Dehydrogenase and method for synthesizing 11 alpha-OH-ADD

Abstract

The invention relates to the technical field of biological medicine, and provides dehydrogenase and a method for synthesizing 11 alpha-OH-ADD.The dehydrogenase is KstD, the nucleotide sequence of the dehydrogenase is shown as SEQ ID NO: 1, and the amino acid sequence of the dehydrogenase is shown as SEQ ID NO: 2. The dehydrogenase KstD can convert a substrate 11 alpha-OH-AD into 11 alpha-OH-ADD, a double-liquid-phase conversion system is adopted, when feeding reaches up to 40 g / L, the conversion rate of about 97% is achieved after reaction is conducted for 23 h, and the dehydrogenase KstD has the effect of rapid and efficient dehydrogenation conversion.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a dehydrogenase and a method for synthesizing 11α-OH-ADD. Background Art

[0002] Steroid drugs have anti-inflammatory, contraceptive, anti-allergic, and anti-toxic effects. Their production is second only to antibiotics and they are widely used in various diseases. Androst-4-ene-3,17-dione (AD) or androst-1,4-diene-3,17-dione (ADD) are important intermediates and can be used as the starting materials for the production of most steroid hormone drugs. Introducing a hydroxyl group at the C11α position of the ADD or AD nucleus can significantly improve the anti-inflammatory activity of steroid drugs.

[0003] To prepare 11α-OH-ADD, two steps are usually required: dehydrogenation of the C1,2 position of the steroid nucleus and hydroxylation of the C11α position. In the prior art (such as CN102827913A, CN108085359A), C11α-hydroxylating microorganisms such as Rhizopus nigromaculus and Aspergillus ochraceus and C1,2 dehydrogenating microorganisms such as Arthrobacter and Mycobacterium are usually used for step-by-step or mixed culture for preparation. These methods have problems such as unstable activity, non-specific conversion, low feed amount, complex reaction time and long time. In addition, filamentous fungi have strict requirements on the structure of the substrate when performing C11α hydroxylation of the steroid position. When the substrate is 4AD, progesterone, and 17-hydroxyprogesterone, it shows a good conversion rate, and the conversion rate is low for other substrates. Therefore, there is an urgent need for a dehydrogenase with high feed amount and high substrate conversion rate and a method for converting 11α-OH-ADD. Summary of the invention

[0004] In view of this, the present invention provides a dehydrogenase and a method for synthesizing 11α-OH-ADD, which has the characteristics of high feed amount and high substrate conversion rate.

[0005] The technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a dehydrogenase, wherein the dehydrogenase is KstD, and its nucleotide sequence is shown in SEQ ID NO:1.

[0006] In a second aspect, the present invention provides a recombinant expression vector, which is a recombinant plasmid pET-28a(+)-KstD containing the dehydrogenase encoding gene shown in claim 1.

[0007] In a third aspect, the present invention provides a recombinant engineered bacterium, wherein the pET-28a(+)-KstD described in claim 2 is transformed into BL21 competent cells, and then inoculated into a culture medium containing kanamycin to induce expression, thereby obtaining a strain of the recombinant engineered bacterium.

[0008] On the basis of the above technical scheme, preferably, the induction expression method is: the recombinant engineered bacterial strain is inoculated into LB medium containing kanamycin resistance, and after overnight culture, it is transferred to fresh LB medium containing kanamycin resistance, and IPTG is added when the bacterial solution OD600nm is 0.6-0.8, and the cells are cultured for 10-20 hours, and the cells are collected by centrifugation, resuspended with Tris-HCl, and the cells are ultrasonically disrupted to observe the protein expression.

