conservation analysis (using the tool https: / / consurf.tau.ac.il / consurf_index.php), the amino acid residues distributed in the middle and left pockets of the ligand in the figure are generally highly conserved, responsible for axial positioning and electron transfer; while the amino acid residues in the right pocket region of the ligand have lower conservation. Figure 1 The area inside the dashed box is roughly divided into regions, which are suitable for pocket reshaping and modification.
[0038] The CAVER 3.0 plugin within the Pymol tool was used to perform channel mining on this non-conservative region, and all mutable amino acids involved were identified as H25, M29, S30, D31, L33, V142, M146, H162, and H205.
[0039] According to the CAST (Combined Active Site Saturation Test) principle, M29 and L33, and V142 and M146 are spatially adjacent amino acid pairs in the side chain. Therefore, two saturation mutations were introduced simultaneously to construct libraries for each of them, and the remaining 5 amino acids were used to construct separate saturation mutation libraries.
[0040] Furthermore, with HO T Using the pET-24a plasmid DNA of the gene as a template, a pair of reverse primers were designed by replacing the original codons at key sites H25, M29 / L33, S30, D31, V142 / M146, H162, and H205 with NNK degenerate codons. Saturation mutations were introduced by PCR using the high-fidelity DNA polymerase primeSTARmax (purchased from TaKaRa Bio). The designed primers are shown in Table 1 below.
[0041] Table 1 Primers for constructing the heme oxygenase mutant library
[0042] PCR reaction system: total volume 20 μL; 1.5 μL each of forward and reverse primers, 2 μL template, 5 μL ddH2O, 10 μL 2X primeSTARmax.
[0043] PCR reaction program: 95℃ for 2 min; 95℃ for 20 s, 56℃ for 20 s, 72℃ for 40 s, repeat for 25 cycles; extend at 72℃ for 2 min.
[0044] The PCR products were treated with DpnI enzyme at 37°C for 1 h and then transformed into Escherichia coli BL21(DE3) competent cells. The cells were then plated on LB solid plates containing a final concentration of 50 mg / L kanamycin resistance and cultured at 37°C for 12 h to obtain 7 different saturation mutant libraries.
[0045] III. High-throughput screening of heme oxygenase mutant libraries
[0046] Directly using HPLC to detect substrates in large-scale mutant screening experiments is inefficient and has relatively weak detection capability for biliverdin products. Each reaction sample requires a large amount of enzyme, making it unsuitable for small-scale high-throughput screening systems. Therefore, a highly sensitive activity comparison and screening method developed by Xiaqing Zhu et al. of Tianjin University (Analytica Chimica Acta 1174 (2021) 338709) was adopted. The principle utilizes a simple and rapid near-infrared fluorescent protein, smURFP, for biliverdin detection as a fluorescent biosensor. Fluorescence is generated through spontaneous binding and covalent linkage with biliverdin, allowing detection by the detector. Within a biliverdin concentration of 25 μM, the fluorescence intensity is basically proportional to the biliverdin concentration. The specific process includes:
[0047] 1. Preparation of smURFP samples
[0048] The amino acid sequence smURFP as shown in SEQ ID NO.7 was synthesized and constructed into the pET-24a expression vector. Protein expression was induced using 0.5 mM IPTG. After harvesting the cells, they were resuspended in PBS (pH 7.5) buffer and subjected to high-pressure cell disruption. The resulting sample was then added with 0.5 M NaCl to obtain the smURFP sample.
[0049] 2. Orifice plate screening process
[0050] 1000 single colonies were selected from each CAST library and 100 single colonies from each single-point saturation mutant library. These were inoculated into 96-well plates containing LB medium with a final concentration of 50 mg / L kanamycin resistance and cultured overnight at 37°C and 220 rpm. A 10% inoculum was then transferred from the overnight culture to fresh 96-well plates containing 1 mL of LB medium with a final concentration of 50 mg / L kanamycin resistance. The plates were cultured with shaking at 37°C and 220 rpm for 3 h, followed by the addition of 0.1 mM IPTG and expression at 25°C for 16 h. The cells were then collected by centrifugation at 4000 rpm as crude enzyme for high-throughput screening.
