Method for synthesizing phycocyanobilin through in-vitro multi-enzyme cascade catalysis
By expressing in E. coli and using the multi-enzyme cascade catalytic action of heme oxygenase, phycocyanobacter reductase and glucose dehydrogenase, the problem of complex process and high cost in phycocyanobacter preparation was successfully solved, and efficient and low-cost phycocyanobacter synthesis was achieved, laying the foundation for large-scale preparation.
Patent Information
- Application Number
- CN202411924925.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-03-28
AI Technical Summary
The existing phycocycebin preparation technology has problems such as complex process, high production costs and limited large-scale preparation. In particular, the insufficient substrate and coenzyme in E. coli seriously limits the efficient preparation of phycocycebin.
The method of in vitro multi-enzyme cascade catalyzing of phycocytobilin is used to express heme oxygenase, phycocytobili reductase and glucose dehydrogenase in Escherichia coli. Through the combined action of these enzymes, heme chloride is used as the substrate to synthesize phycocytobilibin with the assistance of ascorbic acid and methanesulfonic acid deferroamine, and at the same time, glucose is used to promote the regeneration of coenzyme NADPH.
It has achieved efficient synthesis of phycocytobilin, simplified the process flow, reduced production costs, overcome the problem of insufficient substrate and coenzyme in E. coli, and laid the foundation for the large-scale preparation of phycocytobilin.
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Figure CN119709905A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the fields of food and biomedicine, and in particular to a method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro. Background Art
[0002] Phycocyanobilin (PCB) is an open-chain tetrapyrrole compound and a light-harvesting pigment in the phycobilisomes of cyanobacteria. It has a molecular weight of 586.7 g / mol and is easily soluble in organic solvents such as methanol and chloroform. Phycocyanobilin is covalently bound to the two polypeptide chains of phycocyanin in the phycobilisomes through cysteine residues and thioether bonds.
[0003] Due to its photosensitivity and excellent antioxidant properties, phycocyanobilin has broad application prospects and development potential in the fields of food and biomedicine. In the food field, phycocyanobilin, as a natural pigment in algae, can be used as a food colorant. In addition, studies have found that phycocyanobilin plays a vital role in the treatment of neurodegenerative diseases (including Parkinson's and Alzheimer's) by reducing oxidative stress levels. Its unique optical activity also allows it to be used as an immunofluorescence probe for disease diagnosis. In addition, phycocyanobilin can also become a new type of photosensitizer for photodynamic therapy of tumors.
[0004] At present, the preparation of phycocyanobilin mainly includes natural extraction method of spirulina, chemical synthesis and synthesis method of recombinant Escherichia coli. The natural extraction method of spirulina is mainly because the culture period of spirulina is long, and the presence of chlorophyll and other phycobilins affects the purity of phycocyanobilin, so it has the disadvantages of complex process and high production cost. The chemical synthesis method has the disadvantages of complicated synthesis and purification steps, low yield and high cost due to the complex structure of phycocyanin and multiple optical isomer groups. The synthesis of phycocyanobilin by Escherichia coli through genetic engineering technology has the advantages of low cost and easy purification, so it has become a research hotspot in recent years. By cloning the heme oxygenase gene ho1 and the phycocyanobilin reductase gene pcyA and co-expressing them in Escherichia coli, phycocyanobilin is synthesized by using the heme of Escherichia coli itself, and then the bacterial cells of the expression bacteria can be directly lysed with methanol to obtain a methanol solution of phycocyanobilin, which is considered to be a potential method for preparing phycocyanin.
[0005] However, due to the low content of substrate heme and cofactor NADPH in Escherichia coli, the large-scale preparation of phycocyanobilin is severely limited, and the yield of recombinant phycocyanobilin is still at a low level, resulting in high-purity phycocyanobilin on the market. The price is expensive, which limits its application in the fields of food and biomedicine. Therefore, the development of a low-cost and simple process for the preparation of phycocyanobilin will lay the foundation for the widespread application of phycocyanobilin in the fields of food and biomedicine. Summary of the invention
[0006] The present invention aims to provide a simple, rapid and low-cost method for preparing phycocyanobilin and its application in the fields of food and biomedicine.
[0007] In order to achieve the above object, the present invention provides a method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro, comprising the following steps:
[0008] (1) expressing heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase separately or jointly in Escherichia coli;
[0009] (2) disrupting Escherichia coli to obtain crude enzyme solutions or pure enzymes of heme oxygenase, phycocyanobilin reductase, and glucose dehydrogenase;
[0010] (3) Using hemin chloride as a substrate, phycocyanobilin is synthesized by heme oxygenase and phycocyanobilin reductase with the assistance of ascorbic acid and deferoxamine mesylate, while glucose dehydrogenase utilizes glucose to promote the regeneration of the coenzyme NADPH.