[0009] In a fourth aspect, the present invention provides a method for synthesizing 11α-OH-ADD by recombinant engineering bacteria, comprising the following steps:

[0010] S1, inoculating the recombinant engineered bacterial strain into a culture medium containing kanamycin for fermentation, and centrifuging to obtain bacterial sludge;

[0011] S2, two-liquid phase conversion: the reaction system includes 11α-OH-AD, bacterial sludge, buffer, organic solvent and menadione or phenazine methyl sulfate, and the conversion is carried out under the conditions of 30-37°C and 150-200rpm shaking culture. The reaction formula is as follows:

[0012]

[0013] On the basis of the above technical scheme, preferably, a 50 mL reaction system contains 1-2 g of 11α-OH-AD, 20-25 mL of PBS, 25-30 mL of an organic solvent, 5-10 g of bacterial sludge, and 0.01-0.02 g of menadione or phenazine methyl sulfate.

[0014] On the basis of the above technical solution, preferably, the organic solvent is n-octane or butyl acetate.

[0015] On the basis of the above technical solution, preferably, the fermentation method comprises the following steps:

[0016] S1, inoculate the recombinant engineered bacterial strain into LB medium containing kanamycin, culture overnight at 30-37°C and 200-250rpm, then transfer to seed shake flask medium containing kanamycin, culture for 6-8h at 30-37°C and 200-250rpm to obtain fermentation seeds;

[0017] S2, inoculate the fermentation seeds into the fermentation tank, add kanamycin, culture at 30-37°C until the OD600 value reaches 18-22, cool to 20°C, add IPTG and feed medium to induce fermentation, collect the fermentation liquid and centrifuge to obtain bacterial sludge.

[0018] On the basis of the above technical solution, preferably, the components of the seed shake flask culture medium are: LB culture medium containing 8-12wt% peptone, 5-15wt% sodium chloride, and 3-6wt% yeast extract powder.

[0019] On the basis of the above technical scheme, preferably, the components of the fermentation tank culture medium are: 4-6 g / L glycerol, 15-20 g / L peptone, 20-25 g / L yeast extract powder, 2-3 g / L potassium dihydrogen phosphate, 15-18 g / L dipotassium hydrogen phosphate and 0.5-1 g / L defoaming agent silicone oil, and the balance is deionized water.

[0020] Based on the above technical solution, preferably, the components of the feed medium are: 500-700 g / L glycerol and 40-60 g / L diammonium hydrogen phosphate

[0021] Compared with the prior art, the dehydrogenase and the method for synthesizing 11α-OH-ADD of the present invention have the following advantages:

[0022] Beneficial effects:

[0023] (1) The dehydrogenase of the present invention can convert the substrate 11α-OH-AD into 11α-OH-ADD. A two-liquid phase conversion system is used. When the feed is as high as 40 g / L, the conversion rate is about 97% after 23 hours of reaction, providing a new and efficient dehydrogenation conversion preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 is the SDS-PAGE protein expression diagram of dehydrogenase KstD;

[0026] Figure 2 HPLC chart of 11α-OH-ADD prepared in Example 6;

[0027] Figure 3 This is the HPLC chart of 11α-OH-ADD prepared in Comparative Example 1. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In a first aspect, the present invention provides a dehydrogenase KstD, whose nucleotide sequence is shown in SEQ ID NO:1 and amino acid sequence is shown in SEQ ID NO:2.

[0030] In the second aspect, the present invention provides a recombinant engineered bacterium: the above-mentioned dehydrogenase KstD sequence is sent to Sangon Biotech (Shanghai) Co., Ltd. for sequence synthesis, and the recombinant plasmid pET-28a(+)-KstD is constructed, transformed into BL21(DE3) competent cells, and then inoculated into a culture medium containing kanamycin to induce expression, thereby obtaining a strain of the recombinant engineered bacterium.

