A method for improving the storage stability of cytochrome P450 monooxygenase

Abstract

The present application discloses a method for improving the storage stability of cytochrome P450 monooxygenase in the field of biopharmaceutical technology. Adding one or several of glycerol, glucose, sorbitol, fructose, xanthan gum, mannitol, polyethylene glycol or ethylenediaminetetraacetic acid enzyme, and adding one or several of NADH or NADPH can significantly improve its storage stability and the catalytic efficiency of subsequent enzyme conversion reactions. By adding enzyme stabilizers with a final concentration of 1-80%, the storage time of whole cells, crude enzyme solutions, and pure enzymes stored at -100 to 0 °C can be extended to 180 days, and the catalytic performance can be maintained at 50-90%; by adding coenzymes with a final concentration of 1-30 mM to the crude enzyme powder, the catalytic performance of subsequent enzyme conversion reactions can be increased from 0-40% to 50-90%, and its excellent catalytic performance can be maintained for up to 180 days. The present application effectively solves the problem of poor storage stability of P450 enzymes during the production process and provides important technical support for the industrial application of P450 enzymes.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to a method for improving the storage stability of cytochrome P450 monooxygenase. Background Art

[0002] Biocatalytic reactions have mild conditions, good specificity, few side reactions, and avoid problems such as heavy metal residues and environmental pollution during the catalytic process. Therefore, biocatalysis is widely used in drug synthesis.

[0003] As one of the biocatalysts, cytochrome P450 monooxygenase is one of the most catalytically diverse enzymes in nature. It mainly participates in the metabolism of exogenous substances in organisms and the biosynthesis of natural products, and can catalyze various types of chemical reactions with a variety of structurally diverse organic compounds as substrates.

[0004] Although cytochrome P450 monooxygenase has well-known advantages, its successful examples in industrial applications are still limited. This is because most of the enzymes discovered in biocatalysis are proteins, which have many inherent disadvantages, such as poor storage stability, the need for effective coenzyme regeneration, and susceptibility to temperature and pH. Currently, in the study of its storage stability, it is found that most cytochrome P450 monooxygenases can no longer detect substrate consumption after being stored for less than a week, indicating that in less than a week, most cytochrome P450 monooxygenases have been mostly inactivated, which also greatly limits its industrial application. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention designs a method for improving the storage stability of cytochrome P450 monooxygenase with simple operation and low cost.

[0006] A method for improving the storage stability of cytochrome P450 monooxygenase, adding cytochrome P450 monooxygenase to a basic solution, and then adding an enzyme stabilizer or a coenzyme additive, wherein the basic solution is one of PB, PBS, KPB, Tri-HCl, Tri-H 2 SO 4 or Glycine-NaOH; the enzyme stabilizer is one or more of glycerol, glucose, sorbitol, fructose, xanthan gum, mannitol, polyethylene glycol or ethylenediaminetetraacetic acid, and the coenzyme additive is one or more of NADH or NADPH.

[0007] Furthermore, the cytochrome P450 monooxygenase is stored in the form of whole cells, crude enzyme solution, crude enzyme powder or pure enzyme.

[0008] Furthermore, the final concentration of the added enzyme stabilizer is 1% - 80%.

[0009] Furthermore, the final concentration of the coenzyme additive added is 1-30 mM.

[0010] Furthermore, the storage temperature is between -100 °C and 0 °C.

[0011] Furthermore, the concentration of the glycerol is 10%, the concentration of the ethylenediaminetetraacetic acid is 1%, and the concentrations of the remaining enzyme stabilizers are all 2%.

[0012] Furthermore, the storage temperature is -80 °C.

[0013] Furthermore, the coenzyme additive is NADH. The concentration of NADH added to the crude enzyme solution is 3 mM, and the concentration of NADH added to the crude enzyme powder is 10 mM.

[0014] Furthermore, the storage temperature is -20 °C.

