Self-assembly double-enzyme compound, preparation method and application in synthesis of prednisone intermediate

Abstract

The invention discloses a self-assembly double-enzyme compound, a preparation method and application of the self-assembly double-enzyme compound in synthesis of a prednisone intermediate, and the self-assembly double-enzyme compound is obtained by fusing 11 alpha-hydroxylase and 1, 2-dehydrogenase on the basis of a SpyCatcher / SpyTag system. According to the method, a self-assembled double-enzyme compound of 11alpha-hydroxylase and 1, 2-dehydrogenase is constructed through a SpyCatcher / SpyTag system, 17alpha-hydroxyprogesterone is taken as a substrate, double-enzyme cascade one-step catalytic reaction is realized, the reaction process is simplified, and separation and purification operation of an intermediate product is omitted. The self-assembled double-enzyme compound constructed by the invention forms a high-efficiency substrate channel, accelerates the rapid transfer of an intermediate from a catalytic activity center of a first enzyme to a catalytic activity center of a second enzyme, improves the catalytic efficiency, is suitable for industrial application, and has a conversion rate of 99.6%.

Description

(1) Technical field

[0001] The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to a self-assembling double enzyme complex, a preparation method and an application in synthesizing a prednisone intermediate. (2) Background technology

[0002] (11α)-11,17-Dihydroxypregna-1,4-diene-3,20-dione is an important intermediate in the synthesis of the steroidal drug prednisone. Prednisone, also known as cortisone, is a glucocorticoid with anti-inflammatory and anti-allergic properties. It inhibits connective tissue proliferation, reduces capillary wall and cell membrane permeability, reduces inflammatory exudates, and inhibits the formation and release of histamine and other toxic substances. When used in combination with a large number of antimicrobial agents in the setting of severe toxic infections, it has excellent effects in reducing fever, combating toxicity, inflammation, and shock, as well as promoting symptom relief.

[0003] To simplify reaction steps and improve catalytic efficiency, this patent successfully constructed four dual-enzyme molecular components consisting of 11α-hydroxylase (HDE) and 1,2-dehydrogenase (DHE) using SpyCatcher / SpyTag technology. This complex can combine the two biocatalytic steps of 11α-hydroxylation and 1,2-dehydrogenation of 17α-hydroxyprogesterone into a single reaction, thereby simplifying the reaction process and improving the efficiency of biotransformation.

[0004] In the biosynthesis of steroidal compounds, enzyme catalysis stands out for its high selectivity and specificity. Specific enzymes such as cytochrome P450 monooxygenases and hydroxylases can precisely recognize specific sites on the steroid molecule and initiate reactions, eliminating the need for complex chemical synthesis steps and efficiently producing a single active configuration, simplifying the chemical resolution process. Enzyme-catalyzed reactions offer mild conditions and low energy consumption, aligning with green chemistry principles and reducing side reactions and equipment costs. Enzyme catalysis can complete multiple chemical transformations in one go, shortening synthetic pathways and increasing yields. Combined with immobilization technology, enzyme catalysis enables efficient substrate conversion and enzyme reuse. The diverse catalytic functions of enzyme catalysis allow for flexible manipulation of steroidal structure. Combined with microbial fermentation, enzyme catalysis can convert phytosterols into high-value-added products on a large scale. In industrial applications, enzyme catalysis has become a core technology for steroidal drug synthesis due to its environmentally friendly nature. Driven by synthetic biology, enzyme catalysis will continue to promote innovation in the green manufacturing of steroidal compounds, but enzyme stability, substrate solubility, and catalytic efficiency still require improvement.

[0005] In the prior art, the synthesis of (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione primarily involves the hydroxylation of 17α-hydroxyprogesterone at the 11α position under the action of 11α-hydroxylase. Subsequently, under the action of 1,2-dehydrogenase, 11,17-dihydroxyprogesterone undergoes a dehydrogenation reaction at the 1,2 positions to produce (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione. However, this method requires a two-step biotransformation process, resulting in a complex reaction flow and a reduction in substrate conversion efficiency.

[0006] Multi-enzyme cascade catalytic reactions achieve multi-step continuous transformations by integrating multiple enzymes. They are characterized by high integration and simplified steps. They can reduce dependence on cofactors through coenzyme regeneration systems without the need for intermediate separation and complete complex pathway transformations. The improvement in efficiency and yield is due to the substrate-directed channel effect and the inhibition of side reactions. The precise selectivity and high purity are due to the strict sequential control of the cascade reaction and the environmental stability of the system. Green sustainability is reflected in low toxic waste emissions, mild reaction conditions and efficient utilization of biomass resources. Modular design and dynamic control enable it to be flexibly adapted to different synthetic needs. This technology has been applied on an industrial scale to the production of steroid drugs, reducing costs and increasing production capacity. Despite the challenges, through technological advances, multi-enzyme cascade catalysis is driving the innovation of complex molecular manufacturing models.

[0007] The SpyTag / SpyCatcher system was developed by cleaving the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein FbaB. The CnaB2 domain can be split into two parts, allowing protein-peptide linkage via a stable covalent isopeptide bond between the peptide tag (i.e., SpyTag, abbreviated ST) and the protein domain (i.e., SpyCatcher, abbreviated SC). Stable linkage is achieved under mild conditions without the need for additional energy or catalysts. This system is highly specific, irreversible, and allows for modular design. Its advantages include rapid reaction kinetics, controllable multicomponent integration, and good biocompatibility. These properties have led to the SpyCatcher / SpyTag system's widespread application in areas such as protein nanostructure construction, living cell surface engineering, and optimization of multienzyme cascade systems. Compared to traditional methods, the SpyCatcher / SpyTag system provides an efficient and stable molecular "locking" tool suitable for synthetic biology, nanomedicine, and biomanufacturing. However, the application of the SpyCatcher / SpyTag system in dual-enzyme immobilization still faces many challenges. When SpyCatcher and SpyTag form multi-enzyme complexes with enzymes, the SpyCatcher and SpyTag genes can be linked upstream or downstream of the target gene. For example, SpyCatcher can be linked to the upstream (5' end) and downstream (3' end) ends of 11α-hydroxylase (HDE), respectively, to form the HDE-SC and SC-HDE fusion proteins. Similarly, SpyTag can be linked to the upstream (5' end) and downstream (3' end) ends of 1,2-dehydrogenase (DHE), respectively, to form the ST-DHE and DHE-ST fusion proteins. These four fusion proteins, when linked via SpyCatcher / SpyTag, can form four types of dual-enzyme complexes: DHE-ST / HDE-SC, ST-DHE / HDE-SC, DHE-ST / SC-HDE, and ST-DHE / SC-HDE. There are multiple connection sequences when SpyCatcher and SpyTag are connected to two enzyme molecules, and the dual-enzyme complexes formed by multiple connection sequences have different catalytic activities.