[0031] Dehydrogenase KstD induction expression method: Take a fresh single colony of the above-mentioned synthetic recombinant engineering bacterial strain and inoculate it into LB medium containing kanamycin resistance (final concentration of 50 mg / mL), transfer it to fresh LB medium containing kanamycin resistance (final concentration of 50 mg / mL) after overnight culture, and culture it at 37°C until the OD600nm of the bacterial solution is 0.6-0.8. Add IPTG (final concentration of 4.2mM) and culture it at 20°C for about 15h. Collect the bacteria by centrifugation, resuspend the bacteria with Tris-Hcl (50mM pH=8.0), disrupt the cells by ultrasound, and observe the protein expression by SDS-PAGE. The protein is about 95kDa in size and is mainly expressed in the supernatant (see Figure 1 ).

[0032] In a third aspect, the present invention provides a method for synthesizing 11α-OH-ADD by recombinant engineering bacteria, and the specific method is described in detail by the following examples.

[0033] Example 1

[0034] The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria in this embodiment comprises the following steps:

[0035] S1, fermentation of dehydrogenase strains

[0036] S11, pick a newly streaked single colony, inoculate it into 10 mL of LB test tube medium (add kanamycin to a final concentration of 50 mg / mL), culture it at 37°C, 220 rpm overnight, then transfer it to seed shake flask medium (add kanamycin to a final concentration of 50 mg / mL), and culture it at 37°C, 220 rpm for 7 h to obtain fermentation seeds.

[0037] The components of the seed shake flask medium are: LB medium containing 10wt% peptone, 10wt% sodium chloride and 5wt% yeast extract powder, sterilized at 121°C for 30min.

[0038] S12, inoculate the fermenter with bacteria at 10% of the inoculum volume, add kanamycin (final concentration of 50 mg / mL), adjust the air volume and rotation speed in time, culture at 37°C until the OD600 value reaches 20, cool to 20°C, add IPTG (final concentration of 0.42 mM) and feed medium, induce for about 15 hours, collect the fermentation broth and centrifuge to obtain bacterial sludge.

[0039] The components of the fermentation tank culture medium are: the components of the fermentation tank culture medium are: 5 g / L glycerol, 18 g / L peptone, 24 g / L yeast extract powder, 2.3 g / L potassium dihydrogen phosphate, 16 g / L dipotassium hydrogen phosphate and 0.8 g / L defoaming agent silicone oil, and the balance is deionized water.

[0040] The components of the feed medium were: 600 g / L glycerol and 50 g / L diammonium phosphate.

[0041] Before use, the culture medium was adjusted to pH 7.2 with sodium hydroxide and sterilized at 121°C for 30 min.

[0042] S2, two-liquid phase conversion: 50 mL of the reaction system contained 1 g of 11α-OH-AD, 25 mL of PBS (0.1 M, pH 7.4), 25 mL of n-octane, 5 g of bacterial sludge, and 0.01 g of menadione. The conversion was carried out at 37°C and 180 rpm on a shaking table. The enzyme conversion reaction liquid was randomly collected at different time points, and the contents of the substrate 11α-OH-AD and the product 11α-OH-ADD were detected by liquid phase.

[0043] Example 2

[0044] The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria in this embodiment comprises the following steps:

[0045] S1, fermentation of dehydrogenase strains

[0046] S11, pick a newly streaked single colony, inoculate it into 10 mL of LB test tube medium (add kanamycin to a final concentration of 50 mg / mL), culture it at 30°C, 250 rpm overnight, then transfer it to seed shake flask medium (add kanamycin to a final concentration of 50 mg / mL), and culture it at 30°C, 250 rpm for 6 h to obtain fermentation seeds.

[0047] The components of the seed shake flask medium are: LB medium containing 8wt% peptone, 15wt% sodium chloride and 6wt% yeast extract powder, sterilized at 121°C for 30min.

[0048] S12, inoculate the fermenter with bacteria at 10% of the inoculum volume, add kanamycin (final concentration of 50 mg / mL), adjust the air volume and rotation speed in time, culture at 30°C until the OD600 value reaches 18, then cool to 20°C, add IPTG (final concentration of 0.42 mM) and feed medium, induce for about 15 hours, collect the fermentation broth and centrifuge to obtain bacterial sludge.

