A cholesterol oxidase fusion protein and its application

Abstract

This invention provides a cholesterol oxidase fusion protein and its applications. A cholesterol oxidase fusion protein is formed by covalently binding FAD-containing cholesterol oxidase and *Vibrio hygroscopicis* hemoglobin, achieving efficient expression in *Escherichia coli*. The invention also provides an application of the cholesterol oxidase fusion protein in industrial enzyme catalysis.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a cholesterol oxidase fusion protein and its application in industrial enzyme catalysis. Background Technology

[0002] Cholesterol oxidase (COX, EC1.1.3.6) is a monomeric bifunctional enzyme, a type of oxidoreductase with flavin adenine dinucleotide (FAD) as a cofactor. The cholesterol oxidase catalyzes cholesterol reactions involving two steps: (1) Oxidation reaction: With the participation of the FAD cofactor, the 3β-hydroxyl group of cholesterol is oxidized to generate the intermediate product cholester-5-en-3-one, while releasing hydrogen peroxide (H2O2); (2) Isomerization reaction: without the assistance of other enzymes, the cholester-5-en-3-one generated in the previous step is isomerized directly, and finally converted into the stable end product cholester-4-en-3-one.

[0003] Many microorganisms can produce cholesterol oxidase, including *Pseudomonas* sp., *Streptomyces* sp., *Nocardia* sp., *Rhodococcus* sp., *Mycobacterium* sp., *Brevibacterium* sp., *Arthrobacters* sp., and *Burkholderia* sp. However, the enzyme production capacity of these wild-type strains is very low. To increase the yield, studies have used *E. coli* to overexpress microbially derived cholesterol oxidase, followed by purification using a Ni column. The resulting cholesterol oxidase can be used for clinical blood routine tests. Although the amount of cholesterol oxidase expressed in *E. coli* obtained by the above method is higher than that of wild-type bacteria, the cost is high. This is acceptable for high-value-added applications such as clinical blood routine tests, but unacceptable for low-value-added industrial enzyme catalysis applications. If cholesterol oxidase is to be applied in the field of industrial enzyme catalysis, it is necessary to reduce the production cost of cholesterol oxidase and produce cholesterol oxidase that simultaneously meets the three conditions of high activity, high stability, and high expression level.

[0004] Cholesterol oxidase exists in two forms in various bacteria: one where the FAD cofactor is non-covalently bound to the enzyme; and the other where the FAD cofactor is covalently linked to the protein. Therefore, there are two types of cholesterol oxidase: FAD-covalently bound and FAD-non-covalent. Cholesterol oxidases from *Pseudomonas* sp., *Rhodococcus equi.*, *Nostoc punctiforme.*, and *Mycobacterium* sp. are FAD-non-covalent, while those from *Brevibacterium sterolicum.*, *Burkholderia cepacian.*, and *Rhodococcus erythropolis.* are FAD-covalently bound (see Vrielink, A. and Ghisla, S. (2009), Cholesterol oxidase: biochemistry and structural features. The FEBS Journal, 276: 6826-6843.). The non-covalent form of FAD belongs to the glucose-methanol-choline (GMC) oxidoreductase family, while the covalently bound FAD belongs to the vanillyl alcohol oxidase (VAO) family. Studies have shown that the non-covalent form of FAD is prone to detachment under conditions of increased temperature, high concentrations of urea, dilution, and prolonged storage, producing inactive apocosidic proteins. It is easily dissociated and inactivated in the relatively crude industrial enzyme catalysis environment. Under the same conditions, the Tm (melting temperature) of the covalently bound FAD is higher than that of the mutant of the non-covalent FAD with the same backbone (by mutating the amino acid H covalently bound to FAD to A), and it has stronger resistance to pH fluctuations. However, the flavoprotein folding of the covalently bound FAD is more demanding, and it is more likely to form inclusion bodies during heterologous expression (E. coli). This affects the soluble expression of cholesterol oxidase, thus affecting its yield, which limits its application in industrial enzyme catalysis where yield is critical. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in related technologies. To this end, one objective of this invention is to propose a cholesterol oxidase fusion protein and its applications. The fusion of FAD-covalently bound cholesterol oxidase with vitreous hemoglobin (VHb) not only significantly increases expression levels but also broadens substrate scope, enabling efficient 3-position oxidation reactions on non-specific substrates such as β-sitosterol, pregnenolone, epiandrolone, and 17α-methyl-5α-androst-3β,17β-diol. This eliminates the need for the chromic anhydride reagent required in the 3-position oxidation step of steroidal drug intermediate preparations, thus enabling the acquisition of a green and efficient enzyme catalytic element and process.