[0008] In addition, environmental factors such as pH, temperature, and ionic strength in the construction of dual-enzyme complexes have a significant impact on binding efficiency, and the protein scaffold is not stable enough at high temperatures or in organic solvents. Fusion tags may cause conformational changes in the enzyme, thereby reducing activity, while the compact structure produced by immobilization increases the resistance to material transfer. In the process of dual-enzyme co-immobilization, precise control of the enzyme ratio is a challenge, and the optimal conditions for different enzymes are often difficult to coordinate. In addition, the reusability of the dual-enzyme system is limited by the differences in the inactivation of each enzyme.

[0009] Therefore, it is still necessary to explore a suitable catalytic system to improve the synthesis efficiency of (11α)-11,17-dihydroxypregnane-1,4-diene-3,20-dione. (3) Summary of the invention

[0010] The present invention aims to provide a self-assembling dual-enzyme complex, a preparation method, and an application in the synthesis of prednisone intermediates. By utilizing the molecular self-assembly properties of SpyCatcher and SpyTag, 11α-hydroxylase (HDE) and 1,2-dehydrogenase (DHE) are fused to construct a dual-enzyme complex, which catalyzes the one-step synthesis of the substrate 17α-hydroxyprogesterone into (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione, thereby simplifying the reaction process and improving the efficiency of biotransformation, providing a green and efficient path for the synthesis of prednisone intermediates.

[0011] The technical solution adopted in the present invention is:

[0012] The present invention provides a self-assembling dual-enzyme complex. The self-assembling dual-enzyme complex is obtained by fusing 11α-hydroxylase and 1,2-dehydrogenase based on the SpyCatcher / SpyTag system.

[0013] Furthermore, the amino acid sequence of the 11α-hydroxylase is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0014] Furthermore, the amino acid sequence of the 1,2-dehydrogenase is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5.

[0015] Furthermore, in the SpyCatcher / SpyTag system, the amino acid sequence of SpyCatcher is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; the amino acid sequence of SpyTag is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7.

[0016] Furthermore, the self-assembling dual-enzyme complex is obtained by self-assembly of a fusion enzyme formed by connecting SpyCatcher to the N-terminus (5') or C-terminus (3') of 11α-hydroxylase and a fusion enzyme formed by connecting SpyTag to the N-terminus or C-terminus of 1,2-dehydrogenase; the fusion enzyme formed by connecting SpyCatcher to the C-terminus of 11α-hydroxylase is denoted as HDE-SC, and the amino acid sequence of HDE-SC is shown in SEQ ID NO.9; the fusion enzyme formed by connecting SpyCatcher to the N-terminus of 11α-hydroxylase is denoted as SC-HDE, and the amino acid sequence of SC-HDE is shown in SEQ ID NO.10; the fusion enzyme formed by connecting SpyTag to the N-terminus of 1,2-dehydrogenase is denoted as ST-DHE, and the amino acid sequence of ST-DHE is shown in SEQ ID NO.11; the fusion enzyme formed by connecting SpyTag to the C-terminus of 1,2-dehydrogenase is denoted as DHE-ST, and the amino acid sequence of DHE-ST is shown in SEQ ID NO. As shown in IDNO.12.

[0017] The present invention also provides a method for preparing the self-assembling dual-enzyme complex, which comprises the following steps:

[0018] (1) Artificially synthesizing a recombinant plasmid containing SpyCatcher and HDE (preferably pPIC3.5K-SC-HDE and pPIC3.5K-HDE-SC), and introducing the recombinant plasmid into Pichia pastoris competent cells to construct a genetically engineered Pichia pastoris strain;

[0019] (2) artificially synthesizing a recombinant plasmid containing SpyTag and DHE (preferably pPIC3.5K-SC-HDE and pPIC3.5K-HDE-SC), introducing the recombinant plasmid into Escherichia coli competent cells, and constructing genetically engineered Escherichia coli bacteria;

[0020] (3) fermenting the engineered bacteria constructed in steps (1) and (2), crushing the culture solution, and collecting the supernatant by centrifugation to obtain the respective enzyme solutions;

[0021] (4) The enzyme solution in step (3) was adjusted to the same protein concentration and then mixed in equal volumes, and shaken at 37° C. and 180 rpm for 3 h to obtain the self-assembled dual-enzyme complex.

[0022] Furthermore, the Pichia pastoris is preferably Pichia pastoris GS115; and the Escherichia coli is preferably E. coli BL21 (DE3).

[0023] Furthermore, the steps of preparing the enzyme solution by fermentation of genetically engineered Pichia pastoris are as follows:

[0024] 1) Shake flask culture of engineered bacteria

[0025] The genetically engineered Pichia pastoris was inoculated into YPD medium and cultured at 30°C and 220 rpm for 16 hours; then inoculated into BMGY medium at a 5% (v / v) inoculum and cultured under the same conditions for 24 hours; the bacterial solution was transferred to a sterile centrifuge tube, centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed with sterile distilled water to completely remove glycerol from the culture medium, and then centrifuged under the same conditions to collect the cells and discard the supernatant. The cells were transferred to BMMY medium and induced for expression at 30°C and 220 rpm for 12-28 hours.

[0026] 2) Seed solution preparation

[0027] The culture solution after induction expression in step 1) was inoculated on a YPD medium plate, activated at 30°C for 2-3 days, and then a single colony was picked and transferred to BMGY medium. The culture was cultured at 30°C and 220 rpm for 24 hours to a constant OD of 0. 600 When the temperature reaches 2-6, the seed solution is obtained;

[0028] 3) 5L fermenter pretreatment

[0029] A 5-L fermenter was filled with 2 L of BSM medium and calibrated with a pH electrode and a dissolved oxygen electrode. The fermenter was sterilized at 121°C for 20 min. After the fermenter cooled, sterile air was introduced into the fermenter. The fermenter speed was set to 600 rpm, the temperature to 30°C, and the ventilation volume was set to 4 vvm. The pH was adjusted to 5.0 using an acid-base pump. After ventilation for 1 h, the dissolved oxygen (DO) value in this state was set to 100%. BSM medium: H2PO4 26.7 mL / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 mL / L, and the solvent was water.