[0049] The components of the fermentation tank culture medium are: the components of the fermentation tank culture medium are: glycerol 4g / L, peptone 20g / L, yeast extract powder 25g / L, potassium dihydrogen phosphate 2g / L, dipotassium hydrogen phosphate 18g / L and defoaming agent silicone oil 1g / L, and the balance is deionized water.

[0050] The components of the feed medium were: 500 g / L glycerol and 60 g / L diammonium phosphate.

[0051] Before use, the culture medium was adjusted to pH 7.2 with sodium hydroxide and sterilized at 121°C for 30 min.

[0052] S2, two-liquid phase conversion: 50 mL of the reaction system contained 1.5 g of 11α-OH-AD, 20 mL of PBS (pH 7.4), 30 mL of butyl acetate, 7.5 g of bacterial sludge, and 0.015 g of phenazine methyl sulfate (PMS). The conversion was carried out at 35°C and 150 rpm on a shaking table. The enzyme conversion reaction liquid was randomly collected at different time points, and the contents of the substrate 11α-OH-AD and the product 11α-OH-ADD were detected by liquid phase.

[0053] Example 3

[0054] The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria in this embodiment comprises the following steps:

[0055] S1, fermentation of dehydrogenase strains

[0056] S11, pick a newly streaked single colony, inoculate it into 10 mL of LB test tube medium (add kanamycin to a final concentration of 50 mg / mL), culture it at 35°C, 200 rpm overnight, then transfer it to seed shake flask medium (add kanamycin to a final concentration of 50 mg / mL), and culture it at 35°C, 200 rpm for 8 h to obtain fermentation seeds.

[0057] The components of the seed shake flask medium are: LB medium containing 12wt% peptone, 5wt% sodium chloride and 3wt% yeast extract powder, sterilized at 121°C for 30min.

[0058] S12, inoculate the fermenter with bacteria at 10% inoculation volume, add kanamycin (final concentration of 50 mg / mL), adjust the air volume and rotation speed in time, culture at 35°C until the OD600 value reaches 22, then cool to 20°C, add IPTG (final concentration of 0.42 mM) and feed medium, induce for about 15 hours, collect the fermentation broth and centrifuge to obtain bacterial sludge.

[0059] The components of the fermentation tank culture medium are: the components of the fermentation tank culture medium are: 6 g / L glycerol, 15 g / L peptone, 20 g / L yeast extract powder, 3 g / L potassium dihydrogen phosphate, 15 g / L dipotassium hydrogen phosphate and 0.5 g / L defoaming agent silicone oil, and the balance is deionized water.

[0060] The components of the feed medium were: 700 g / L glycerol and 40 g / L diammonium phosphate.

[0061] Before use, the culture medium was adjusted to pH 7.2 with sodium hydroxide and sterilized at 121°C for 30 min.

[0062] S2, two-liquid phase conversion: 50 mL of the reaction system contains 2 g of 11α-OH-AD, 25 mL of PBS (pH 7.4), 25 mL of n-octane, 10 g of bacterial sludge, and 0.02 g of menadione. The conversion was carried out at 30°C and 200 rpm on a shaking table. The enzyme conversion reaction liquid was randomly collected at different time points, and the contents of the substrate 11α-OH-AD and the product 11α-OH-ADD were detected by liquid phase.

[0063] Conversion rate determination: After the conversion solution is diluted to an appropriate concentration, the supernatant is centrifuged and filtered with a 0.22 μm organic filter membrane, and the substrate and product are detected by the high performance liquid chromatography area normalization method. Conversion rate calculation: Conversion rate = [C1 / (C1+C2)] × 100%, C1 is the peak area content % measured by the product, and C2 is the peak area content % measured by the substrate.

[0064] Table 1 Conversion rate of Examples 1-3

[0065]

[0066] As shown in Table 1, the conversion rate of Examples 1-3 of the present invention can reach more than 95% after reacting for 23 hours, indicating that the dehydrogenase of the present invention has the effect of rapidly and efficiently converting 11α-OH-AD.