[0006] Specifically, the present invention provides the following technical solution:

[0007] A first aspect of the present invention provides a cholesterol oxidase fusion protein comprising FAD covalently bound cholesterol oxidase and vitreous hemoglobin.

[0008] Furthermore, the amino acid sequence of the cholesterol oxidase fusion protein is shown in SEQ ID NO: 6.

[0009] Furthermore, the amino acid sequence of the FAD covalently bound cholesterol oxidase is referenced to the wild-type sequence of Prescottella equiATCC 33707 or its homologous sequence. The wild-type sequence or its homologous sequence is the sequence shown in SEQ ID NO: 1 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the sequence shown in SEQ ID NO: 1. The sequence of SEQ ID NO: 1 is specifically:

[0010]

[0011]

[0012] A third aspect of the present invention provides a recombinant vector comprising the nucleotide sequence of the cholesterol oxidase fusion protein described above, which is a common and frequently used vector in the art and can be purchased from a legitimate company.

[0013] Furthermore, the expression vector of the recombinant vector is pET28a(+).

[0014] A fourth aspect of the present invention provides a recombinant bacterium, the recombinant bacterium comprising the recombinant vector described above.

[0015] Furthermore, the recombinant bacteria uses Escherichia coli BL21(DE3) as the host bacterium, or can use cells that are common and frequently used in the field and can be purchased from a legitimate company.

[0016] The fifth aspect of the present invention provides the application of the above-described cholesterol oxidase fusion protein in industrial enzyme catalysis.

[0017] Furthermore, the cholesterol oxidase fusion protein can perform 3-position oxidation on various steroidal drug precursors with a hydroxyl group at the 3-position to generate a 3-position ketone group. These 3-position hydroxyl group steroidal drug precursors include β-sitosterol, pregnenolone, epiandrolone, and 17α-methyl-5α-androst-3β,17β-diol.

[0018] A sixth aspect of the present invention provides a method for increasing the expression level of FAD covalently bound cholesterol oxidase, the method being to fuse FAD covalently bound cholesterol oxidase with vitreous hemoglobin.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention provides a cholesterol oxidase fusion protein and its applications. The cholesterol oxidase fusion protein provided by this invention fuses FAD-covalently bound cholesterol oxidase with vitreous hemoglobin (VHb). The fusion protein achieves heterologous and efficient expression in *E. coli*, reducing industrial production costs and solving the problem of inclusion body formation in existing heterologous expression methods. Experiments through examples demonstrate that both the activity and expression level of cholesterol oxidase are significantly improved. Furthermore, it becomes a broad-spectrum cholesterol oxidase, capable of efficiently oxidizing non-specific substrates such as β-sitosterol, pregnenolone, epiandrolone, and 17α-methyl-5α-androst-3β,17β-diol at the 3-position. This eliminates the need for the environmentally polluting chromic anhydride reagent required in the 3-position oxidation step of steroid drug intermediate preparation, demonstrating good environmental friendliness and promising prospects for industrial enzyme catalysis applications. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Results of the analysis of the source cholesterol oxidase secretion signal peptide of Prescottella equi ATCC 33707;

[0023] Figure 2 SDS-PAGE analysis of pET28a-COX expression strain after induction under three temperature conditions;

[0024] Figure 3 SDS-PAGE analysis of pET28a-COX-N expression strain after induction under three temperature conditions and two culture medium conditions;

[0025] Figure 4 SDS-PAGE analysis of pET28a-VHb-COX expression strain after induction under three temperature conditions;

[0026] Figure 5 SDS-PAGE analysis of pET28a-COX-N and pET28a-VHb-COX expression strains under high-density fermentation conditions;

[0027] Figure 6 TLC image of the oxidation of epiandrolone at the 3-position catalyzed by cholesterol oxidase VHb-COX;

[0028] Figure 7 TLC image of the oxidation of β-sitosterol at the 3-position catalyzed by cholesterol oxidase VHb-COX;

[0029] Figure 8 TLC image of the oxidation of pregnenolone at the 3-position catalyzed by cholesterol oxidase VHb-COX;

[0030] Figure 9 TLC diagram of the oxidation reaction of 17α-methyl-5α-androst-3β,17β-diol at the 3-position catalyzed by cholesterol oxidase VHb-COX. Detailed Implementation

[0031] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The present invention will be specifically described below with reference to specific embodiments.