[0051] Add 0.5 mL of 5 mg / L heme chloride (diluted with PBS (pH 7.5)) to the well plate, resuspend thoroughly, and incubate at 30°C with gentle shaking for 1 h. After centrifugation again, add 100 μL of the supernatant to a 96-well plate, then add the same 100 μL of smURFP sample and mix gently. Incubate in the dark at room temperature for 10 min, then use a PerkinElmer EnSpire plate. ®A multi-mode plate detector was used to measure the fluorescence intensity of all mutant samples (excitation wavelength: 642 nm; emission wavelength: 670 nm). Results were obtained starting with the mutant HO. T0 The relative activity was compared as 100%.
[0052] The optimal mutant for each library was obtained and validated three times in a shake-flask experiment. The results are shown in Table 2 below.
[0053] Table 2. Statistics on the relative activity of mutants
[0054] Subsequently, based on the mutant V142N / M146Y with the highest activity enhancement, mutation sites with an activity greater than 1 were selected and introduced into specific mutation sites sequentially using the same PCR construction method described above for iterative combinations. Specifically, V142N / M146Y was first combined with the remaining site H205T, which had the highest activity. If the activity increased compared to either of the two, it was retained, and this process was repeated for the next round of iterations; if there was no increase, the mutation site was discarded. Finally, the optimal mutant HO, containing five mutation sites (M29S, L33V, V142N, M146Y, H205T), was determined. T5 HO T5 The enzyme activity is HO T0 2.1 times the enzyme activity, HO T5 The amino acid sequence is shown in SEQ ID NO.3, HO T5 The DNA sequence is shown in SEQ ID NO.4.
[0055] Example 2: SUMO-HO T5 Enzyme construction
[0056] Because the helix content in the heme oxygenase protein structure is extremely high, it may increase the difficulty of prokaryotic expression. Therefore, this embodiment provides a SUMO-HO protein. T5 Enzymes, compared to HO T5 It increased the soluble expression level.
[0057] in HO T5 Common soluble tags SUMO, NT11, and NUSA are fused to the N-terminus of the protein sequence to enhance soluble expression. The three gene sequences are constructed upstream of the target gene by PCR.
[0058] This embodiment provides a method for using HO T5 SUMO-HO was constructed by fusing a soluble tag SUMO to the N-terminus of a protein sequence. T5 Enzymatic method: The amino acid sequence of the SUMO tag is shown in SEQ ID NO.8. Enzymatic methods are employed by designing a tag containing pET24a-HO. T5The homologous arm primers were used to amplify the DNA sequence of the SUMO protein from the cloning vector via PCR, and then this fragment was used as a large primer, pET24a-HO. T5 Using a vector as a template, the SUMO gene fragment was recombined into the start codon and HO region via PCR. T5 Between gene sequences (constructed expression vectors such as Figure 2 (As shown). Among them, pET24a-HO T5 The forward primer DNA sequence of the homologous arm is shown in SEQ ID NO.23, and the reverse primer DNA sequence is shown in SEQ ID NO.24.
[0059] Subsequently, after treatment with DpnI enzyme, the cells were transformed into E. coli BL21(DE3) competent cells for expression, yielding SUMO-HO. T5 Enzymes.
[0060] Activity comparisons showed that, compared to HO, the addition of SUMO, NT11, and NUSA tags resulted in [a significant improvement]. T5 The activities were 200%, 178%, and 33%, respectively, with the fusion of the SUMO tag showing the best effect, making it effective in HO. T5 It has been increased by 2 times on top of its activity.
[0061] The HO obtained in Example 1 T5 The induced expression cells served as a control. Cell disruption was performed using an ultrasonic disruptor, and the supernatant was directly collected after centrifugation for SDS-PAGE verification of expression. Figure 3 The protein gel results from the two expression supernatants show that, compared to unlabeled HO... T5 Enzyme (expected protein molecular weight approximately 24.2 KD), fused with SUMO-HO expressed using the SUMO tag. T5 The soluble expression level of the enzyme (protein molecular weight approximately 35.3 KD, its amino acid sequence is shown in SEQ ID NO.5, and its DNA sequence is shown in SEQ ID NO.6) was significantly improved.