[0030] 4) Inoculation and basic growth stage

[0031] The seed solution from step 2) was inoculated into the fermenter pretreated in step 3) at a 5% (v / v) inoculum, and a PTM1 basal salt solution with a final volume concentration of 0.435% (v / v) was added. The culture was incubated at 30°C and 600 rpm for 24 hours, during which the aeration rate was adjusted to stabilize the DO value above 20%. After 24 hours, due to the depletion of glycerol in the BSM medium, the DO value rapidly soared to 100%, marking the end of the basal growth phase, and the wet weight of the bacteria reached 90 to 150 g·L. -1PTM1 basic salt solution: CuSO4·5H2O 6.0 g / L, sodium iodide 0.08 g / L, MnSO4·H2O 3.0 g / L, Na2MoO4·2H2O 0.2 g / L, H3BO3 0.02 g / L, CoCl2 0.5 g / L, ZnCl2 20.0 g / L, FeSO4·7H2O 65.0 g / L, biotin 0.2 g / L, sulfuric acid 5.0 mL / L, distilled water 1000 mL;

[0032] 5) Glycerol feeding stage

[0033] At 36.3 mL·h -1 Glycerol feed medium was added at a rate of 100 μg / L and the DO value was maintained above 20%. When the wet weight of the bacterial cells exceeded 180 g·L -1 The glycerol feed was stopped at 1 ℃ and maintained for 1 hour to allow the glycerol in the fermenter to be completely consumed. The fermentation broth was centrifuged at 8000 rpm for 20 minutes and the cells were collected. Glycerol feed medium: 0.5 g / L glycerol aqueous solution containing 1.2% (v / v) PTM1 basal salt solution;

[0034] 6) Preparation of enzyme solution

[0035] The cells obtained in step 5) were homogenized and disrupted (preferably using an AH-basic homogenizer at 1100-1200 bar for 4 times) to obtain an enzyme solution containing cell fragments. The supernatant was obtained by centrifugation, and the protein content was detected using Nanodrop. The protein concentration of the enzyme solution was adjusted to 5 g / L using 0.2 mM phosphate buffer, pH 8.0, to obtain SC-HDE and HDE-SC enzyme solutions.

[0036] Furthermore, the steps of preparing enzyme solution by fermentation of genetically engineered Escherichia coli are as follows:

[0037] The genetically engineered E. coli bacteria were inoculated into a conical flask containing LB liquid medium and incubated at 37°C and 180 rpm until the OD600 value reached 1.6 to obtain a seed solution. The seed solution was inoculated into a 5 L fermentor containing 2 L fermentation medium at 4% (v / v), stirred at 37°C and 900 rpm, with a ventilation volume of 6 vvm and a pH of 7.0. Feed medium was added through a flow system to maintain a dissolved oxygen level of 30%. When the OD600 value reached 80, IPTG was added at a final concentration of 0.5 mM. , induction at 25°C for 20 hours, and fermentation was stopped when the bacterial concentration reached 150 g / L; the fermentation broth was centrifuged at 8000 rpm for 20 minutes to collect the bacterial cells; the bacterial cells were homogenized and disrupted (preferably using an AH-basic homogenizer at 1100-1200 bar for 2 times), and the supernatant was obtained by centrifugation. The protein content was detected using Nanodrop, and the enzyme solution was adjusted to a protein concentration of 5 g / L with 0.2 mM phosphate buffer pH 8.0 to obtain ST-DHE and DHE-ST enzyme solutions, respectively;

[0038] Fermentation medium: yeast extract 12.0 g / L, peptone 20.0 g / L, NaCl 1.0 g / L, (NH4)2SO4 6.0 g / L, MgSO4·7H2O 2.0 g / L, glycerol 7.5 mL / L, trace element solution 1 mL / L, KH2PO4 3 g / L, Na2HPO4·12H2O 8.2 g / L;

[0039] Trace element solution: FeCl3·4H2O 22.87g / L, ZnCl2 1.31g / L, CoCl2·6H2O 2.0g / L, CaCl2 0.76g / L, CuCl2·2H2O 1.25g / L, H3BO3 0.5g / L, MnSO4·H2O 2.0g / L, 2mol / L HCl 100mL / L, solvent is water.

[0040] The present invention also provides an application of the self-assembling dual enzyme complex in catalyzing 17α-hydroxyprogesterone to prepare a steroid drug prednisolone intermediate (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione.

[0041] Furthermore, the application method is as follows: using a self-assembled dual enzyme complex as a catalyst, 17α-hydroxyprogesterone as a substrate, adding metal ions, cyclodextrin and a cosolvent, and using 0.2 mM phosphate buffer at pH 7-9 as a reaction medium to form a conversion system, reacting at 25-45° C. and 100-200 rpm for 12-108 h (preferably 33° C. and 160 rpm for 96 h), separating and purifying the reaction solution to obtain (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione; the cosolvent includes dimethyl sulfoxide (DMSO), methanol, ethanol, isopropanol or acetone, preferably dimethyl sulfoxide; the metal ions include K + , Mg 2+ , Ca 2+ , Fe 2+ , preferably Fe 2+ .

[0042] Furthermore, the volume concentration of the cosolvent is 2-6%, preferably 4%; the concentration of the substrate is 40-100 g / L, preferably 90 g / L; the amount of the catalyst is 0.1-2 g / L based on protein content, preferably 1.7 g / L; the concentration of the metal ion is 0.5 mM; and the final concentration of cyclodextrin is 20 g / L.

[0043] Furthermore, the cyclodextrin includes α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), methyl-β-cyclodextrin (Me-β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD), preferably methyl-β-cyclodextrin.

[0044] Furthermore, the buffer is preferably a phosphate buffer with a pH of 8.0 and a 0.2 mM concentration.

[0045] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0046] The present invention constructs a self-assembled dual-enzyme complex of 11α-hydroxylase and 1,2-dehydrogenase through the SpyCatcher / SpyTag system, takes 17α-hydroxyprogesterone as a substrate, realizes a dual-enzyme cascade one-step catalytic reaction, simplifies the reaction process, and omits the separation and purification operation of the intermediate product.

[0047] The self-assembling dual-enzyme complex constructed by the present invention forms an efficient substrate channel, accelerates the rapid transfer of intermediates from the catalytic active center of the first enzyme to the catalytic active center of the second enzyme, improves the catalytic efficiency, is suitable for industrial applications, and has a conversion rate of 99.6%. (IV) Description of the accompanying drawings

[0048] Figure 1 Flowchart of the preparation of prednisone from 17α-hydroxyprogesterone catalyzed by 11α-hydroxylase and 1,2-dehydrogenase, a dual-enzyme complex.

[0049] Figure 2 , HPLC chromatogram of the reaction solution under the optimized conditions of Example 11. (V) Specific implementation methods

[0050] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0051] Example 1. Preparation of free enzyme HDE and two fusion enzymes HDE-SC and SC-HDE enzyme solutions

[0052] The culture medium formula involved in this embodiment is as follows:

[0053] YPD medium: yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L, agar 20 g / L, solvent is water.