[0067] Compared with Example 1, the reaction system of two-liquid phase transformation in Example 4 is different, specifically: 11α-OH-AD: 1 g (20 g / L), PBS (0.1 M pH 7.4): 25 mL, n-octane: 25 mL (50%), 5 times complete cell sludge: 5 g, PMS: 0.01 g.

[0068] Example 5

[0069] Compared with Example 1, the reaction system of two-liquid phase transformation in Example 5 is different, specifically: 11α-OH-AD: 1.5 g (30 g / L), PBS (pH 7.4): 20 mL, n-octane: 30 mL (60%), 5 times complete cell sludge: 7.5 g, and menadione: 0.015 g.

[0070] Example 6

[0071] Compared with Example 1, the reaction system of two-liquid phase transformation in Example 6 is different, specifically: 11α-OH-AD: 2 g (40 g / L), PBS (pH 7.4): 20 mL, n-octane: 30 mL (60%), 5 times complete cell sludge: 10 g, and menadione: 0.02 g.

[0072] Table 2 Effect of menadione and PMS on conversion rate

[0073]

[0074] The results in Table 2 show that the use of menadione or PMS in the reaction system has little effect on the conversion, and both can achieve a conversion rate of about 97%.

[0075] Table 3 Effect of substrate feed amount on conversion rate

[0076]

[0077]

[0078] The results in Table 3 show that when the feed is increased to 30-40 g / L, the dehydrogenase can still be converted to a level of about 97%. The HPLC test results of 40 g / L-73 h in Example 6 are shown in Table 3. Figure 2 .

[0079] Based on the above test results: using the n-octane two-phase conversion system, when the substrate 11α-OH-AD feed is 20-40g / L, the n-octane addition amount is between 50%-60%, using 5 times fermented dehydrogenase complete cell sludge, and adding 1% of the substrate amount of menadione, a conversion rate of about 97% can be achieved.

[0080] Comparative Example 1

[0081] Comparative Example 1 Compared with Example 1, the conversion system is different, and aqueous phase conversion is adopted. The specific method is as follows: 1g substrate 11α-OH-AD and 45mL Tris-HCl (50mM, pH 8.0) are added to a 250mL shaking flask, and the substrate is treated with microwave ultrasound to make it uniformly dispersed, and then 4g fermentation complete cell mud, 0.01g menadione, and 5mL of isopropanol are added. The conversion is carried out overnight at 37°C and 180rpm on a shaking table. The enzyme conversion reaction liquid is randomly collected at different time points, and the content of the substrate 11α-OH-AD and the product 11α-OH-ADD therein is detected by liquid phase. The results are shown in Tables 4 and 4 below. Figure 3 .

[0082] Table 4 Conversion rate of comparative example 1

[0083] Product content% Substrate content% Conversion Rate % 23h 24.79 69.36 26.33032395 47h 28.13 66.07 29.86199575 71h 30.88 61.8 33.31894691

[0084] As shown in Table 4, as the conversion time increases, the conversion level increases to a certain extent, but the final conversion rate is still only 33% in 71 hours (see Figure 3 ).

[0085] Comparative Example 2

[0086] Effect of different organic solvents on conversion rate:

[0087] 50mL reaction system: 1g (20g / L) substrate, 35mL PBS (10mM, pH7.4), 15mL (30%) organic solvent, 4-fold fermentation whole cell sludge (4g), 0.01g menadione.

[0088] Conversion conditions: overnight conversion on a shaker at 37°C and 180 rpm. The enzyme conversion reaction solution was randomly collected at different time points, and the contents of substrate 11α-OH-AD and product 11α-OH-ADD therein were detected by liquid phase. The detection results are shown in Table 5.