[0062] Example 3: Detection of reaction products and comparative analysis of their activities
[0063] This embodiment provides SUMO-HO T5 Enzymes and HO T5 Methods and results of comparative analysis of enzyme activity.
[0064] Weigh an excess of the substrate heme chloride powder and dissolve it in 10 mL of 0.1 M NaOH solution as the stock solution (substrate concentration 10 g / L). Then dilute it 100 times and dissolve it in PBS buffer (pH 7.5) to achieve a final concentration greater than 0.1 g / L. Subsequently, add SUMO-HO at a mass ratio of 1:2.T5 After enzyme expression, the wet bacterial cells were incubated at 30°C with uniform shaking for 1 h. The reaction was then terminated by diluting the sample 10-fold with methanol. The diluted reaction product sample was then allowed to dissolve completely for another 1 h before being filtered through a 22 μm organic filter membrane for HPLC analysis and calculation of the biliverdin conversion rate.
[0065] Detection: The chromatographic column was an Agilent 1260 C18 column (250 mm × 4.6 mm); the mobile phase was acetonitrile / 1% acetic acid aqueous solution = 95:5 (v / v); the column temperature was 30℃; the flow rate was 1.0 mL / min; and the detection wavelength was 450 nm. Each enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol of product per minute under the above conditions.
[0066] The above detection method is also used to detect bilirubin: after mixing 0.01 g / L biliverdin with bilirubin at the same concentration, the injection volume is set to 10 μL, and the results are as follows. Figure 4 As shown, the retention times for bilirubin and biliverdin are 2.6 min and 10.3 min, respectively.
[0067] Since each experiment tests crude enzyme activity, different batches of the same mutant may have natural errors. Therefore, data comparison is only performed within the same batch of experiments, and the conversion rate is usually converted into relative activity.
[0068] will HO T0 and SUMO-HO T5 The mutant strain was cultured and expressed in 1 L shake flasks, and the cells were collected. Experiments were performed according to the above reaction and detection procedures, using the starting strain HO... T0 The activity was 100%, and the calculated data results are shown in the table below.
[0069] Table 3 SUMO-HO T5 Enzymes and HO T5 Comparative analysis results of enzyme activities
[0070] Therefore, the final mutant SUMO-HO T5 Compared to the starting enzyme HO T0 The enzyme activity increased by 4.22 times.
[0071] Example 4: SUMO-HO T5 Enzyme-catalyzed preparation of bilirubin
[0072] This embodiment provides SUMO-HO T5This method describes the enzymatic preparation of bilirubin. The conversion process requires the introduction of two additional enzymes: biliverdin reductase and glucose dehydrogenase (GDH). The former catalyzes the conversion of biliverdin to bilirubin, and this catalysis requires the coenzyme NADPH. Since NADPH is relatively expensive, the latter is introduced, along with a small amount of NADP. + NADPH can then be recycled and regenerated.
[0073] The method includes: using an ultrasonic lysate to break down SUMO-HO T5 After enzyme expression, the wet bacterial cells were mixed with PBS (pH=7.5) at a mass ratio of 1:2, and the cells were lysed to obtain SUMO-HO. T5 Cytolytic enzyme solution; after centrifugation at 5000 rpm, the supernatant was collected for later use. The reaction was then carried out in a 50 mL jacketed reactor.
[0074] Reaction system: SUMO-HO T5 20 mL of supernatant from the cytolytic enzyme solution; 0.5 g of crude biliverdin reductase powder; NADP + Coenzyme 0.1 g; glucose 0.5 g; crude GDH enzyme powder 0.5 g; PBS (pH=7.5) buffer 20 mL; the substrate heme chloride first needs to be dissolved in 0.02 M NaOH to make the stock solution concentration 10 g / L, and then added to the reaction system by feeding at a flow rate of 0.2 mL / h, and the addition is completed in 20 h (final concentration 1 g / L), and the reaction continues for 24 h. The reaction conditions are 30℃, 300 rpm, and the pH is controlled at 7.5 by adding NaOH dropwise using a pH meter throughout the process.