[0054] MD medium: glucose 20 g / L, amino-free yeast nitrogen base (YNB) 13.4 g / L, agar 20 g / L, solvent is water.

[0055] BMGY medium: yeast powder 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 13.4 g / L, 500× biotin 1 mL / L, glycerol 10 mL / L, solvent is water.

[0056] BMMY medium: yeast powder 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 13.4 g / L, 500× biotin 1 mL / L, methanol 10 mL / L, solvent is water.

[0057] BSM medium: H2PO4 26.7 mL / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 mL / L, solvent is water.

[0058] Ammonia feed medium: 50% (v / v) ammonia.

[0059] Glycerol feed medium: 0.5 g / L glycerol aqueous solution containing 1.2% (v / v) PTM1 basal salt solution.

[0060] Methanol feed medium: methanol containing 1.2% (v / v) PTM1 basal salt solution and 4 g / L 17α-hydroxyprogesterone.

[0061] PTM1 basic salt solution: CuSO4·5H2O 6.0 g / L, sodium iodide 0.08 g / L, MnSO4·H2O 3.0 g / L, Na2MoO4·2H2O 0.2 g / L, H3BO3 0.02 g / L, CoCl2 0.5 g / L, ZnCl2 20.0 g / L, FeSO4·7H2O 65.0 g / L, biotin 0.2 g / L, sulfuric acid 5.0 mL / L, distilled water 1000 mL.

[0062] 1. Construction of recombinant plasmid

[0063] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the HDE recombinant plasmid pPIC3.5K-HDE containing free enzyme, pPIC3.5K-SC-HDE containing SC fusion enzyme connected to the 5' end of HDE, and pPIC3.5K-HDE-SC containing SC fusion enzyme connected to the 3' end of HDE, wherein the nucleotide sequence of HDE is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2, the nucleotide sequence of SC is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4, the amino acid sequence of HDE-SC is shown in SEQ ID NO.9, and the amino acid sequence of SC-HDE is shown in SEQ ID NO.10.

[0064] SEQ ID NO.2

[0065] 。

[0066] SEQ ID NO.4:

[0067] GDSATHIKFSKREDDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG.

[0068] 2、construction of bacteria

[0069] The recombinant plasmids pPIC3.5K-HDE, pPIC3.5K-SC-HDE, and pPIC3.5K-HDE-SC constructed in step 1 were linearized with the restriction endonuclease Sac I at 37°C for 2 h and recovered using a DNA purification kit (Biyuntian). Transformants were introduced into Pichia pastoris GS115 competent cells by electroporation. Transformants were inoculated onto MD medium plates and cultured at 30°C for 2 days until colonies emerged, resulting in the engineered strains P. pastoris GS115-pPIC3.5K-HDE, P. pastoris GS115-pPIC3.5K-SC-HDE, and P. pastoris GS115-pPIC3.5K-HDE-SC.

[0070] 3. Fermentation of engineered bacteria and preparation of HDE, SC-HDE, and HDE-SC enzyme solutions

[0071] (1) Shake flask culture of engineered bacteria

[0072] Transfer the transformants from the MD medium to 10 mL of YPD medium and incubate at 30°C, 220 rpm for 16 h. Inoculate the transformed cells at a 5% (v / v) inoculum into a 250 mL shake flask containing 40 mL of BMGY medium and incubate under the same conditions for 24 h. Transfer the bacterial suspension to a sterile 50 mL centrifuge tube, centrifuge at 5000 rpm for 5 min, discard the supernatant, and wash the cells with 40 mL of sterile distilled water to completely remove the glycerol in the culture medium. Collect the cells by centrifugation under the same conditions and discard the supernatant. Transfer the cells to a 250 mL shake flask containing 40 mL of BMMY medium and induce expression at 30°C, 220 rpm for 12-28 h.

[0073] (2) Seed solution preparation

[0074] The culture solution after induction expression in step (1) was inoculated on a YPD medium plate, activated at 30°C for 2-3 days, and then a single colony was picked and transferred to a 250 mL shake flask containing 100 mL of BMGY medium. The culture was cultured at 30°C and 220 rpm for 24 h to a OD of 0. 600 When the temperature reaches 2 to 6, the seed solution is obtained.

[0075] (3) 5L fermenter pretreatment

[0076] A 5-L fermenter was filled with 2 L of BSM medium and calibrated with a pH electrode and a dissolved oxygen electrode. The fermenter was sterilized at 121°C for 20 min. After the fermenter cooled, sterile air was introduced into the fermenter. The fermenter speed was set to 600 rpm, the temperature to 30°C, and the ventilation volume to 4 vvm. The pH was adjusted to 5.0 using an acid-base pump.

[0077] (4) Inoculation and basic growth stage

[0078] The seed solution from step (2) was inoculated into the fermenter pretreated in step (3) at a 5% (v / v) inoculum, and a PTM1 basal salt solution with a final volume concentration of 0.435% (v / v) was added. The culture was incubated at 30°C, 600 rpm for 24 h, during which the ventilation rate was adjusted to stabilize the DO value above 20%. After 24 h, due to the depletion of glycerol in the BSM medium, the DO value quickly soared to 100%, marking the end of the basal growth phase, and the wet weight of the bacteria reached 150 g·L. -1 .

[0079] (5) Glycerol feeding stage

[0080] At 36.3 mL·h -1 Glycerol feed medium was added at a rate of 100 μg / L for about 5 h, and the DO value was maintained above 20%. -1 The glycerol feed was stopped at 1 ℃ and maintained for 1 hour to allow the glycerol in the fermenter to be completely consumed. The fermentation broth was centrifuged at 8000 rpm for 20 minutes and the cells were collected.

[0081] (6) Bacterial cell disruption and preparation of HDE, SC-HDE, and HDE-SC enzyme solutions

[0082] The bacteria obtained in step (5) were broken into cells four times using an AH-basic homogenizer at 1100-1200 bar to obtain an enzyme solution containing cell fragments. The supernatant was obtained by centrifugation, and the protein content was detected using a Nanodrop microspectrophotometer. The protein concentration was adjusted with 0.2 mM phosphate buffer pH 8.0 to obtain HDE, SC-HDE, and HDE-SC enzyme solutions with a protein concentration of 5 g / L.

[0083] Example 2, Preparation of HDE, ST-DHE and DHE-ST Enzyme Solutions

[0084] The culture medium formula involved in this embodiment is as follows:

[0085] LB liquid medium: peptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, solvent is water.

[0086] LB solid medium composition: peptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, agar 20 g / L, solvent is water.