[0032] Example 1: Preliminary heterologous expression of cholesterol oxidase from Prescottella equi ATCC 33707

[0033] The complete wild-type sequence of cholesterol oxidase from Prescottella equi ATCC 33707 is shown in SEQ ID NO: 1, and was obtained via a signal peptide prediction website. https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / Predicting secretion signal peptides was performed, using Gram-positive bacteria. The prediction results were as follows: Figure 1 As shown, a TAT signal peptide is present, with the signal peptide cleavage site between amino acids 39 and 40. The amino acid sequence formed after cleaving the signal peptide sequence is shown in SEQ ID NO: 2. The sequence of SEQ ID NO: 2 was reverse transcribed to form a DNA sequence, and codon optimization for E. coli was performed. The optimized nucleotide sequence is shown in SEQ ID NO: 3. The nucleotide sequence of SEQ ID NO: 3 was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for gene synthesis and inserted into the Nco I and Xho I restriction sites of the pET28a plasmid to form the pET28a-COX expression plasmid. This plasmid was transformed into BL21(DE3) competent cells and plated on kanamycin-resistant LB plates. The single colony that grew after overnight incubation at 37°C is the pET28a-COX expression strain.

[0034] The pET28a-COX expression strain was inoculated into LB agar containing 50 μg / mL kanamycin to cultivate seed culture, and then transferred to LB agar containing 50 μg / mL kanamycin for expansion culture. OD 600 When the pH reached approximately 0.8, IPTG was added to a final concentration of 0.8 mM, and induction was performed at 18℃, 25℃, and 32℃ for 18 h. One bottle was kept without IPTG and cultured at 25℃ and 220 rpm. After induction, the bacterial cells were collected by centrifugation. 1 g of the expression cells were weighed and resuspended in 10 mL of 50 mM phosphate buffer (pH 7.0). After sonication, the cells were centrifuged at 12000 rpm for 30 min. The supernatant was collected to obtain the crude enzyme supernatant sample. The centrifuged precipitate was resuspended in 10 mL of 50 mM phosphate buffer (pH 7.0) to obtain the precipitate sample. The pET28a-COX crude enzyme solution and the precipitate sample were simultaneously subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown, from Figure 2 It can be seen that no target protein was expressed at any of the three induction temperatures.

[0035] Example 2: Expression after N-terminus truncation

[0036] The N-terminus of SEQ ID NO: 2 was further truncated by removing the VPAGSAGSLGSLG sequence to form SEQ ID NO: 4. The sequence of SEQ ID NO: 4 was reverse transcribed to form a DNA sequence and optimized for E. coli codons. The optimized nucleotide sequence is shown in SEQ ID NO: 5. The nucleotide sequence of SEQ ID NO: 5 was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for gene synthesis and inserted into the Nco I and Xho I restriction sites of the pET28a plasmid to form the pET28a-COX-N expression plasmid. This plasmid was transformed into BL21(DE3) competent cells and plated on kanamycin-resistant LB plates. The single colony that grew after overnight incubation at 37°C was the pET28a-COX-N expression strain.

[0037] The pET28a-COX-N expression strain was inoculated into LB agar containing 50 μg / mL kanamycin to cultivate seed culture, and then transferred to LB agar containing 50 μg / mL kanamycin for expansion culture. OD 600 When the pH reached approximately 0.8, IPTG was added to a final concentration of 0.8 mM, and induction was performed at 18℃, 25℃, and 32℃ for 18 h at a rotation speed of 220 rpm. Simultaneously, the culture was expanded by transferring the culture medium to TB containing 50 μg / mL kanamycin. OD 600 When the pH reached approximately 0.8, IPTG was added to a final concentration of 0.8 mM and induced at 25℃ for 18 h at 220 rpm. After induction, the bacterial cells were collected by centrifugation. 1 g of the expressed bacterial cells were weighed and resuspended in 10 mL of 50 mM phosphate buffer (pH 7.0). After ultrasonic disruption, the cells were centrifuged at 12000 rpm for 30 min. The supernatant was collected to obtain the crude enzyme supernatant sample. The centrifuged precipitate was resuspended in 10 mL of 50 mM phosphate buffer (pH 7.0) to obtain the precipitate sample. The pET28a-COX-N crude enzyme solution and the precipitate sample were simultaneously subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 3 As shown, from Figure 3 It can be seen that a small amount of the target protein was solublely expressed at induction temperatures of 18℃ and 25℃. When the induction temperature was increased to 32℃, the target protein formed inclusion bodies, and the expression level of the inclusion bodies was also very low. When the culture medium was changed to TB medium, the expression level of the target protein was significantly improved. However, on the one hand, the expression level was still relatively low, which is not conducive to industrial enzyme catalysis. On the other hand, due to cost reasons, the TB medium-induced fermentation process cannot be industrialized (industrialization generally uses the lower-cost inorganic salt high-density fermentation process).