[0075] The above-mentioned crude biliverdin reductase powder and crude GDH powder are commonly used commercially available enzyme preparations. In this example, mouse enzymes are used. Mus musculus The crude biliverdin reductase powder was purchased from Shanghai Bailang Biotechnology Co., Ltd., product number BLE008-1BGDH; the Bacillus megaterium used in this example is... Bacillus megaterium The crude GDH powder was purchased from Jiangsu Meike Biotechnology Co., Ltd., and its trade name is GB31.
[0076] HPLC analysis showed that the bilirubin conversion rate was 100%, indicating that the SUMO-HO mutant achieved a bilirubin conversion rate of 100% according to the above reaction method. T5 The enzyme can catalyze the complete conversion of 1 g / L substrate heme chloride to bilirubin.
[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A heme oxygenase mutant HO T5 Its characteristics are, The heme oxygenase mutant HO T5 The amino acid sequence of heme oxygenase, as shown in SEQ ID NO.1, is obtained by mutating methionine at position 29 to proline, leucine at position 33 to phenylalanine, valine at position 142 to glycine, methionine at position 146 to lysine, and histidine at position 205 to threonine. The heme oxygenase mutant HO T5 The amino acid sequence is shown in SEQ ID NO.
3.
2. The heme oxygenase mutant HO according to claim 1 T5 Its characteristics are, The heme oxygenase mutant HO T5 The N-terminus also includes a soluble tag selected from SUMO, TN11 and NUSA.
3. The heme oxygenase mutant HO according to claim 1 or 2 T5 Its characteristics are, The heme oxygenase mutant HO T5 The N-terminus also includes a soluble tag, which is SUMO, and the amino acid sequence of SUMO is shown in SEQ ID NO.
8.
4. The heme oxygenase mutant HO according to claim 1 or 2 T5 Its characteristics are, The heme oxygenase mutant HO T5 The expression host is Escherichia coli BL21(DE3) competent cells.
5. Encoding the heme oxygenase mutant HO as described in claim 1 T5 The gene is characterized by, The DNA sequence of the gene is shown in SEQ ID NO.
4.
6. A heme oxygenase mutant as described in claim 3, characterized in that, The heme oxygenase mutant is SUMO-HO. T5 Enzyme; the SUMO-HO T5 The amino acid sequence of the enzyme is shown in SEQ ID NO.
5.
7. Encoding the heme oxygenase mutant as described in claim 3 or the SUMO-HO mutant as described in claim 6. T5 The gene for an enzyme is characterized by, The DNA sequence of the gene is shown in SEQ ID NO.
6.
8. The heme oxygenase mutant HO as described in any one of claims 1-4 T5 Or as described in claim 6, SUMO-HO T5 Application of enzymes in the preparation of bilirubin or products containing bilirubin.
9. A method for preparing bilirubin, characterized in that, Using the heme oxygenase mutant HO as described in any one of claims 1-4 T5 Or as described in claim 6, SUMO-HO T5 The enzyme, the preparation method of which includes: Dissolve heme chloride in NaOH solution to obtain the reaction mother liquor; will HO T5 or SUMO-HO T5 After enzyme expression, the wet bacterial cells were mixed with PBS to lyse the cells and obtain the lysed enzyme solution. The supernatant of the lysed enzyme solution was collected by centrifugation. Add the supernatant of the cytolytic enzyme solution, crude biliverdin reductase powder, and NADP to the reaction system. + Coenzyme, glucose, crude GDH enzyme powder, and PBS buffer are added to the reaction system via feedstock, and the reaction proceeds to obtain the final product.
10. The preparation method according to claim 9, characterized in that, The concentration of the mother liquor is 8-12 g / L, and the concentration of the NaOH solution is 0.15-0.25 M. The mass ratio of the wet bacterial cells to PBS is 1:1.5-2; The volume ratio of the cytolytic enzyme supernatant to PBS buffer is 1:1-1.2; The crude biliverdin reductase dry powder, NADP + The mass ratio of coenzyme, glucose, and crude GDH enzyme powder is (4.5-5.5):(1-1.2):(4.5-5.5):(4.5-5.5). The flow rate is 0.15-0.3 mL / h, and the flow duration is 18-22 h; The final concentration of the mother liquor in the reaction system is 0.8-1.2 g / L; The reaction conditions are 25-35℃, stirring at 280-320 rpm, and a total reaction time of 24-25 h.