[0087] Fermentation medium (YT-M9): yeast extract 12.0 g / L, peptone 20.0 g / L, NaCl 1.0 g / L, (NH4)2SO4 6.0 g / L, MgSO4·7H2O 2.0 g / L, glycerol 7.5 mL / L, trace element solution 1 mL / L, KH2PO4 3 g / L, Na2HPO 4· MgSO4·7H2O and trace element solution were sterilized separately under flame protection and then added to the fermenter. The initial volume of basal medium in each tank was 2 L.

[0088] Trace element solution: FeCl3·4H2O 22.87g / L, ZnCl2 1.31g / L, CoCl2·6H2O 2.0g / L, CaCl2 0.76g / L, CuCl2·2H2O 1.25g / L, H3BO3 0.5g / L, MnSO4·H2O 2.0g / L, 2mol / L HCl 100mL / L, solvent is water.

[0089] Feed medium: glycerol 500 g / L, yeast extract 10 g / L, vitamin C 0.45 g / L, solvent is water.

[0090] Preparation of 2 mol / L HCl: In a fume hood, add 20 mL of HCl to a sterile feeding bottle containing 110 mL of pure water for pH control during the culture process.

[0091] Preparation of 25% ammonia: Add analytical grade ammonia to a 500 mL feed bottle sterilized at 121°C for 30 minutes in a fume hood. This is used for pH control during the tank culture. All sterilization conditions are 121°C for 30 minutes. Sterilize the feed and pH adjustment reagents simultaneously with the fermentor.

[0092] 1. Construction of recombinant plasmid

[0093] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the recombinant plasmid pET-28(a)-DHE containing the free enzyme DHE, pET-28(a)-ST-DHE containing the fusion enzyme ST connected to the 5' end of DHE, and pET-28(a)-DHE-ST containing the fusion enzyme ST connected to the 3' end of DHE, wherein the nucleotide sequence of DHE is shown in SEQ ID NO.5 and the amino acid sequence is shown in SEQ ID NO.6, wherein the nucleotide sequence of ST is shown in SEQ ID NO.7 and the amino acid sequence is shown in SEQ ID NO.8, the amino acid sequence of ST-DHE is shown in SEQ ID NO.11, and the amino acid sequence of DHE-ST is shown in SEQ ID NO.12.

[0094] SEQ ID NO.6 (Amino acid sequence of DHE):

[0095] MPFFTGLLEIYHSLILDNPVQTLSTIIVLAAAYWLATLQPSDLPELNPAKPFEFTNRRRVHEFVENSKSLLARGRELHGHEPYRLMSEWGSLIVLPPECADELRNDPRMDFETPTTDDSHGYIPGFDALNADPNLTKVVTKYLTKALNKLTAPISHEASIAMKAVLGDDPDWREIYPARDLLQLVARMSTRVFLGEEMCNNQDWIQTSSQYAALAFGVGDKLRIYPRMIRPIVHWFMPSCWELRRSLRRCRQILTPYIHKRKSLKGTTDEQGKPLMFDDSIEWFERELGPNHDAVLKQVTLSIVAIHTTSDLLLQAMSDLAQNPKVLQAVREEVVRVLSTEGLSKVSLHSLKLMDSALKESQRLRPTLLGSFRRQATNDIKLKSGFVIKKGTRVVIDSTHMWNPEYYTDPLQYDGYRYFNKRQTPGEDKNALLVSTSANHMGFGHGVHACPGRFFASNEIKIALCHIILNYEWRLPDGFKPQPLNIGMTYLADPNTRMLIRPRKAEIDMASLTV

[0096] SEQ ID NO.8 (Amino acid sequence of ST):

[0097] AHIVMVDAYKPTK

[0098] 2. Construction of engineered bacteria

[0099] The recombinant plasmids pET-28(a)-DHE, pET-28(a)-ST-DHE, and pET-28(a)-DHE-ST were mixed with competent E. coli BL21 cells at a volume ratio of 1:100. The mixture was placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, and quickly transferred back to ice for 2 minutes. An appropriate amount of LB liquid medium was added and the cells were shaken and incubated at 37°C for 1 hour to allow the cells to recover. The recovered cells were plated onto LB medium supplemented with 50 μg / mL kanamycin and incubated at 37°C overnight to obtain engineered E. coli BL21-pET-28a(+)-DHE, E. coli BL21-pET-28a(+)-ST-DHE, and E. coli BL21-pET-28a(+)-DHE-ST. These cells were then mixed with equal volumes of 50% glycerol for storage.

[0100] 3. Fermentation of engineered bacteria E. coli BL21-pET-28a(+)-DHE, E. coli BL21-pET-28a(+)-ST-DHE, and E. coli BL21-pET-28a(+)-DHE-ST and preparation of DHE, ST-DHE, and DHE-ST enzyme solutions

[0101] (1) 5L fermenter pretreatment

[0102] A 5-L fermenter was filled with 2 L of YT-M9 medium and a calibrated pH and dissolved oxygen electrode. The fermenter was sterilized at 121°C for 20 min. After the fermenter cooled, sterile air was introduced into the fermenter. The fermenter speed was set to 600 rpm, the temperature to 30°C, and the ventilation volume to 4 vvm. The pH was then adjusted to 5.0 using an acid-base pump.

[0103] (2) Cultivation of engineered bacteria in 5L fermentation tank

[0104] The engineered bacteria stored in glycerol in step 2 were inoculated into a conical flask containing 100 mL of LB liquid medium and incubated at 37°C and 180 rpm until the OD 600 The value reaches 1.6, and the seed solution is obtained. The seed solution is inoculated into the fermentation tank of step (1) at 4% (v / v), stirred at 37°C and 900 rpm, with a ventilation volume of 6 vvm and a pH of 7.0. Feed medium is added through the flow system to maintain a dissolved oxygen level of 30%. When the OD 600 When the value reached 80, IPTG was added to a final concentration of 0.5 mM and induced at 25°C for 20 hours. Fermentation was stopped when the bacterial concentration reached 150 g / L. The fermentation broth was centrifuged at 8000 rpm for 20 minutes and the bacterial cells were collected.

[0105] (3) Preparation of DHE, ST-DHE and DHE-ST enzyme solutions

[0106] The cells obtained in step (2) were broken twice using an AH-basic homogenizer at 1100-1200 bar, and the supernatant was obtained by centrifugation. The protein content was detected using Nanodrop, and the protein concentration was adjusted with 0.2 mM phosphate buffer pH 8.0 to obtain DHE, ST-DHE and DHE-ST enzyme solutions with a protein concentration of 5 g / L.

[0107] Example 3 Preparation of four dual enzyme complexes and comparison of their biocatalytic performance with DHE / HDE free enzymes

[0108] (1) Preparation of DHE-SpyTag / HDE-SpyCatcher Dual Enzyme Complex

[0109] 5 g / L HDE-SC enzyme solution prepared in Example 1 and 5 g / L DHE-ST enzyme solution prepared in Example 2 were mixed in equal volumes, shaken at 37°C and 180 rpm for 3 h to obtain a DHE-SpyTag / HDE-SpyCatcher self-assembled dual enzyme complex enzyme solution with a protein concentration of 5 g / L.