[0015] Compared with the prior art, by adding enzyme stabilizers with a final concentration of 1%-80%, the storage times of whole cells, crude enzyme solutions, and pure enzymes stored at -100 °C to 0 °C can be extended to 180 d, and the catalytic performance can be maintained at 50%-90%; by adding a coenzyme additive with a final concentration of 1-30 mM to the crude enzyme powder, the catalytic performance of the subsequent enzyme conversion reaction can be increased from 0%-40% to 50%-90%, and its excellent catalytic performance can be maintained for up to 180 d. This invention effectively solves the key scientific problem of poor storage stability of cytochrome P450 enzymes during the production process and provides important technical support for the industrial application of cytochrome P450 enzymes.

[0016] The method of the present invention is simple to operate, low in cost, and convenient for popularization and utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram showing the effects of no enzyme stabilizer on the ee value and yield of P450 enzyme at different storage temperatures;

[0018] Figure 2 It is a schematic diagram showing the protective effects of 15% glycerol as an enzyme stabilizer on the ee value and yield of P450 enzyme at different storage temperatures;

[0019] Figure 3 It is a schematic diagram showing the protective effects of different concentrations of glycerol as an enzyme stabilizer on the ee value and yield of P450 enzyme at -80 °C;

[0020] Figure 4 It is a schematic diagram showing the promoting effects of glycerol, sorbitol, mannitol, ethylenediaminetetraacetic acid, and glycerol combined with sorbitol, glycerol combined with mannitol, and glycerol combined with ethylenediaminetetraacetic acid as enzyme stabilizers on the ee value and yield of P450 enzyme;

[0021] Figure 5 Schematic diagram of the protective effects of glycerol, ethylenediaminetetraacetic acid, and glycerol and mannitol as enzyme stabilizers on the ee value and yield of P450 enzyme

[0022] Figure 6 Schematic diagram of the protective effects of 15% glycerol as an enzyme stabilizer on the ee value and yield of P450 enzyme in the form of whole cells and crude enzyme powder

[0023] Figure 7 Schematic diagram of the improvement of the catalytic performance of crude enzyme solution by coenzyme additives NADH at different concentrations

[0024] Figure 8 Schematic diagram of the improvement of the catalytic performance of crude enzyme powder by coenzyme additives NADH at different concentrations

[0025] Figure 9 Schematic diagram of the protective effects of 10 mM coenzyme additive NADH on the ee value and yield of P450 enzyme of crude enzyme powder stored at -20°C

[0026] Figure 10 Control schematic diagram of the ee value and yield of P450 enzyme of crude enzyme powder stored at -20°C without coenzyme additive and with 10 mM coenzyme additive NADH

[0027] Figure 11 Reaction yield curve graph and CO difference spectrum scanning result graph of P450 enzyme, where (b) stored for 0 d; (c) stored for 5 d; (d) stored for 30 d Detailed implementation mode

[0028] The following is a further detailed description through specific implementation modes:

[0029] The P450 enzyme involved in the examples is derived from Deinococcus apachensis

[0030] Strain culture: Pick an activated single colony from the TB solid medium plate and inoculate it into 50 mL of TB liquid medium, add Kan antibiotic, and culture at 37°C and 250 rpm for 8 h. Transfer the bacterial liquid into 100 mL of TB medium at a volume ratio of 2%, and after culturing at 37°C and 250 rpm for 3 h, add IPTG to induce protein expression. The final concentration of the inducer is 0.2 mM, and induce at 25°C and 250 rpm for 10 - 12 h

[0031] Whole cell catalytic reaction: Take a small amount of induced bacterial liquid and dilute it 10 times for OD 600 value detection, then use a high-speed refrigerated centrifuge to recover the bacterial cells, centrifuge at 9000 rpm for 3 min at 4°C, discard the supernatant and retain the bacterial cells, and according to the measured OD 600Relationship between value and cell concentration: Add an appropriate volume of PB (50 mM, pH 8.0, 15% v / v Glycerol) as a buffer solution, and vortex to resuspend and mix the cells evenly. The cell concentration is 20 g cdw / L, 2 mM 1-chloro-4-phenyl-3-butene is used as the substrate, and the reaction conditions are 10 °C, 250 rpm, 24 h.

[0032] Reaction systems for crude enzyme solution and pure enzyme: 5 mL of PB (50 mM, pH 8.0, 15% v / v Glycerol) is used as the buffer solution, the amount of P450 enzyme is 20 g cdw / L, 2 mM 1-chloro-4-phenyl-3-butene is used as the substrate, and the reaction conditions are 10 °C, 250 rpm, 24 h.

