C7-site hydroxylated monooxygenase for efficiently catalyzing BA and application of C7-site hydroxylated monooxygenase

Abstract

The invention relates to C7-site hydroxylated monooxygenase for efficiently catalyzing BA and application of the C7-site hydroxylated monooxygenase, and belongs to the technical field of synthetic biology. The amino acid sequence of the C7-site hydroxylated monooxygenase is as shown in SEQ ID NO: 2. Meanwhile, the invention also provides a gene sequence for coding the C7-hydroxylated monooxygenase and a recombinant genetically engineered bacterium for expressing the C7-hydroxylated monooxygenase. Compared with an original enzyme, the structurally modified mutant disclosed by the invention has the advantages that the catalytic ability and the protein stability are remarkably improved, the conversion rate of a substrate BA (22-hydroxy-23, 24-norcholest-4-ene-3-ketone) is increased to 95%, the Tm value of zymoprotein is increased to 66.51 degrees, the stability is remarkably improved, and the mutant has the advantages of being high in yield, good in stability and good in stability. The method has the advantages that the method is simple and convenient to operate, the product 7-OH-DBA (7, 22-dihydroxy-23, 24-norcholest-1, 4-diene-3-ketone) can be efficiently generated, and the efficient utilization of the substrate is realized.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a highly efficient C7-hydroxylation monooxygenase that catalyzes BA and its applications. Background Technology

[0002] The method of introducing a hydroxyl group (-OH) into the steroid molecule during microbial treatment is the most important and successful of all transformation methods. With further research, microbial hydroxylation has become an important production method for steroid drugs, effectively replacing some complex steps in chemical synthesis reactions and thus greatly improving production efficiency.

[0003] C-7 hydroxylated steroidal compounds, such as 7-OH-AD (7-hydroxy-4-androstenedione), 7-OH-BA (7,22-dihydroxy-23,24-norchos-4-en-3-one), and 7-OH-DHEA (7-hydroxy-dehydroepiandrosterone), are important precursors for the synthesis of various steroidal drug molecules. The chemical synthesis of C-7 hydroxylated steroidal compounds is cumbersome and costly. Currently, due to the difficulty in overcoming the problems of low selectivity, numerous byproducts, and environmental pollution, there is still a lack of biotransformation methods for producing C-7 hydroxylated steroidal drug intermediates in industry.

[0004] By relying on biosynthesis to synthesize C-7 hydroxylated steroids, which specifically recognize substrates and carry out efficient hydroxylation reactions, production costs can be significantly reduced and environmental pollution problems can be solved.

[0005] C7-hydroxylase (PtmO6-0) is a modified α-ketoglutarate-dependent dioxygenase (Ptmo6), derived from the C7-hydroxylase PtmO6 gene in the ptm gene cluster of Streptomyces CB00739. C7-hydroxylase (PtmO6-0) catalyzes the C7-hydroxylation of 22-hydroxy-23,24-norcholest-4-en-3-one (BA) to generate 7,22-dihydroxy-23,24-norcholest-1,4-dien-3-one (7-OH-DBA) (e.g., ...). Figure 1 (As shown). C-7 hydroxylase PtmO6 has difficulty converting BA to 7-OH-DBA.

[0006] The C7-hydroxylase (PtmO6-0) has two large loop structures, such as Figure 2 As shown, they are named Flex 1 and Flex 2, respectively. The presence of a large loop structure is detrimental to enzyme stability, which is a key factor affecting its conversion efficiency. Therefore, it is necessary to improve the enzyme's structure to enhance its substrate conversion efficiency. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a highly efficient C7-hydroxylation monooxygenase for BA and its application, which addresses the shortcomings of the existing technology. The amino acid sequence of the enzyme is SEQ ID NO: 2.

[0008] This invention modifies the structure of the C7-hydroxylase PtmO6-0 by truncating the Flex 2 of PtmO6-0, resulting in an enzyme mutant named PtmO6-2-4.

[0009] The present invention also provides a gene sequence expressing PtmO6-2-4, the gene sequence of which corresponds to SEQ ID NO: 3.

[0010] The present invention also provides an expression vector, wherein the expression vector comprises a gene sequence SEQ ID NO: 3.