[0089] Table 5 Effect of different organic solvents on conversion rate

[0090] Types of organic solvents Product content% Substrate content% Conversion rate % at 66h n-octane 84.64 14.02 85.78958038 Tert-Butanol 0.14 97.11 0.143958869 n-Hexanol 7.23 88.6 7.544610247 n-Hexane 52.02 45.65 53.26098085 Ethyl acetate 4.61 90.57 4.843454507 Butyl acetate 64.2 13.62 82.49807247 Dichloromethane 10 84.26 10.60895396 Chloroform 13.97 80.92 14.72231004

[0091] The results in Table 5 show that n-octane has the highest conversion efficiency, followed by butyl acetate.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dehydrogenase, characterized in that The dehydrogenase is KstD, whose nucleotide sequence is shown in SEQ ID NO:1, and whose amino acid sequence is shown in SEQ ID NO:

2.

2. A recombinant expression vector, characterized in that: The recombinant expression vector is a recombinant plasmid pET-28a(+)-KstD containing the dehydrogenase encoding gene shown in claim 1.

3. A recombinant engineered bacterium, characterized in that: The pET-28a(+)-KstD described in claim 2 is transformed into BL21 competent cells, and then inoculated into a culture medium containing kanamycin to induce expression, thereby obtaining a recombinant engineered bacterial strain.

4. A recombinant engineered bacterium according to claim 3, characterized in that: The induction expression method is as follows: the recombinant engineered bacterial strain is inoculated into LB medium containing kanamycin resistance, and after overnight culture, it is transferred to fresh LB medium containing kanamycin resistance, and IPTG is added when the bacterial solution OD600nm is 0.6-0.8 to culture for 10-20 hours, the bacteria are collected by centrifugation, the bacteria are resuspended with Tris-Hcl, the cells are ultrasonically disrupted and the protein expression is observed.

5. The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria according to claim 3 or 4, characterized in that: The following steps are involved: S1, inoculating the recombinant engineered bacterial strain into a culture medium containing kanamycin for fermentation, and obtaining bacterial sludge by centrifugation; S2, two-liquid phase transformation: the reaction system includes 11α-OH-AD, bacterial sludge, buffer, organic solvent and menadione or phenazine methyl sulfate, and the transformation is carried out under the conditions of 30-37°C and 150-200rpm shaking culture.

6. The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria according to claim 5, characterized in that: Each 50 mL reaction system contains 1-2 g of 11α-OH-AD, 20-25 mL of PBS, 25-30 mL of an organic solvent, 5-10 g of bacterial mud, and 0.01-0.02 g of menadione or phenazine methyl sulfate.

7. The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria according to claim 5, characterized in that: The organic solvent is n-octane or butyl acetate.

8. The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria according to claim 5, characterized in that: The fermentation method comprises the following steps: S1, inoculate the recombinant engineered bacterial strain into LB medium containing kanamycin, culture overnight at 30-37°C and 200-250rpm, then transfer to seed shake flask medium containing kanamycin, culture for 6-8h at 30-37°C and 200-250rpm to obtain fermentation seeds; S2, inoculate the fermentation seeds into the fermentation tank, add kanamycin, culture at 30-37°C until the OD600 value reaches 18-22, cool to 20°C, add IPTG and feed medium to induce fermentation, collect the fermentation liquid and centrifuge to obtain bacterial sludge.

9. The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria according to claim 8, characterized in that: The components of the seed shake flask culture medium are: LB culture medium containing 8-12wt% peptone, 5-15wt% sodium chloride and 3-6wt% yeast extract powder.

10. The method for synthesizing 11α-OH-ADD by recombinant engineering bacteria according to claim 8, characterized in that: The components of the fermentation tank culture medium are: 4-6 g / L glycerol, 15-20 g / L peptone, 20-25 g / L yeast extract powder, 2-3 g / L potassium dihydrogen phosphate, 15-18 g / L dipotassium hydrogen phosphate and 0.5-1 g / L defoamer, and the balance is deionized water; The components of the feed medium are: 500-700 g / L glycerol and 40-60 g / L diammonium phosphate.

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