[0033] Reaction system for crude enzyme powder: 5 mL of PB (50 mM, pH 8.0, 15% v / v Glycerol) is used as the buffer solution, 100 mg of crude enzyme powder is dissolved in 5 mL of buffer solution, 2 mM 1-chloro-4-phenyl-3-butene is used as the substrate, and the reaction conditions are 10 °C, 250 rpm, 10 h.

[0034] Sample preparation: When the biocatalytic reaction reaches the reaction time, add an equal volume of ethyl acetate containing 20 mM phenylpropanol internal standard to the reaction solution for extraction. Centrifuge the mixture at 12,000 rpm until it is layered. Take 1 mL of the organic phase and transfer it to a 1.5 mL EP tube containing an appropriate amount of anhydrous sodium sulfate to dry the water. Filter the supernatant through a 0.22 μm organic membrane, and transfer the filtrate into a clean liquid phase analysis sample bottle.

[0035] The prepared sample test solution is analyzed using a Shimadzu LC-20D high-performance liquid chromatograph. After integrating the chromatogram, the product concentration is obtained by substituting the ratio of the peak areas of the internal standard and the generated product into the prepared standard curve, and the ee and yield are calculated.

[0036] Cultivate using TB medium. After collecting the cells, prepare a suspension with a cell concentration of 20 g cdw / L using PB (50 mM, pH 8.0, 15% v / v Glycerol) as the buffer solution. Take a part for enzyme amount determination and a part for biotransformation at a substrate concentration of 2 mM. Calculate TTN based on the final reaction yield and the amount of P450 enzyme, and calculate TOF for the period with the fastest reaction rate among each time period.

[0037] Determination of P450 enzyme protein concentration by CO difference spectrum scanning method: The sample was equally divided and dispensed into 3 quartz cuvettes of 3 mL. One cuvette was placed in the sample cell, and the instrument was set to perform baseline correction at 400 - 500 nm. About 3 mg of sodium dithionite was added to the second cuvette, mixed well, and used as a baseline sample for scanning. The third cuvette was introduced with CO gas in a fume hood for 3 min. After adding about 3 mg of sodium dithionite to reduce the P450 enzyme, multiple scans were performed at 400 - 500 nm until the peak at 450 nm no longer increased. The obtained curve was the CO binding spectrum of the reduced P450 enzyme. The soluble expression of the P450 enzyme was judged according to the absorbance changes at 420 nm and 450 nm between the baseline and the CO binding spectrum, and the enzyme amount was calculated based on the CO binding spectrum after subtracting the baseline.

[0038] Biotransformation reaction: Take 5 mL of the suspension with a protein concentration of 20 g cdw / L and place it in a 25 mL stoppered ground - glass Erlenmeyer flask. Divide it into 11 groups, with three replicates in each group. Add 50 μL of the prepared substrate stock solution (100 mM) to each. After tightly stoppered, place it in a constant - temperature shaker and react at 10 °C and 250 rpm. Samples were taken at 20 min, 40 min, 1 h, 2 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, and 24 h of the reaction. Use 5 mL of ethyl acetate solution containing 20 mM phenylpropanol for extraction and sample preparation. Analyze the reaction samples by high - performance liquid chromatography. Plot the yield against time to obtain a line graph, and calculate the TOF value for the time period with the fastest reaction rate. Calculate the TTN based on the yield at 24 h of the reaction.

[0039] Calculation method of P450 enzyme concentration: P450 (μM) = (ΔA450 - ΔA490) / 0.091, and the molar extinction coefficient ε of the P450 enzyme = 0.091 μM -1 cm -1 , and ΔA490 was used as the baseline value to calculate the peak height at 450 nm.

[0040] Calculation method of turnover frequency (TOF): TOF (min -1 ) = C p / (t×C e ), where C p is the product concentration, C e is the P450 enzyme concentration, and t is the reaction time.

[0041] Calculation method of total turnover number (TTN): TTN = C p / C e , where C p is the final product concentration, and C e is the P450 enzyme concentration.

[0042] Example 1

[0043] Use PB (50 mM, pH 8.0) as the storage solvent and store it at -80 °C, -20 °C, 4 °C, and 10 °C for 0 d, 1 d, 2 d, 5 d, 10 d, 15 d, and 30 d.