[0011] In addition, the present invention also provides an expression system, wherein the expression system comprises the above-mentioned gene sequence.

[0012] The present invention also provides a recombinant genetically engineered bacterium, comprising the recombinant expression vector described above, or having the gene sequence described above integrated into its genome.

[0013] The genetically engineered bacteria is Escherichia coli.

[0014] The application of the C7-hydroxylating monooxygenase, the gene sequence, the recombinant expression vector, or the recombinant genetically engineered bacteria in catalyzing the C7-hydroxylation reaction of steroidal compounds.

[0015] The steroidal compound is 22-hydroxy-23,24-norcholest-4-en-3-one, and the product of the hydroxylation reaction is 7,22-dihydroxy-23,24-norcholest-1,4-dien-3-one.

[0016] The reaction conditions for the C7-hydroxylation reaction are: 30℃, pH 8.0, and shaking culture for 2 to 6 days.

[0017] This invention also provides the application of the above-mentioned enzyme in the preparation of C7-hydroxylated steroidal compounds, and the synthesized 7-OH-DBA will provide strong support for the development of new processes for the production of steroidal drugs.

[0018] The present invention achieves the following technical effects

[0019] This invention successfully obtained a high-performance enzyme mutant, PtmO6-2-4, by rationally modifying the original C7-hydroxylase PtmO6-0. The T... mThe value was 66.51°, which was significantly improved compared to PtmO6-0, proving that the structural modification strategy of the present invention has a significant effect on improving the stability of the enzyme protein; the enzyme mutant PtmO6-2-4 has a conversion rate of up to 95.1% for substrate BA, which is greatly improved compared to PtmO6-0.

[0020] The enzyme mutant of this invention enables one-step catalytic conversion of the substrate BA into the target product 7-OH-DBA. Compared with traditional chemical synthesis methods, the reaction does not involve complex chemical synthesis steps and has unique advantages such as high efficiency, specificity, and mildness.

[0021] This invention enables the efficient and green biosynthesis of C7-hydroxylated steroidal compounds (such as 7-OH-DBA), which are important precursors for the synthesis of various steroidal drugs. This provides strong technical support and new ideas for developing new processes for the production of steroidal drugs. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the reaction catalyzed by the C7-hydroxylase PtmO6-0 to produce the product 7-OH-DBA from the substrate BA.

[0023] Figure 2 This is a schematic diagram of the three-dimensional structure of the original enzyme PtmO6-0, with the two loop structures Flex 1 and Flex 2 marked in the figure.

[0024] Figure 3 This is a schematic diagram of the Flex 2 structural modification of PtmO6-2-4. Yellow represents the original structure; purple represents the structure after removing VSLDGRNVGV SVAEGM.

[0025] Figure 4 SDS-PAGE electrophoresis analysis of each mutant enzyme (M is protein standard marker; 1 is blank; 2 and 3 are the precipitate and supernatant of PtmO6-2-4 after 5 minutes of lysis, respectively; induction conditions: 18℃, 0.4 mM IPTG, induction for 14 h).

[0026] Figure 5 The HPLC (High Performance Liquid Chromatography) analysis of the modified enzyme PtmO6-2-4 for BA conversion is shown, along with the HPLC detection chromatogram of the substrate BA standard sample. The retention time of the substrate BA is approximately 13 minutes, and the retention time of the product 7-OH-DBA is approximately 6 minutes. Detailed Implementation

[0027] The following examples will further illustrate this point. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0028] The C7-hydroxylating monooxygenase PtmO6-2-4 described in this embodiment of the invention is based on the C7-hydroxylase PtmO6-0 sequence (SEQ ID NO:1). The C7-hydroxylase (PtmO6-0) is a modified α-ketoglutarate-dependent dioxygenase (PtmO6). The PtmO6 enzyme is derived from the C-7 hydroxylase PtmO6 gene in the ptm gene cluster of Streptomyces CB00739.

[0029] The C7-hydroxylase PtmO6-0 has two loop structures, named Flex 1 and Flex 2, respectively. The gene for the C7-hydroxylated monooxygenase PtmO6-2-4 is a truncated design of Flex 2.

[0030] The amino acid sequence of the C7-hydroxylated monooxygenase PtmO6-2-4 is shown in SEQ ID NO.2.