[0011] The heme oxygenase in step (1) of the present invention is preferably selected from the enzymes numbered ALJ66630.1, UWU51119.1, AFY85345.1, QLE40117.1, and BCU10973.1 in Genbank.
[0012] The phycocyanobilin reductase in step (1) of the present invention is preferably selected from the enzymes numbered ALJ68839.1, UWU50912.1, AFY84909.1, QLE39349.1, and BCU12592.1 in Genbank.
[0013] The glucose dehydrogenase in step (1) of the present invention is from Bacillus subtilis and is numbered as WP_041340171.1, WP_032726518.1, WP_128473595.1, WP_128992411.1, and WP_041340171.1.
[0014] The Escherichia coli described in step (1) of the present invention is preferably Escherichia coli BL21 (DE3) or Escherichia coli C41 (DE3).
[0015] The heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase in step (1) of the present invention are expressed separately in different Escherichia coli BL21 cells or simultaneously expressed jointly in the same Escherichia coli BL21 cell.
[0016] The heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase in step (2) of the present invention are crude enzyme liquid obtained by crushing the bacterial cells or pure enzymes obtained by purification.
[0017] In step (3) of the present invention, glucose dehydrogenase utilizes glucose to generate coenzyme NADPH, thereby further promoting the synthesis of phycocyanobilin.
[0018] In step (3) of the present invention, ascorbic acid and deferoxamine mesylate need to be added as an auxiliary.
[0019] The reaction temperature in step (3) of the present invention is 37° C. and the pH is 7.3.
[0020] Compared with the prior art, the beneficial effects of the present invention include:
[0021] (1) The present invention utilizes genetic engineering technology to express two key enzymes for synthesizing phycocyanobilin in Escherichia coli, and screens out highly active heme oxygenase and phycocyanobilin reductase, further promoting the synthesis of phycocyanobilin in vivo and in vitro.
[0022] (2) Compared with the natural extraction of phycocyanin from algae, the method of the present invention has a short production cycle, is not interfered by other pigments such as chlorophyll and carotenoids, has a simple purification process and is low in cost.
[0023] (3) Compared with the production of phycocyanobilin by recombinant Escherichia coli, the method of the present invention overcomes the serious influence of insufficient substrate and coenzyme in the production of phycocyanobilin by Escherichia coli, directly uses hemin chloride as a substrate, synthesizes phycocyanobilin under the catalysis of heme oxygenase and phycocyanobilin reductase, and at the same time, glucose dehydrogenase uses glucose to generate the coenzyme NADPH, further improving the production level of phycocyanobilin and laying a foundation for the large-scale preparation of phycocyanobilin. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Schematic diagram of the in vitro multi-enzyme cascade catalytic synthesis of phycocyanobilin.
[0025] Figure 2 This is a diagram showing the results of the in vitro multi-enzyme catalytic synthesis of phycocyanobilin in Example 3.
[0026] Figure 3 This is a graph showing the effect of adding NADPH on the enzyme-catalyzed synthesis of phycocyanobilin in Example 4.
[0027] Figure 4 This is a graph showing the effect of hemin concentration on the enzyme-catalyzed synthesis of phycocyanobilin in Example 5.
[0028] Figure 5 This is a graph showing the effect of glucose concentration on the enzyme-catalyzed synthesis of phycocyanobilin in Example 6.
[0029] Figure 6 This is a graph showing the effect of reaction time on the enzyme-catalyzed synthesis of phycocyanobilin in Example 7. DETAILED DESCRIPTION
[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments of the specification. The following embodiments are only used to illustrate the present invention, but not to limit the scope of protection of the present invention.
[0031] The method for synthesizing phycocyanobilin by multi-enzyme cascade in vitro is as follows: first, heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase are expressed separately or jointly in Escherichia coli; then, the Escherichia coli cells are broken to obtain crude enzyme solutions or pure enzymes of heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase; with hemin chloride as substrate, phycocyanobilin is synthesized by a two-step enzymatic cascade of heme oxygenase and phycocyanobilin reductase with the assistance of ascorbic acid and deferoxamine mesylate, while glucose dehydrogenase utilizes glucose to promote the regeneration of coenzyme NADPH. Figure 1 Schematic diagram of the in vitro multi-enzyme cascade catalytic synthesis of phycocyanobilin.
[0032] Example 1: Joint expression of heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase in Escherichia coli.