[0110] (2) Preparation of SpyTag-DHE / HDE-SpyCatcher Dual Enzyme Complex

[0111] 5 g / L HDE-SC enzyme solution prepared in Example 1 and 5 g / L ST-DHE enzyme solution prepared in Example 2 were mixed in equal volumes, shaken at 37°C and 180 rpm for 3 h to obtain a SpyTag-DHE / HDE-SpyCatcher self-assembled dual enzyme complex enzyme solution with a protein concentration of 5 g / L.

[0112] (3) Preparation of DHE-SpyTag / SpyCatcher-HDE dual enzyme complex

[0113] 5 g / L SCHDE enzyme solution prepared in Example 1 and 5 g / L DHE-ST enzyme solution prepared in Example 2 were mixed in equal volumes, shaken at 37°C and 180 rpm for 3 h to obtain a DHE-SpyTag / SpyCatcher-HDE self-assembled dual enzyme complex enzyme solution with a protein concentration of 5 g / L.

[0114] (4) Preparation of SpyTag-DHE / SpyCatcher-HDE Dual Enzyme Complex

[0115] 5 g / L SC-HDE enzyme solution prepared in Example 1 and 5 g / L ST-DHE enzyme solution prepared in Example 2 were mixed in equal volumes, shaken at 37°C and 180 rpm for 3 h to obtain a SpyTag-DHE / SpyCatcher-HDE self-assembled dual enzyme complex enzyme solution with a protein concentration of 5 g / L.

[0116] (5) Preparation of DHE / HDE free enzyme mixture

[0117] 5 g / L of the free enzyme HDE solution prepared in Example 1 and 5 g / L of the free enzyme DHE solution prepared in Example 2 were mixed in equal volumes, and shaken at 37° C. and 180 rpm for 3 h to obtain a DHE / HDE free enzyme mixture with a protein concentration of 5 g / L.

[0118] (6) Comparison of biocatalytic performance of four self-assembled dual enzyme complexes and DHE / HDE free enzyme mixture

[0119] To 10 mL of pH 8.0, 0.2 mM phosphate buffer, add 50 g / L of substrate 17α-hydroxyprogesterone, 2 mL of self-assembled dual enzyme complex enzyme solution or DHE / HDE free enzyme mixture (the amount of enzyme solution used is 0.8 g / L based on protein content), and then add 20 g / L of methyl-β-cyclodextrin, 4% DMSO, and 0.5 mM Fe 2+ A 12 mL reaction system was constructed, and biotransformation was performed at 35°C and 160 rpm for 96 h. After the reaction, the conversion solution was filtered, and 5 mg of the filter cake was dissolved in 20 mL of acetonitrile. High-performance liquid chromatography (HPLC) was used to analyze the contents of the substrate and product (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione in the conversion solution based on standard curves of their respective peak areas and concentrations. The conversion rate was then calculated, as shown in Table 1. The results showed that the biocatalytic activities of the four self-assembled dual-enzyme complexes were superior to those of the two free enzyme mixtures. The DHE-SpyTag / SpyCatcher-HDE self-assembled dual-enzyme complex exhibited the highest biocatalytic activity, with a conversion rate of 98.2%.

[0120] Table 1 Comparison of biocatalytic performance of four self-assembled dual enzyme complexes and DHE / HDE free enzyme

[0121]

[0122] Liquid chromatograph: Agilent Technologies 1260 Infinity Series. UV detector: Agilent Technologies 1200 Infinity Series. Column: Agela C18 (250 mm × 4.6 mm, 5 μm). Mobile phase: methanol: water = 80:20 (v / v). Detection wavelength: 254 nm. Flow rate: 1 mL / min. Column temperature: 35°C. Injection volume: 10 μL of sample solution. The elution times of the substrate and product were 6.422 and 4.193 min, respectively.

[0123] Example 4: Effect of biotransformation time on the catalytic cascade reaction of the DHE-SpyTag / SpyCatcher-HDE self-assembled dual enzyme complex

[0124] 10 mL of pH 8.0, 0.2 mM phosphate buffer was added with a final concentration of 50 g / L of substrate 17α-hydroxyprogesterone, 2 mL of DHE-SpyTag / SpyCatcher-HDE type self-assembled dual enzyme complex enzyme solution (the amount of enzyme solution was 0.8 g / L based on protein content), and then 20 g / L of methyl-β-cyclodextrin, 4% DMSO, and 0.5 mM Fe 2+ A 12 mL reaction system was constructed and biotransformed at 35°C and 160 rpm for 12, 24, 36, 48, 72, 96, and 108 h. After completion of the reaction, the conversion solution was filtered, and 5 mg of the filter cake was dissolved in 20 mL of acetonitrile. High-performance liquid chromatography (HPLC) was used to analyze the contents of the substrate and product (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione in the conversion solution based on standard curves comparing their peak areas and concentrations. The conversion rate was then calculated. The results are shown in Table 2.

[0125] The results showed that the conversion rate gradually increased with the extension of biotransformation time. When the conversion time reached 96 hours, the conversion rate reached 98.2%. When the reaction time was further extended to 108 hours, the conversion rate did not increase much. Therefore, the optimal biotransformation time was 96 hours.

[0126] Table 2 Effect of biotransformation time on conversion rate

[0127]

[0128] Example 5: Effect of reaction pH on the catalytic cascade reaction of DHE-SpyTag / SpyCatcher-HDE self-assembled dual enzyme complex

[0129] The conversion time in Example 4 was changed to 96 h, and the pH of the 0.2 mM phosphate buffer was changed to 7.0, 7.5, 8.0, 8.5 and 9.0, respectively. Other operations were the same. The results are shown in Table 3.

[0130] The results showed that the conversion rate reached 98.2% when the pH was 8.0. Therefore, the optimal pH value of the bioconversion system was 8.0.

[0131] Table 3 Effect of reaction system pH on conversion rate

[0132]

[0133] Example 6: Effect of reaction temperature on the catalytic cascade reaction of DHE-SpyTag / SpyCatcher-HDE self-assembled dual enzyme complex

[0134] The conversion time in Example 4 was changed to 96 h, and the reaction temperatures were changed to 25, 30, 35, 40 and 45° C., respectively. Other operations were the same. The results are shown in Table 4.