[0044] The results showed that when stored for 0 d to 5 d, the storage effect was best at 4 °C; as the storage time prolonged, the storage at 4 °C was quite unstable. The storage at -80 °C and -20 °C was relatively stable, but the yield decreased significantly (such as Figure 1 ).

[0045] Example 2

[0046] Add glycerol with a final concentration of 15% enzyme stabilizer to PB (50 mM, pH 8.0) as the storage solvent and store it at -80 °C, -20 °C, 4 °C, and 10 °C for 0 d, 1 d, 2 d, 5 d, 10 d, 15 d, and 30 d.

[0047] The results showed that when stored at -80 °C, the yield not only did not decrease, but instead increased. Moreover, when stored for 10 d, it was still higher than that at 0 d. When stored for 15 d, although it decreased, the amplitude was very small; when stored for 30 d, the yield still had an upward trend, indicating that the addition of the enzyme stabilizer effectively improved the storage stability of cytochrome P450 monooxygenase (such as Figure 2 ).

[0048] Example 3

[0049] Suspend the whole cells in PB (50 mM, pH 8.0) buffer containing different concentrations of glycerol and store them at -80 °C for 0 d, 1 d, 2 d, 5 d, 15 d, and 30 d.

[0050] The results showed that in the PB (50 mM, pH 8.0) control group, when stored for 1 d, the yield decreased sharply to 10.2%. However, after adding only 5% glycerol, the ee value and the yield could basically be maintained at a good level. Considering the comprehensive storage results for 30 d, the optimal addition amount of glycerol was determined to be 10% (such as Figure 3 ).

[0051] Example 4

[0052] Add enzyme stabilizers such as glycerol, sorbitol, mannitol, ethylenediaminetetraacetic acid, and glycerol with sorbitol, glycerol with mannitol, and glycerol with ethylenediaminetetraacetic acid to the whole cell reaction system.

[0053] The results showed that after adding the enzyme stabilizer, the ee value and yield catalyzed by P450 enzyme could be improved. It is worth mentioning that when 2% ethylenediaminetetraacetic acid was added, its ee value and yield were significantly improved, with the ee value up to 96.0% and the yield up to 59.5%, and the yield was 22.6% higher than that of the PB (50 mM, pH 8.0) control group. Then, we screened the concentration of ethylenediaminetetraacetic acid and found that when 1% ethylenediaminetetraacetic acid was added, although the ee value decreased slightly, the yield increased to 66.0% (as Figure 4 ).

[0054] Example 5

[0055] After the whole cells were suspended in PB (50 mM, pH 8.0) buffer containing different enzyme stabilizers, they were stored at -80 °C for 0 d, 1 d, 2 d, 5 d, 10 d, 15 d, and 30 d.

[0056] The results showed that adding glycerol, ethylenediaminetetraacetic acid, and glycerol with mannitol could improve the ee value and yield catalyzed by P450 enzyme and maintain good stability. It was found that the group with the synergistic effect of glycerol, ethylenediaminetetraacetic acid, and glycerol with mannitol showed better catalytic effect; the group with the synergistic effect of glycerol and mannitol showed better catalytic results compared with the glycerol group and the mannitol group. It was speculated that the synergistic effect in the enzyme stabilizer might be better than that of a single enzyme stabilizer (as Figure 5 ).

[0057] Example 6

[0058] In the form of whole cells and crude enzyme solution, 15% enzyme stabilizer glycerol was added to PB (50 mM, pH 8.0) as the storage solvent and stored at -80 °C for 0 d, 1 d, 2 d, 5 d, 15 d, and 30 d.

[0059] The results showed that whether stored in the form of whole cells or crude enzyme solution, good catalytic performance could be maintained and good storage stability was achieved (as Figure 6 ).

[0060] Example 7

[0061] 0 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM coenzyme additive NADH were added to the crude enzyme solution reaction system.

[0062] The results showed that from 0 mM to 3 mM, the ee value and yield showed an upward trend, and the highest value was reached at 3 mM, with the yield up to 82.4%, which was 35.4% higher than that at 0 mM (as Figure 7 ).