[0033] The selected heme oxygenase gene ho1 of Spirulina platensis and the phycocyanobilin reductase gene pcyA from Microcystis aeruginosa were connected to the two expression frames of the pETDuet-1 vector by genetic engineering to construct the recombinant plasmid pETDuet-ho1-pcyA. The glucose dehydrogenase gene gdh from Bacillus subtilis was connected to the pCDFDuet-1 vector to construct the recombinant plasmid pCDFDuet-gdh. The recombinant plasmids pETDuet-ho1-pcyA and pCDFDuet-gdh were co-transformed into Escherichia coli BL21 (DE3) competent cells to obtain the recombinant Escherichia coli strain S1 that can efficiently express the three enzymes under the drive of the T7 promoter. Single colonies of the recombinant strain on the solid plate were randomly picked and inoculated into LB medium and cultured at 37°C and 170rpm. The cultured seed liquid was inoculated into TB medium at an inoculation ratio of 1:100 and cultured at 37°C and 170rpm. When the bacteria grew to OD 600 =0.6-0.8, add the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mmol / L for induction, and continue culturing at 18°C and 170 rpm for 12 hours before stopping.
[0034] Example 2: Preparation of heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase.
[0035] After the culture of recombinant bacteria S1 expressing heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase was completed, the E. coli cells were collected by centrifugation at 8500g for 10 min at 4°C, washed twice with 0.9% NaCl solution, resuspended in 50mM HEPES breaking buffer, and adjusted to OD 600 to 20. After adding 1mM PMSF, use an ultrasonic disruptor to disrupt in an ice bath (power 150W, work 5s, stop 5s) for 5min. Keep the cells in a low temperature environment (0℃) throughout the disruption process. After the disruption is completed, centrifuge at 9000g for 10min at 4℃, collect the supernatant, and obtain the crude enzyme solution containing heme oxygenase, phycocyanin reductase and glucose dehydrogenase.
[0036] Example 3: Construction of an in vitro multi-enzyme catalytic synthesis system of phycocyanobilin.
[0037] The crude enzyme solution of multi-enzyme expression bacteria S1 was used as a catalyst to construct an in vitro multi-enzyme catalytic synthesis of phycocyanobilin system. 5 mL of crude enzyme solution, 0.1 mM hemin, 3 mM ascorbic acid and 1 mM deferoxamine mesylate were added to a centrifuge tube. The reaction conditions were set at pH 7.3 and temperature at 37 °C. When the reaction was terminated, 500 μL of the sample was added to 500 μL of acidic methanol (containing 5% concentrated hydrochloric acid), centrifuged at 11000 g for 10 min, and the supernatant was taken to measure its UV-visible absorption spectrum. According to A 680 Calculate the yield of phycocyanin. Figure 2 This is a diagram showing the results of in vitro multi-enzyme catalytic synthesis of phycocyanobilin.
[0038] Example 4: Effect of adding NADPH on enzyme-catalyzed synthesis of phycocyanobilin.
[0039] The crude enzyme solution of multi-enzyme expression bacteria S1 was used as a catalyst to further optimize the in vitro multi-enzyme catalytic synthesis of phycocyanobilin system. 5 mL of crude enzyme solution, 0.1 mM hemin, 0.1 mM NADPH, 3 mM ascorbic acid and 1 mM deferoxamine mesylate were added to the centrifuge tube. The reaction conditions were set at pH 7.3 and temperature at 37 °C. When the reaction was terminated, 500 μL of the sample was added to 500 μL of acidic methanol (containing 5% concentrated hydrochloric acid), centrifuged at 11000 g for 10 min, and the supernatant was taken to measure its UV-visible absorption spectrum. According to A 680 Calculate the yield of phycocyanin. Figure 3 This is a diagram showing the effect of NADPH on enzyme-catalyzed synthesis of phycocyanin.
[0040] Example 5: Effect of hemin concentration on enzyme-catalyzed synthesis of phycocyanobilin.
[0041] The crude enzyme solution of multi-enzyme expression bacteria S1 was used as a catalyst to further optimize the concentration of hemin in the in vitro multi-enzyme catalytic synthesis of phycocyanobilin system. The hemin concentrations in the reaction system were set to 0.1, 0.2, 0.4, 0.6 and 1 mM, respectively. The reaction system also included 5 mL of crude enzyme solution, 0.1 mM NADPH, 3 mM ascorbic acid and 1 mM deferoxamine mesylate. The reaction conditions were set at pH 7.3 and temperature at 37 °C. When the reaction was terminated, 500 μL of the sample was added to 500 μL of acidic methanol (containing 5% concentrated hydrochloric acid), centrifuged at 11000 g for 10 min, and the supernatant was taken to measure its UV-visible absorption spectrum. According to A 680 Calculate the yield of phycocyanin. Figure 4 This graph shows the effect of hemin concentration on enzyme-catalyzed synthesis of phycocyanobilin.
[0042] Example 6: Effect of glucose concentration on enzyme-catalyzed synthesis of phycocyanobilin.