[0038] Example 3: Expression of fusion protein

[0039] Some fusion proteins can increase the expression level of target proteins. Commonly used fusion tags in the industry include SUMO, sfGFP, GST, and MBP tags. Based on past experience, GST and MBP tags, with their large molecular weights, may result in increased expression levels after fusion, but a significant increase in the molecular weight of the fusion protein can lead to a substantial decrease in protein specific activity. While SUMO and sfGFP tags have small molecular weights, their functions are not related to cholesterol oxidase. This embodiment combines the catalytic functions of cholesterol oxidase and Vibrio hygroscopicis hemoglobin (VHb). The former catalyzes the reaction of oxygen and cholesterol to produce hydrogen peroxide and 4-cholesten-3-one, while the latter binds to and transfers oxygen to cholesterol oxidase under hypoxic conditions, while simultaneously hydrolyzing the hydrogen peroxide byproduct of the former into oxygen and water. Furthermore, literature reports that VHb can significantly enhance the growth rate, protein synthesis, and yield of metabolites (such as antibiotics and enzymes) of the host bacteria during hypoxic fermentation.

[0040] The *Vibrio hygroscopicus* hemoglobin (VHb) gene was fused with a truncated cholesterol oxidase gene using the linker peptide GTGGGG. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 6. The sequence of SEQ ID NO: 6 was reverse transcribed to form a DNA sequence, and codon optimization was performed on *E. coli*. The optimized nucleotide sequence is shown in SEQ ID NO: 7. The nucleotide sequence of SEQ ID NO: 7 was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for gene synthesis and inserted into the Nco I and Xho I restriction sites of the pET28a plasmid to form the pET28a-VHb-COX expression plasmid. This plasmid was transformed into BL21(DE3) competent cells and plated on kanamycin-resistant LB plates. The single colony that grew after overnight incubation at 37°C is the pET28a-VHb-COX expression strain. The pET28a-VHb-COX expression strain was inoculated into LB agar containing 50 μg / mL kanamycin to cultivate seed culture, and then transferred to LB agar containing 50 μg / mL kanamycin for expansion culture. OD 600When the pH reached approximately 0.8, IPTG was added to a final concentration of 0.8 mM, and induction was performed at 18℃, 25℃, and 32℃ for 18 h at a rotation speed of 220 rpm. After induction, the bacterial cells were collected by centrifugation. 1 g of the expression cells were weighed and resuspended in 10 mL of 50 mM phosphate buffer (pH 7.0). After ultrasonic disruption, the cells were centrifuged at 12000 rpm for 30 min. The supernatant was collected to obtain the crude enzyme supernatant sample. The centrifuged precipitate was resuspended in 10 mL of 50 mM phosphate buffer (pH 7.0) to obtain the precipitate sample. The pET28a-VHb-COX crude enzyme solution and the precipitate sample were simultaneously subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 4 As shown, from Figure 4 It can be seen that the expression level of the target protein is significantly increased under all three induction temperature conditions.