[0135] The results showed that within the reaction temperature range of 25-35°C, the conversion rate increased with increasing reaction temperature. At 35°C, the conversion rate reached a maximum of 98.2%. However, when the temperature exceeded 35°C, the conversion rate dropped rapidly, indicating that high temperatures significantly affect enzyme activity and may lead to enzyme denaturation and inactivation. Therefore, the optimal conversion temperature is 35°C.

[0136] Table 4 Effect of reaction temperature on conversion rate

[0137]

[0138] Example 7: Effect of cosolvent on catalytic cascade reaction of DHE-SpyTag / SpyCatcher-HDE self-assembled dual enzyme complex The conversion time in Example 4 was changed to 96 h, and the cosolvent was changed to that shown in Table 5. Other operations were the same, and the results are shown in Table 5.

[0139] The results showed that a suitable cosolvent helped dissolve the substrate and improve the conversion rate. The best cosolvent was 4% DMSO, with a conversion rate of 98.2%.

[0140] Table 5 Effect of cosolvent type on conversion rate

[0141]

[0142]

[0143] Example 8: Effect of cyclodextrin on the catalytic cascade reaction of DHE-SpyTag / SpyCatcher-HDE self-assembled dual enzyme complex

[0144] The conversion time in Example 4 was changed to 96 h, methyl-β-cyclodextrin (Me-β-CD) was changed to α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), and hydroxypropyl-β-cyclodextrin (HP-β-CD), and the other operations were the same. The results are shown in Table 6.

[0145] The results showed that the type of cyclodextrin affected the conversion rate, with the conversion rate reaching 98.2% when 20 g / L of methyl-β-cyclodextrin was added. Therefore, 20 g / L of methyl-β-cyclodextrin was the optimal cyclodextrin to add to the conversion process, which was beneficial for improving the conversion rate.

[0146] Table 6 Effect of cyclodextrin type on conversion rate

[0147]

[0148] Example 9: Effect of Metal Ions on the Catalytic Cascade Reaction of the DHE-SpyTag / SpyCatcher-HDE Self-Assembled Dual Enzyme Complex

[0149] The conversion time in Example 4 was changed to 96 h, and the metal ion Fe 2+ The final concentration of metal ions (K + , Mg 2+ , Ca 2+ , Cu 2+ ,Zn 2+ , Mn 2+ , blank as control), other operations were the same, and the results are shown in Table 7.

[0150] The results showed that the addition of appropriate metal ions helped to obtain higher conversion rates. 2+ The conversion rate can reach 98.2%. Therefore, the optimal metal ion is 0.5mM Fe 2+ .

[0151] Table 7 Effect of metal ions on conversion rate

[0152]

[0153] Example 10: Effect of enzyme dosage on catalytic cascade reaction of DHE-SpyTag / SpyCatcher-HDE self-assembled dual enzyme complex

[0154] The conversion time in Example 4 was changed to 96 h, and the amount of the self-assembled dual-enzyme complex enzyme solution was changed to 1, 2, 3, 4, and 5 mL. The corresponding enzyme protein concentrations in the reaction solution were 0.5, 0.8, 1.2, 1.4, and 1.7 g / L. Other operations were the same. The results are shown in Table 8.

[0155] The results showed that the conversion rate gradually increased with the increase of enzyme dosage. When the enzyme dosage in the reaction system exceeded 0.8g / L, the conversion rate did not increase much. Therefore, when the enzyme dosage was 0.8g / L, the optimal conversion rate was 98.2%.

[0156] Table 8 Effect of enzyme amount on conversion rate

[0157]

[0158] Example 10: Effect of substrate amount on catalytic cascade reaction of DHE-SpyTag / SpyCatcher-HDE self-assembled dual enzyme complex

[0159] The conversion time in Example 4 was changed to 96 h, the substrate dosage was changed to 40, 50, 60, 70, 80, 90 and 100 g / L, and other operations were the same. The results are shown in Table 9. The results show that with the increase in the amount of substrate added, the conversion rate gradually decreases.

[0160] Table 9 Effect of substrate addition amount on conversion rate

[0161]

[0162] Example 11: Optimization of enzyme amount when increasing substrate processing capacity

[0163] The conversion time in Example 4 was changed to 96 h, the substrate dosage was changed to 90 g / L, the enzyme solution was changed to 2, 3, 4, 5, and 6 mL, and the corresponding protein addition concentrations were 0.8, 1.2, 1.4, 1.7, and 1.9 g / L. Other operations were the same. The results are shown in Table 10.

[0164] The results showed that when the substrate amount was increased to 90 g / L, the conversion rate increased with the increase of enzyme dosage, and when the enzyme dosage reached 1.9 g / L, the conversion rate reached 99.6%, indicating that the ideal conversion rate can still be achieved by increasing the substrate amount and the enzyme dosage.

[0165] Table 10 Optimization of enzyme amount when increasing substrate processing capacity

[0166]

[0167] Summarize

[0168] Through condition optimization, 90 g / L substrate 17α-hydroxyprogesterone, 5 mL DHE-SpyTag / SpyCatcher-HDE type self-assembled dual enzyme complex enzyme solution (enzyme solution dosage based on protein content is 1.7 g / L), final concentration of 20 g / L methyl-β-cyclodextrin, final concentration of 4% DMSO, and final concentration of 0.5 mM Fe were added to 10 mL, pH 8.0 0.2 mM phosphate buffer. 2+, 35℃, 160rpm biotransformation 96h, HPLC chromatogram is shown in Figure 2 , the conversion rate reached 99.6%.

Claims

1. A self-assembling dual enzyme complex, characterized in that The self-assembling dual-enzyme complex is obtained by fusing 11α-hydroxylase and 1,2-dehydrogenase based on the SpyCatcher / SpyTag system.

2. The self-assembling dual-enzyme complex according to claim 1, wherein The amino acid sequence of the 11α-hydroxylase is shown in SEQ ID NO.2, and the amino acid sequence of the 1,2-dehydrogenase is shown in SEQ ID NO.

6.

3. The self-assembling dual-enzyme complex according to claim 1, wherein In the SpyCatcher / SpyTag system, the amino acid sequence of SpyCatcher is shown in SEQ ID NO.4, and the amino acid sequence of SpyTag is shown in SEQ ID NO.

8.

4. The self-assembling dual-enzyme complex according to claim 1, wherein The self-assembling dual enzyme complex is obtained by self-assembly of a fusion enzyme formed by connecting SpyCatcher to the N-terminus or C-terminus of 11α-hydroxylase and a fusion enzyme formed by connecting SpyTag to the N-terminus or C-terminus of 1,2-dehydrogenase.