[0043] The crude enzyme solution of multi-enzyme expression bacteria S1 was used as a catalyst to further optimize the concentration of glucose in the in vitro multi-enzyme catalytic synthesis of phycocyanobilin system. Grape dehydrogenase can use glucose to generate coenzyme NADPH, promoting the synthesis of phycocyanobilin. The glucose concentrations in the reaction system were set to 0, 0.1, 0.5, 1 and 2 mM, respectively. The reaction system also included 5 mL of crude enzyme solution, 0.2 mM hemin, 0.1 mM NADPH, 3 mM ascorbic acid and 1 mM deferoxamine mesylate. The reaction conditions were set at pH 7.3 and temperature at 37 °C. When the reaction was terminated, 500 μL of the sample was added to 500 μL of acidic methanol (containing 5% concentrated hydrochloric acid), centrifuged at 11000 g for 10 min, and the supernatant was taken to measure its UV-visible absorption spectrum. According to A 680 Calculate the yield of phycocyanin. Figure 5 This graph shows the effect of glucose concentration on enzyme-catalyzed synthesis of phycocyanobilin.
[0044] Example 7: Effect of reaction time on enzyme-catalyzed synthesis of phycocyanobilin.
[0045] The crude enzyme solution of multi-enzyme expression bacteria S1 was used as a catalyst to investigate the effect of reaction time on the enzyme-catalyzed synthesis of phycocyanobilin under optimal conditions. The reaction system included 5 mL of crude enzyme solution, 0.2 mM hemin, 0.1 mM NADPH, 0.1 m glucose, 3 mM ascorbic acid and 1 mM deferoxamine mesylate. The reaction conditions were set at pH 7.3 and temperature at 37 °C. At 0, 1, 2, 2.5, 3, 3.5, and 4 h of the reaction, 500 μL of the sample was added to 500 μL of acidic methanol (containing 5% concentrated hydrochloric acid), centrifuged at 11000 g for 10 min, and the supernatant was taken to measure its UV-visible absorption spectrum. According to A 680 Calculate the yield of phycocyanin. Figure 6This graph shows the effect of reaction time on enzyme-catalyzed synthesis of phycocyanobilin.
[0046] As can be seen from the above examples, the basic feature of the present invention is to use heme oxygenase and phycocyanobilin reductase to catalyze the synthesis of phycocyanobilin in vitro, and promote the regeneration of coenzyme NADPH by grape dehydrogenase. This method not only avoids the problems of long algae culture cycle, high cost and complex purification process, but also avoids the problem of insufficient substrate and coenzyme in vivo when recombinant Escherichia coli synthesizes phycocyanobilin, and synthesizes phycocyanobilin more simply and quickly, laying a foundation for the large-scale preparation of phycocyanobilin.
Claims
1. A method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro, characterized in that: The main steps include: (1) expressing heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase separately or jointly in Escherichia coli; (2) disrupting Escherichia coli to obtain crude enzyme solutions or pure enzymes of heme oxygenase, phycocyanobilin reductase, and glucose dehydrogenase; (3) Using hemin chloride as a substrate, phycocyanobilin is synthesized by heme oxygenase and phycocyanobilin reductase with the assistance of ascorbic acid and deferoxamine mesylate, while glucose dehydrogenase utilizes glucose to promote the regeneration of the coenzyme NADPH.
2. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: The heme oxygenase described in step (1) is preferably selected from the enzymes numbered ALJ66630.1, UWU51119.1, AFY85345.1, QLE40117.1, and BCU10973.1 in Genbank.
3. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: The phycocyanobilin reductase described in step (1) is preferably selected from the enzymes numbered ALJ68839.1, UWU50912.1, AFY84909.1, QLE39349.1, and BCU12592.1 in Genbank.
4. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: The glucose dehydrogenase described in step (1) is from Bacillus subtilis, and the enzymes are numbered WP_041340171.1, WP_032726518.1, WP_128473595.1, WP_128992411.1, and WP_041340171.
1.
5. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: The Escherichia coli described in step (1) is preferably Escherichia coli BL21 (DE3) or Escherichia coli C41 (DE3).
6. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: The heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase described in step (1) are expressed separately in different Escherichia coli BL21 cells or simultaneously expressed in the same Escherichia coli BL21 cell.
7. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: The heme oxygenase, phycocyanobilin reductase and glucose dehydrogenase described in step (2) are crude enzyme solutions obtained by crushing the bacterial cells or pure enzymes obtained by purification.
8. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: In step (3), glucose dehydrogenase utilizes glucose to generate the coenzyme NADPH, thereby further promoting the synthesis of phycocyanobilin.
9. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: In step (3), ascorbic acid and deferoxamine mesylate need to be added as an aid.
10. The method for synthesizing phycocyanobilin by multi-enzyme cascade catalysis in vitro according to claim 1, characterized in that: The reaction temperature of step (3) is 37° C. and the pH is 7.3.