[0031] SEQ ID NO.1 (PtmO6-0 amino acid sequence):

[0032] MAGLVWEPTS AGWGARVGLD LGAPLGAESA AALVELFRAR HLLVFSGQDF

[0033] SLEEQIRFMG YLGPVLHEEG SGIGFVSNVK EGAALGVSEL SFHSDTGYCA

[0034] VPLEAVSLFA EDVEGSVTST RFANVAAAYG RLPAGLRSRV ASLVSENAMP

[0035] VSLDGRNVGV SVAEGMPRAE HPVVWRHPVS GEPGLMVNAN ETTRIVGLED

[0036] AESRELLEEL FSVMYAEDAV YEHSWRQGDV VVWHNLAVQH ARGGLEGNGR

[0037] RTLRRVALGE KGFWEQCPTL RYADFKNQQN TAA.

[0038] SEQ ID NO.2 (PtmO6-2-4 amino acid sequence):

[0039] MAGLVWEPTS AGWGARVGLD LGAPLGAESA AALVELFRAR HLLVFSGQDF

[0040] SLEEQIRFMG YLGPVLHEEG SGIGFVSNVK EGAALGVSEL SFHSDTGYCA

[0041] VPLEAVSLFA EDVEGSVTST RFANVAAAYG RLPAGLRSRV ASLVSENAM

[0042] PRAE HPVVWRHPVS GEPGLMVNAN ETTRIVGLED

[0043] AESRELLEEL FSVMYAEDAV YEHSWRQGDV VVWHNLAVQH ARGGLEGNGR

[0044] RTLRRVALGE KGFWEQCPTL RYADFKNQQN TAA.

[0045] This invention also discloses a nucleic acid encoding the C7-hydroxylated monooxygenase PtmO6-2-4, enabling the expression of the encoded enzyme mutant. The sequence encoding the nucleic acid is shown in SEQ ID NO: 3.

[0046] SEQ ID NO.3 (gene sequence encoding PtmO6-2-4):

[0047] TCCGCTGGACTGGTATGGGAACCCACATCAGCGGGTTGGGGTGCGCGTGTCGGTCTGGACCTGGGTGCACCGCTGGGCGCGGAGTCCGCGGCTGCGTTGGTAGAATTGTTCCGCGCGCGCCACTTGCTGGTTTTTTCCGGTCAGGATTTCAGCCTGGAGGAACAGATTCGTTTTATGGGTTACCTGGGTCCGGTGCTGCATGAAGAGGGCAGCGGCATCGGATTCGTGTCCAATGTTAAAGAAGGTGCGGCTCTGGGCGTCTCTGAATTAAGCTTCCACAGCGATACCGGTTATTGCGCTGTGCCGCTGGAAGCAGTTTCCTTGTTCGCGGAAGACGTCGAGGGGTCCGTTACTTCAACCCGTTTTGCGAACGTGGCGGCCGCGTATGGTCGTCTGCCGGCAGGTTTGCGTAGCCGTGTGGCGAGCCTGGTTTCCGAAAACGCCATGCCGCGTGCGGAGCACCCTGTGGTGTGGCGCCACCCGGTCTCTGGCGAACCGGGCCTGATGGTTAATGCAAACGAAACCACCCGAATTCTGGGCCTGGAGGACGCAGAGAGCCGTGAATTGCTTGAGGAGTTGTTCAGCGTTATGTACGCCGAGGACGCCGTGTATGAACACTCTTGGCGTCAGGGAGATGTGGTGGTTTGGCATAATCTGGCAGTCCAGCATGCTCGTGGTGGTTTAGAGGGCAATGGTCGCCGTACGCTGCGCCGGGTTGCGCTTGGCGAGAAAGGTTTTTGGGAACAATGCCCGACGCTGCGCTACGCTGATTTTAAGAACCAACAAAACACCGCAGCTTAG。

[0048] This invention cloned, expressed, structurally verified, and tested the catalytic activity of the gene encoding the C7-hydroxylated monooxygenase PtmO6-2-4. The mutant genes were obtained by PCR amplification, digested and ligated into the pSZD expression vector, and then introduced into *E. coli* DH5α strain (for the construction and amplification of recombinant plasmids) and BL21(DE3) strain (for the induction of recombinase expression; induction conditions: 18℃, 0.4 mM IPTG, induction for 14 h). After expression, the expression of the recombinase was verified by SDS-PAGE, and the recombinase was used to conduct an in vitro transformation reaction with substrate BA to systematically verify the catalytic performance of the mutants.