[0063] Example 8

[0064] Add 0 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM coenzyme additive NADH to the crude enzyme powder reaction system.

[0065] The results show that from 0 mM to 10 mM, the ee value and the yield show an upward trend, and the yield increases significantly. After 10 mM, it tends to be stable. The yield can reach 71.6% at 10 mM, which is 65.9% higher than that at 0 mM (as Figure 8 ).

[0066] Example 9

[0067] After storing the crude enzyme powder at -20 °C for 0 d, 5 d, 10 d, 20 d, 30 d, 40 d, 50 d, 60 d, 90 d, add 10 mM NADH to catalyze the reaction.

[0068] The results show that with the extension of the storage time, the ee value and the yield change little. Even after storing for 90 d, the yield is still as high as 67%, indicating that the crude enzyme powder has good storage stability (as Figure 9 ).

[0069] Example 10

[0070] After storing the crude enzyme powder at -20 °C for 0 d, 5 d, 10 d, 20 d, 30 d, 40 d, 50 d, 60 d, 90 d, directly catalyze the reaction and add 10 mM NADH to catalyze the reaction.

[0071] The results show that when 10 mM NADH is added, the yield increases from 8% to 70%. It shows that the coenzyme additive NADH plays a crucial role in this reaction (as Figure 10 ).

[0072] Example 11

[0073] Determine the P450 enzyme protein concentration according to the CO difference spectrum scanning results; and sample and monitor at 20 min, 40 min, 1 h, 2 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 24 h of the reaction. Plot the yield against time to obtain the line graph, and calculate the turnover frequency (TOF) value for the time period with the fastest reaction rate, and calculate the total turnover number (TTN) based on the yield at 24 h of the reaction.

[0074] The results show that the P450 enzyme protein concentrations stored for 0 d, 5 d, 30 d are 0.47 μM, 0.47 μM, 0.54 μM; the TOF values are 79.05 min -1 , 79.05 min -1 , 68.80 min -1; The TTN values are 3173.94, 3173.94, and 2762.50. It indicates that the P450 enzyme can still basically maintain its original protein concentration after being stored for 30 days, and both the TOF value and the TTN value change little (such as Figure 11 ).

[0075] The above are only the embodiments of the present invention, and common knowledge such as the specific structures and characteristics known in the solution is not described in detail here. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicability of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to interpret the content of the claims.

Claims

1. A method for improving the storage stability of cytochrome P450 monooxygenase, characterized in that, cytochrome P450 monooxygenase is added to a basic solution, and then an enzyme stabilizer and / or a coenzyme additive are added. Among them, the basic solution is one of PB, PBS, KPB, Tri-HCl, Tri-H2SO4 or Glycine-NaOH; the enzyme stabilizer is one or more of glycerol, mannitol or ethylenediaminetetraacetic acid, and the coenzyme additive is NADH, and the storage temperature is between -100°C and 0°C.

2. The method for improving the storage stability of cytochrome P450 monooxygenase according to claim 1, characterized in that: the cytochrome P450 monooxygenase is stored in the form of whole cells, crude enzyme solution, crude enzyme powder or pure enzyme.

3. The method for improving the storage stability of cytochrome P450 monooxygenase according to claim 2, characterized in that: the final concentration of the added enzyme stabilizer is 1% - 80%.

4. The method for improving the storage stability of cytochrome P450 monooxygenase according to claim 2, characterized in that: the final concentration of the added coenzyme additive is 1 - 30 mM.

5. The method for improving the storage stability of cytochrome P450 monooxygenase according to claim 3, characterized in that: the concentration of glycerol is 10%, the concentration of ethylenediaminetetraacetic acid is 1%, and the concentrations of the remaining enzyme stabilizers are all 2%.

6. The method for improving the storage stability of cytochrome P450 monooxygenase according to claim 5, characterized in that: the storage temperature is -80°C.

7. The method for improving the storage stability of cytochrome P450 monooxygenase according to claim 4, characterized in that: the coenzyme additive is NADH, the concentration of NADH added to the crude enzyme solution is 3 mM, and the concentration of NADH added to the crude enzyme powder is 10 mM.

8. The method for improving the storage stability of cytochrome P450 monooxygenase according to claim 7, characterized in that: the storage temperature is -20°C.

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