5. A method for preparing the self-assembling dual-enzyme complex according to claim 1, characterized in that: The method comprises the following steps: (1) Artificially synthesizing a recombinant plasmid containing SpyCatcher and 11α-hydroxylase, and introducing the recombinant plasmid into Pichia pastoris competent cells to construct a genetically engineered Pichia pastoris strain; (2) Artificially synthesizing a recombinant plasmid containing SpyTag and 1,2-dehydrogenase, and introducing the recombinant plasmid into competent Escherichia coli cells to construct genetically engineered Escherichia coli; (3) fermenting the engineered bacteria constructed in steps (1) and (2), crushing the culture solution, and collecting the supernatant by centrifugation to obtain the respective enzyme solutions; (4) The enzyme solution in step (3) was adjusted to the same protein concentration and then mixed in equal volumes, and shaken at 37° C. and 180 rpm for 3 h to obtain the self-assembled dual-enzyme complex.

6. The method according to claim 5, wherein The steps for preparing enzyme solution by fermentation of genetically engineered Pichia pastoris are as follows: 1) Shake flask culture of engineered bacteria The genetically engineered Pichia pastoris was inoculated into YPD medium and cultured at 30°C and 220 rpm for 16 hours; then inoculated into BMGY medium at a volume concentration of 5% and cultured under the same conditions for 24 hours; the bacterial solution was transferred to a sterile centrifuge tube, centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. The bacteria were washed with sterile distilled water to completely remove glycerol in the culture medium, and the bacteria were collected by centrifugation under the same conditions. The bacteria were transferred to BMMY medium and induced for expression at 30°C and 220 rpm for 12-28 hours. 2) Seed solution preparation The culture solution after induction expression in step 1) was inoculated on a YPD medium plate, activated at 30°C for 2-3 days, and then a single colony was picked and transferred to BMGY medium. The culture was cultured at 30°C and 220 rpm for 24 hours to a constant OD of 0. 600 When the temperature reaches 2 to 6, the seed solution is obtained; 3) 5L fermenter pretreatment A 5-L fermenter was filled with 2 L of BSM medium and calibrated with a pH electrode and a dissolved oxygen electrode. The fermenter was sterilized at 121°C for 20 min. After the fermenter cooled, sterile air was introduced into the fermenter. The fermenter speed was set to 600 rpm, the temperature to 30°C, and the ventilation volume to 4 vvm. The pH was adjusted to 5.0 using an acid-base pump. After ventilation for 1 h, the dissolved oxygen value in this state was set to 100%. 4) Inoculation and basic growth stage The seed solution from step 2) was inoculated into the fermenter pretreated in step 3) at a volume concentration of 5%, and a basal salt solution of PTM1 with a final volume concentration of 0.435% was added. The culture was incubated at 30°C and 600 rpm for 24 hours, during which the aeration rate was adjusted to stabilize the DO value above 20%. After 24 hours, due to the depletion of glycerol in the BSM medium, the DO value rapidly soared to 100%, marking the end of the basal growth phase, and the wet weight of the bacteria reached 90 to 150 g·L. -1 ; 5) Glycerol feeding stage At 36.3 mL·h -1 Glycerol feed medium was added at a rate of 100 μg / L and the DO value was maintained above 20%. When the wet weight of the bacterial cells exceeded 180 g·L -1 The glycerol feed was stopped at 1 ℃ and maintained for 1 hour to allow the glycerol in the fermenter to be completely consumed. The fermentation broth was centrifuged at 8000 rpm for 20 minutes and the cells were collected. The glycerol feed medium consisted of a 0.5 g / L glycerol aqueous solution containing a 1.2% PTM1 basal salt solution by volume. 6) Preparation of enzyme solution The bacterial cells obtained in step 5) are homogenized and broken to obtain an enzyme solution containing cell fragments, which is then centrifuged to obtain a supernatant, and the protein concentration is adjusted with 0.2 mM phosphate buffer at pH 8.0 to obtain the enzyme solution.

7. The method according to claim 5, wherein The steps for preparing enzyme solution by fermentation of genetically engineered Escherichia coli are as follows: The genetically engineered Escherichia coli bacteria were inoculated into a conical flask containing LB liquid culture medium and incubated at 37°C and 180 rpm until the OD600 value reached 1.6 to obtain a seed solution; the seed solution was inoculated into a 5L fermentor containing 2L fermentation medium at a volume concentration of 4%, and stirred at 37°C and 900 rpm with a ventilation volume of 6 vvm and a pH of 7.0; feed medium was added through a flow system to maintain a dissolved oxygen level of 30%; when the OD600 value reached 80, IPTG was added at a final concentration of 0.5 mM and induced at 25°C for 20 hours. Fermentation was stopped when the bacterial concentration reached 150 g / L; the fermentation broth was centrifuged at 8000 rpm for 20 minutes to collect the bacterial cells; the bacterial cells were homogenized and crushed, and the supernatant was obtained by centrifugation, and the protein concentration was adjusted with 0.2 mM phosphate buffer at pH 8.0 to obtain the enzyme solution; Fermentation medium: yeast extract 12.0 g / L, peptone 20.0 g / L, NaCl 1.0 g / L, (NH4)2SO4 6.0 g / L, MgSO4·7H2O 2.0 g / L, glycerol 7.5 mL / L, trace element solution 1 mL / L, KH2PO4 3 g / L, Na2HPO4·12H2O 8.2 g / L; Trace element solution: FeCl3·4H2O 22.87 g / L, ZnCl2 1.31 g / L, CoCl2·6H2O 2.0 g / L, CaCl2 0.76 g / L, CuCl2·2H2O 1.25 g / L, H3BO3 0.5 g / L, MnSO4·H2O 2.0 g / L, 2 mol / L HCl 100 mL / L, solvent is water.

8. Use of the self-assembling dual-enzyme complex according to claim 1 in catalyzing the preparation of a steroid drug prednisolone intermediate from 17α-hydroxyprogesterone.

9. The use according to claim 8, characterized in that The application method comprises the following steps: using a self-assembled dual enzyme complex as a catalyst, using 17α-hydroxyprogesterone as a substrate, adding metal ions, cyclodextrin and a cosolvent, using a 0.2 mM phosphate buffer solution with a pH of 7-9 as a reaction medium to form a conversion system, reacting at 25-45° C. and 100-200 rpm for 12-108 hours, separating and purifying the reaction solution to obtain (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione; the cosolvent comprises dimethyl sulfoxide, methanol, ethanol, isopropanol or acetone; the metal ions comprise K + , Mg 2+ , Ca 2+ , Fe 2+ The cyclodextrin includes α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

10. The use according to claim 9, characterized in that In the conversion system, the volume concentration of the cosolvent is 2-6%; the concentration of the substrate is 40-100 g / L; the amount of the catalyst is 0.1-2 g / L based on protein content; the concentration of the metal ion is 0.5 mM; and the final concentration of cyclodextrin is 20 g / L.

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