[0049] Primitive PtmO6-0 enzyme protein T m The value is 48.94°, and the present invention relates to the T value of the modified mutant. m The values ​​of all enzymes were significantly improved, including PtmO6-2-4 and T. m The value was increased to 66.51°, and the stability was significantly improved.

[0050] The original PtmO6-0 was induced to express in the E. coli expression system, with a substrate conversion rate of about 75%. The enzyme PtmO6-2-4, which was structurally optimized in this invention, has enhanced the enzyme's conversion activity for BA, with a conversion rate as high as 95%, which greatly improves the conversion rate and enhances the utilization of the substrate.

[0051] This invention optimizes the PtmO6-2-4 enzyme catalytic system, achieving a conversion rate of 95% in a 50 mL shake flask reaction using 2 g / L BA as the raw material. Compared to traditional chemical synthesis methods, enzyme catalysis saves four reaction steps, significantly reducing cumbersome synthesis steps and pollution waste. Biosynthesis of steroid drugs is an economical and environmentally friendly method and technology.

[0052] The modified enzyme exhibits C7-hydroxylation activity on substrate BA, and its transformation has unique advantages such as high efficiency, specificity, and mildness, providing new ideas and directions for the synthesis of steroidal drugs.

[0053] The present invention provides a C7-hydroxylated monooxygenase PtmO6-2-4, which efficiently converts 7-OH-DBA using BA as a substrate.

[0054] The present invention will be described below with reference to specific embodiments to provide a better understanding of it.

[0055] Example 1: Construction of recombinase plasmid and expression vector

[0056] A new fragment is introduced into the target sequence using a two-step fusion PCR method, as follows.

[0057] Design forward and reverse mutation primers based on the selected mutation sites. The specific primer base sequences are shown in Table 1.

[0058] Table 1

[0059]

[0060] Using pSZD-PtmO6-0 plasmid as a template, T7-F as the front primer, and mutant primer 2-4-R as the back primer, the pre-fragment was amplified by PCR and denoted as U; the post-fragment was amplified by PCR using mutant primer 2-4-F as the front primer and T7-R as the back primer and denoted as D. The fusion PCR reaction systems U and D and their procedures are shown in Tables 2, 3, and 4 below.

[0061] Table 2

[0062]

[0063] Table 3

[0064]

[0065] Table 4

[0066]

[0067] After the PCR reaction, the size of the amplified bands was identified by 1% agarose gel electrophoresis. The theoretical size of amplified band U should be the length from the T7-F sequence on the recombinant plasmid to the target mutant fragment; the theoretical size of amplified band D should be the length from the mutation site to the DR sequence on the recombinant plasmid. Reaction solutions with band sizes matching these values ​​were selected, and the target fragment was recovered using a universal gel extraction and purification kit, as follows:

[0068] a. Add 50 μL of PC solution to the PCR reaction solution and mix by pipetting and aspiration in equal proportion;

[0069] b. Pipette the reaction solution onto the wall of the column and add it to the recovery column. Place the recovery column in the collection tube, cover it, and centrifuge at 12000 rpm for 1 min. Remove the recovery column, discard the liquid in the collection tube, and put the recovery column back into the collection tube. Add 600 μL of PW solution to the recovery column and centrifuge at 12000 rpm for 1 min. Remove the collection tube and discard the effluent. Then repeat the PW elution once.

[0070] c. After elution twice, put the recovery column back into the collection tube, centrifuge at 12000 rpm for 2 min, pour out the liquid in the collection tube, and remove the recovery column to let it stand and dry.

[0071] d. Take a bottle of UP water and preheat it at a constant temperature of 65℃. Place the dried recovery column into a 1.5 mL EP tube, and then add 30 μL of the previously preheated 65℃ UP water to the recovery column. After adding the water, let the recovery column stand at room temperature for 2 min, then cover it and centrifuge it together with the EP tube at 12000 rpm for 1 min.