[0041] Example 4: Determination of cholesterol oxidase COX and its activity during high-density fermentation in a 5L tank

[0042] Glyceryl strains of pET28a-COX-N and pET28a-VHb-COX expression strains were inoculated into seed culture in LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm for 15 h until OD was reached. 600 Transplantation should begin at 2.0-3.0. Transfer to LB broth containing 50 μg / mL kanamycin for culture expansion. Incubate at 37°C with shaking at 200 rpm for 5 hours, until OD reaches 2.0. 600 Inoculate the fermenter at a ratio of 1:100 when the OD reaches 1.0-3.0. Pre-sterilize 2.5L of M9 medium in a 5L fermenter and incubate at 37℃, using glycerol as the feed throughout the process. 600 At approximately 25-30 minutes, the temperature was lowered to 25°C, and IPTG was added to a final concentration of 0.1 mM for isothermal induction of protein expression. After 16 hours, the mixture was transferred to two separate tanks. The bacterial sludge was collected by centrifugation from both tanks. 1 g of the sludge was resuspended in 10 mL of 50 mM phosphate buffer (pH 7.0), ultrasonically disrupted, and then centrifuged at 12000 rpm for 30 min. The supernatant was collected to obtain the crude enzyme supernatant sample for activity determination. Simultaneously, the pET28a-COX-N and pET28a-VHb-COX supernatant enzyme solutions were diluted 10-fold and 20-fold, respectively, and 10 μL of each solution was loaded for SDS-PAGE electrophoresis analysis to determine expression.

[0043] Unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μM H2O2 per minute. The principle of activity measurement is that 1M cholesterol is catalyzed by cholesterol oxidase to produce 1M 4-cholesten-3-one and 1M H2O2, and 2M hydrogen peroxide is catalyzed by horseradish peroxidase in the presence of 4-aminoantipyrine and TOOS to produce 1 mole of quinone imine. Quinone imine is detected by spectrophotometer at 555 mM.

[0044] Activity calculation (U / mL) = (ΔA × Vt × df × 60) / (39.2 × Vs × 1.0 × 0.5).

[0045] Where: Vt: total reaction volume; Vs: enzyme solution volume; 1.0: optical path length; df: enzyme solution dilution factor; 0.5: 0.5M quinone imine generated from 1M hydrogen peroxide; 39.2: extinction coefficient of quinone imine at 555 nM wavelength.

[0046] The results are shown in Table 1 and Figure 5 As shown in Table 1, the fragmentation supernatant activity of pET28a-VHb-COX is 2.5 times that of pET28a-COX-N. Figure 5 It is speculated that the expression level of pET28a-VHb-COX per unit of bacterial sludge under high-density fermentation conditions may be more than four times that of pET28a-COX-N. The discrepancy between the expression level data and the activity data may be due to VHb possessing catalase activity, which can compete with horseradish peroxidase for hydrogen peroxide, resulting in a lower measured value than the actual activity.

[0047] Table 1. Activity assay results of crude enzyme supernatant samples from pET28a-COX-N and pET28a-VHb-COX expression strains.

[0048] sample Activity (U / mL) pET28a-COX-N crushed supernatant 29 pET28a-VHb-COX crushing supernatant 72

[0049] Example 5: Oxidation of epiandrolone at the 3-position catalyzed by cholesterol oxidase VHb-COX

[0050] Reaction pathway:

[0051]

[0052] Reaction protocol: Prepare a 500mL reaction flask, add 1g epiasterone, 100mL 0.1M pH 7.0 phosphate buffer, and 1mL VHb-COX enzyme solution (200 U / mL). Aerate at 2 L / min and stir at 30℃. Monitor the reaction progress by TLC. Results are shown below. Figure 6 As shown, from Figure 6 It can be seen that after 23 hours of reaction, the substrate epiandrogenes was completely converted into the product 5α-androstanedione. Here, S represents the substrate epiandrogenes, P represents the product 5α-androstanedione, and the samples from 4 hours and 23 hours are the samples from 4 hours and 23 hours of reaction, respectively.

[0053] Example 6: Cholesterol oxidase VHb-COX catalyzes the oxidation of β-sitosterol at the 3-position.

[0054] Reaction pathway:

[0055]

[0056] Reaction protocol: Prepare a 500 mL reaction flask, add 1 g β-sitosterol, 100 mL 0.1 M pH 7.0 phosphate buffer, and 1 mL VHb-COX enzyme solution (200 U / mL). Aerate at 2 L / min and stir at 30 °C. Monitor the reaction progress by TLC. Results are shown below. Figure 7 As shown, from Figure 7 It can be seen that after 23 hours of reaction, the substrate β-sitosterol was completely converted into the product β-sitosterone. Here, S represents the substrate β-sitosterol, and 5h and 23h represent samples reacted for 5 hours and 23 hours, respectively.