[0072] e. Remove the EP tube and observe that the eluent has been centrifuged to the bottom of the tube. Discard the recovery column, cap the EP tube, and then use NanoDrop to determine the DNA concentration and purity in the eluent. DNA solutions with excessively high protein content are not suitable for subsequent experiments. After labeling the concentration, store the DNA tube at -20°C. Label the recovered DNA fragments as U and D for later use.

[0073] Using fragments U and D as templates, T7-F as the front primer and T7-R as the back primer, fragment fusion PCR was performed. The PCR procedure is shown in Table 4 above, and the PCR system is shown in Table 5 below.

[0074] Table 5

[0075]

[0076] After the PCR reaction, the size of the amplified bands was identified by 1% agarose gel electrophoresis. The theoretical size of the amplified bands should be the length of the T7-F to T7-R sequences on the recombinant plasmid vector. A reaction solution with the expected band size was selected, and the target fragment was recovered using a universal gel extraction kit. The recovered fragment was designated PtmO6-2-4. Fragment PtmO6-2-4 and the pSZD plasmid were double-digested with BamHI and KpnI at 37℃ for 3 h. The digestion was then purified using a Tiangen universal DNA purification and recovery kit, and the resulting product was mixed with T4 DNA ligase at 16℃ for 4 h. The digestion and ligation reaction systems are shown in Tables 6 and 7 below.

[0077] Table 6

[0078]

[0079] Table 7

[0080]

[0081] After transformation, the plate containing the ligation product should show uniformly distributed single colonies; otherwise, it may be contaminated by other microorganisms. Several clear, appropriately sized single colonies were picked from the plate using a pipette tip and used as templates for colony validation PCR. PCR reactions were performed using the vector pre-primer T7-F and post-primer T7-R to verify whether they were positive transformants. Simultaneously, the pipette tips containing the picked single colonies were transferred to 100 μL of kanamycin-resistant LB broth for amplification. The colony PCR reaction system and PCR procedure are shown in Tables 3 and 4 above. After the PCR reaction was completed, the size of the amplified bands from each colony was identified by 1% agarose gel electrophoresis. The theoretical size of the amplified bands in this study was expected to be around 1000 bp. According to the marker bands, the bacterial cultures corresponding to the positive transformants whose band sizes met the expectations were sent to Sangon Biotech Co., Ltd. for gene sequencing. The bacterial cultures with correct sequencing were preserved. The volume ratio of glycerol to bacterial culture was 3:7. After mixing, the mixture was added to a glycerol tube and labeled as DH5α-pSZD-PtmO6-2-4. After expansion culture, plasmids were extracted using the Tiangen Plasmid Mini-Prep Kit. The extracted plasmids were then transformed into E. coli BL21(DE3) competent cells. Positive transformants were selected using the same method and labeled as BL21-pSZD-PtmO6-2-4. After expansion culture, the strain was preserved by adding 30% glycerol and storing it in a -80°C freezer.

[0082] Example 2: Enzyme Expression and Crude Enzyme Solution Preparation

[0083] 100 μL of the constructed BL21-pSZD-PtmO6-2-4 glycerol tube culture was added to a test tube containing 3 mL of kanamycin-resistant LB liquid medium and incubated at 37°C and 200 rpm for 6 h on a shaker. The culture was then serially expanded to 50 mL Erlenmeyer flasks, and the 50 mL culture was then poured into 1 L of 0.1% kanamycin-resistant LB liquid medium. The culture was incubated at 37°C and 200 rpm until OD... 600When the pH reached 0.6, the temperature was lowered from 37℃ to 20℃, and isopropyl thio-β-D-galactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce protein expression. After 14 h of shaking culture, the cells were collected. The cells were centrifuged at 6000 rpm for 20 min at 4℃ in a refrigerated centrifuge, and the supernatant was discarded to collect the bacterial sludge. The cells were resuspended in an appropriate amount of 0.1 M Tris-HCl buffer (pH=8.0) and homogenized using a high-pressure homogenizer at 4℃ and 1200 bar for 6 cycles. The cell lysate was collected in a centrifuge cup, precisely balanced, and centrifuged at 12000 rpm for 30 min at 4℃ in a floor-standing refrigerated centrifuge. The supernatant after centrifugation was the crude enzyme solution of PtmO6-2-4 enzyme, used for fermentation and substrate transformation.