[0057] Example 7: Oxidation of pregnenolone at the 3-position catalyzed by cholesterol oxidase VHb-COX

[0058] Reaction pathway:

[0059]

[0060] Reaction protocol: Prepare a 500mL reaction flask, add 1g pregnenolone, 100mL 0.1M pH 7.0 phosphate buffer, and 1mL VHb-COX enzyme solution (200 U / mL). Aerate at 2 L / min and stir at 30℃. Monitor the reaction progress by TLC. Results are shown below. Figure 8 As shown, from Figure 8 It can be seen that the substrate pregnenolone was completely converted into the product after 5 hours of reaction. S represents the substrate pregnenolone, P represents the product progesterone, and the samples at 5 hours and 23 hours are the samples after 5 hours and 23 hours of reaction, respectively.

[0061] Example 8: Cholesterol oxidase VHb-COX catalyzes the oxidation of 17α-methyl-5α-androst-3β,17β-diol at the 3-position.

[0062] Reaction pathway:

[0063]

[0064] Reaction protocol: Prepare a 500 mL reaction flask, add 1 g of 17α-methyl-5α-androst-3β,17β-diol, 100 mL of 0.1 M pH 7.0 phosphate buffer, and 1 mL of VHb-COX enzyme solution (200 U / mL). Aerate at 2 L / min and stir at 30 °C. Monitor the reaction progress by TLC. Results are as follows: Figure 9 As shown, from Figure 9 It can be seen that the substrate pregnenolone was completely converted into the product after 23 hours of reaction. S is the substrate 17α-methyl-5α-androst-3β,17β-diol, P is the product mexilonolone, and 5h and 23h are samples after 5h and 23h of reaction, respectively.

[0065] The above description is merely a description of preferred embodiments of the present invention and is not intended to limit the scope of the present invention in any way. Any changes or modifications made by those skilled in the art based on the above disclosure shall fall within the protection scope of the claims.

Claims

1. A cholesterol oxidase fusion protein, characterized in that, The cholesterol oxidase fusion protein includes FAD covalently bound cholesterol oxidase and vitreous hemoglobin.

2. The cholesterol oxidase fusion protein according to claim 1, characterized in that, The amino acid sequence of the cholesterol oxidase fusion protein is shown in SEQ ID NO:

6.

3. The cholesterol oxidase fusion protein according to claim 1, characterized in that, The amino acid sequence of the FAD covalently bound cholesterol oxidase is referenced to the wild-type sequence of Prescottella equi ATCC 33707 or its homologous sequence, wherein the wild-type sequence or its homologous sequence is the sequence shown in SEQ ID NO: 1 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the sequence shown in SEQ ID NO:

1.

4. The cholesterol oxidase fusion protein according to claim 3, characterized in that, The amino acid sequence of the FAD covalently bound cholesterol oxidase is shown in SEQ ID NO:

4. The amino acid sequence of the FAD covalently bound cholesterol oxidase is obtained by truncating the signal peptide sequence of SEQ ID NO: 1 and the N-terminal VPAGSAGSLGSLG sequence, wherein the signal peptide cleavage site is between amino acids 39 and 40.

5. A nucleotide sequence of a cholesterol oxidase fusion protein as described in any one of claims 1-4, characterized in that, The nucleotide sequence of the cholesterol oxidase fusion protein is shown in SEQ ID NO:

7.

6. A recombinant vector, characterized in that, The recombinant vector comprises the nucleotide sequence of the cholesterol oxidase fusion protein according to claim 5.

7. The recombinant vector according to claim 6, characterized in that, The expression vector for the recombinant vector is pET28a(+).

8. A recombinant bacterium, characterized in that, The recombinant bacteria include the recombinant vector as described in claim 6 or 7.

9. The application of the cholesterol oxidase fusion protein according to any one of claims 1-4 in industrial enzyme catalysis, characterized in that, The cholesterol oxidase fusion protein can oxidize a variety of steroidal drug precursors with a hydroxyl group at the 3-position to generate a 3-keto group. The 3-hydroxyl steroidal drug precursors include β-sitosterol, pregnenolone, epiandrolone, and 17α-methyl-5α-androst-3β,17β-diol.

10. A method for increasing the expression level of FAD-covalently bound cholesterol oxidase, characterized in that, The method involves fusing FAD-covalently bound cholesterol oxidase with vitreous hemoglobin.

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