[0084] After centrifugation, the supernatant and precipitate were sampled separately to prepare protein samples, and SDS-PAGE was used to detect protein expression.

[0085] The results are as follows Figure 4 As shown, the size of PtmO6-2-4 is approximately 28.8 kDa;

[0086] LB liquid culture medium formula (1 L): 10 g peptone; 5 g yeast extract; 10 g sodium chloride.

[0087] Example 3: Purification of enzyme protein and T m Determination of value

[0088] Take 30 μL of the constructed BL21-pSZD-PtmO6-2-4 glycerol tube culture and add it to a test tube containing 3 mL of kanamycin-resistant LB liquid medium. Incubate overnight at 37°C and 200 rpm on a shaker. Then, add 500 μL of the bacterial culture and 50 μL of kanamycin-resistant medium to a 50 mL Erlenmeyer flask and incubate for 5 to 12 hours. Finally, pour 50 mL of the cultured bacterial culture into 1 L of 0.1% kanamycin-resistant LB liquid medium and incubate at 37°C and 200 rpm until OD reaches 0.5%. 600When the nm value reached 0.6, the temperature was lowered from 37℃ to 20℃, and isopropyl thio-β-D-galactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce protein expression. After 14 h of shaking culture, the bacterial cells were collected. The cells were centrifuged at 4000 rpm for 20 min at 4℃ using a refrigerated centrifuge, and the supernatant was discarded to collect the bacterial sludge. The sludge was resuspended in buffer A (pH=8.0), and after lysing the *E. coli*, the supernatant obtained by centrifugation was used as the sample for column loading. Purification was first performed using a HisTrap™ HP nickel column. Before passing the nickel column through the column, it needed to be pretreated to regenerate. First, the residual protein in the nickel column was flushed with 6 M urea for approximately 3 column volumes, then flushed with UP water for approximately 3 column volumes, and finally flushed with 0.5 M EDTA (ethylenediaminetetraacetic acid) for approximately 2 column volumes. The EDTA eluent was recovered. At this point, the nickel column changed from green to milky white, indicating that the nickel ions had been flushed away. Finally, rinse the column with approximately 6 column volumes of UP water and approximately 2 column volumes of 20% ethanol solution, then circulate 1 M NiSO4 solution onto the nickel column for approximately 15 min. When using a nickel column for column purification, first rinse the nickel column with 3 column volumes of UP water, then rinse and equilibrate the nickel column with 8 column volumes of Ni-NTA Buffer A. The pre-column sample prepared earlier is then slowly pumped into the nickel column using a peristaltic pump. Subsequently, the program is set up using the ÄKTA protein purification system's interface, and gradient elution is performed using Ni-NTA Buffer B. The target protein is collected when the peak appears on the instrument interface.

[0089] Place the white flat-capped eight-row tubes on a PCR plate and pre-cool on ice. Add the following components in sequence to construct a 26 μL reaction system (set up at least 3 replicates for each system):

[0090] (1) Add purified enzyme protein (final concentration 100 μM).

[0091] (2) Add ligand BA (final concentration 100 μM);

[0092] (3) Add 4 μL of 62.5× SYPRO Orange dye;

[0093] (4) Add PBS to 25 μL. Gently mix by pipetting, then seal and incubate in the dark for 15 min. The cobas z480 fully automated real-time PCR analyzer was used for programmed temperature ramping: the instrument was pre-cooled to 20 °C, the reaction plate was loaded, and the temperature was increased from 20 °C to 95 °C at a rate of 1.8 °C / min. During this period, 20 fluorescence signals were collected at every °C interval under the conditions of 498 nm excitation wavelength and 580 nm emission wavelength. As the ambient temperature rises to a certain range, these hydrophobic groups will gradually be exposed to the solvent, thereby specifically binding to the SYPRO Orange fluorescent dye and generating a detectable fluorescence signal. The thermal stability of the protein can be quantitatively assessed by monitoring changes in fluorescence intensity. Its characteristic S-shaped curve can be used to estimate the melting temperature (T). m T m The statistical results are shown in Table 8 below:

[0094] Table 8

[0095]

[0096] Table 8 shows that the T of PtmO6-2-4 m The value was significantly improved compared to PtmO6-0, T m The value increased to 66.51°, indicating a significant improvement in the stability of the enzyme protein, which is beneficial for subsequent conversion and utilization of the enzyme protein.

[0097] Example 4: Construction of a shake-flask enzyme-to-BA reaction system

[0098] We used the crude enzyme solution obtained from the preparation to test the catalytic activity of the enzyme on the steroid substrate BA, and constructed an in vitro transformation system. The enzyme used in the in vitro transformation system of this study was prepared according to Example 2. The reaction system is shown in Table 9 below. The reaction was carried out in 50 mL Erlenmeyer flasks in a shaker at 200 rpm at 30°C and pH 8.0 for 2 to 6 days. Under the same conditions, a protein-free reaction mixture was used as a negative control. Samples were taken every 1 day. 1 mL of the reaction solution was taken from the Erlenmeyer flask, and the same volume of ethyl acetate was added to extract the reaction product. The extract was centrifuged at 12000 rpm for 10 min. At this time, the lipid-soluble steroid substrate and product dissolved in the supernatant. After pipetting the supernatant, the ethyl acetate was dried at 65°C to obtain the powder or oily reaction product.

[0099] Table 9

[0100]

[0101] Example 5: Detection of products from the enzyme conversion of BA

[0102] The sample to be analyzed must be dissolved in chromatographic grade methanol and filtered through a 0.22 μm filter membrane. The sample loading volume is set to 10 μL. A detector emits 254 nm ultraviolet light to detect the UV absorbance of the sample passing through the chromatographic column. A Sunfire C18 reverse-phase column (5 μm, 4.6 × 150 mm, Waters) is used. The mobile phase is methanol:water = 7:3, the column temperature is 30℃, and the flow rate is 1 mL / min. If a standard curve is established for the sample to be analyzed, HPLC can quantify the concentration of the sample.

[0103] Test results as follows Figure 5 As shown, the elution time of BA is about 12 min, and the elution time of 7-OH-DBA is about 6 min.

[0104] The conversion rate statistics are shown in Table 10 below.

[0105] Table 10

[0106]

[0107] As shown in Table 10, the catalytic efficiency of the 7-hydroxylated product was significantly improved by PtmO6-2-4 compared to PtmO6-0, with a conversion rate of 95%, achieving efficient utilization of the substrate.

[0108] Those skilled in the art should understand that the above embodiments are merely exemplary embodiments, and various changes, substitutions, and modifications can be made without departing from the spirit and scope of this application.

Claims

1. A mutant of a highly efficient catalytic C7-hydroxylation monooxygenase for BA, characterized in that, The mutant is named PtmO6-2-4, and its amino acid sequence is shown in SEQ ID NO:

2.

2. The mutant according to claim 1, characterized in that, The mutant was obtained by structural modification of the enzyme PtmO6-0, which has the amino acid sequence shown in SEQ ID NO:

1.

3. A gene sequence encoding the mutant of claim 1, characterized in that, The gene sequence is shown in SEQ ID NO:

3.

4. A recombinant expression vector, characterized in that, It contains the gene sequence as described in claim 3.

5. A recombinant genetically engineered bacterium, characterized in that, It is obtained by conversion from the recombinant expression vector described in claim 3.

6. The recombinant genetically engineered bacteria according to claim 5, characterized in that, The genetically engineered bacteria is Escherichia coli.

7. The use of a mutant as described in claim 1, a gene sequence as described in claim 3, a recombinant expression vector as described in claim 4, or a recombinant genetically engineered bacterium as described in claim 5 or 6 in catalyzing the C7-hydroxylation reaction of steroidal compounds.

8. The application according to claim 7, characterized in that, The steroidal compound is 22-hydroxy-23,24-norcholest-4-en-3-one, and the product of the hydroxylation reaction is 7,22-dihydroxy-23,24-norcholest-1,4-dien-3-one.

9. The application according to claim 8, characterized in that, The reaction conditions for the C7-hydroxylation reaction are: 30℃, pH 8.0, and shaking incubation for 2 to 6 days.

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