CN111471736B - Method for preparing C1, 2-dehydrogenation steroid compound - Google Patents

Abstract

The invention provides a method for preparing a C1, 2-dehydrogenation steroid compound, which comprises the following steps: constructing an expression vector for expressing the recombinant KstD enzyme, wherein the amino acid sequence of the recombinant KstD enzyme is SEQ ID.3; expressing the recombinant KstD enzyme by using the expression vector and a cell culture mode to obtain a crude enzyme solution; adding a substrate androst-4-ene-3, 17-dione (4-AD) into the crude enzyme solution, and reacting to obtain the C1, 2-dehydrosteroid compound. The invention obtains a soluble KstD fusion enzyme by modifying the KstD211 enzyme of mycobacterium HGMS 2; the KstD fusion enzyme is prepared by utilizing the KstD fusion enzyme efficiently expressed by recombinant escherichia coli and adopting high-density fermentation, and the specific enzyme activity reaches 31.6U/mg.

Description

Method for preparing C1, 2-dehydrogenation steroid compound

Technical Field

The invention relates to the field of biochemistry, in particular to a method for preparing a C1, 2-dehydrogenation steroid compound.

Background

Steroid hormone drugs are clinically important drugs, have particularly significant anti-inflammatory activity, and are widely used for preventing and treating various diseases. In addition, changes in the steroid parent nucleus structure may lead to more potent steroid drugs. For example, when unsaturation is introduced into the 1, 2-position of Hydrocortisone Acetate (HA) by Δ 1-dehydrogenation, the anti-inflammatory activity of the product Prednisolone Acetate (PA) increases three to four times. However, due to the complexity of the steroid structure, the conventional chemical method is difficult to specially modify the steroid intermediate, and the chemical method has the defects of low conversion rate, more byproducts, environmental pollution and the like. Thus, enzymatic conversion has attracted much attention due to its mild reaction conditions, high efficiency and specificity (regioselectivity and stereoselectivity). Today, enzymatic conversion technology has proven to be a new, efficient and economical method for the production of new steroid drugs and active pharmaceutical ingredients.

The 3-ketosteroid-delta 1-dehydrogenase (KstD) catalyzes the dehydrogenation of androstenedione (4-AD) at C1,2 position to form 1, 4-Androstenedione (ADD) (FIG. 1). ADD, converted from 4-AD, is an important precursor for the synthesis of high-end steroid drugs, such as contraceptives, estrogens and progestins. Although the chemical dehydrogenation process is already used in the steroid pharmaceutical industry, compared with the multi-step chemical synthesis of hormone synthesis, the enzymatic process has high efficiency, no byproduct accumulation and high product purity, so that a 'green' high-efficiency enzymatic formula is developed by heterologously expressing KstDThe preparation of steroid intermediates has now begun to attract attention. M. smegmatis mc²155 MstStD 1 makes it possible to achieve a cortisol yield of 90% in a concentration of 6g/L within 3 hours. KstD2 of the novel Mycobacterium DSM 1381 gave an almost complete conversion of 30 g/L4-AD. However, the low substrate concentrations and low conversion rates described above limit the commercial application of the KstD enzyme process.

The KstD enzymatic reaction belongs to redox reaction, and 3-sterone-1, 2-dehydrogenase (KstD) is a flavoprotein dependent dehydrogenase which takes Flavin Adenine Dinucleotide (FAD) as a cofactor in the catalytic reaction process and can catalyze the dehydrogenation of a carbon-carbon single bond (C-C) at the 1,2 position of the A ring of a 3-sterone mother nucleus into a carbon-carbon double bond (C ═ C). The cost of industrially regenerating coenzyme FAD is very high, and the development of efficient and inexpensive electron acceptors or the opening of efficient and inexpensive coenzyme regeneration systems is required, which is a key technology limiting the industrial application of the KstD enzyme process.

KstD is an inducible enzyme and is also a membrane protein. Although many examples of heterologous expression are provided, membrane proteins mostly exist in the form of inclusion bodies due to the poor solubility of the membrane proteins, and a technical problem to be solved in the industrial application is urgently needed.

The KstD enzymes from different microorganisms have different specificities for steroid substrates, because the amino acid sequences of proteins of different KstD have differences, which determine their conformational differences in local regions, and the screening or designing of KstD enzymes with substrate specificity, or the screening of a KstD enzyme with a large number of substrates in common, is also a key requirement for the industrialization of steroid enzyme processes.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for preparing C1, 2-dehydrosteroid compounds, comprising: constructing an expression vector for expressing the recombinant KstD enzyme, wherein the amino acid sequence of the recombinant KstD enzyme is SEQ ID.3; expressing the recombinant KstD enzyme by using the expression vector and a cell culture mode to obtain a crude enzyme solution; adding a substrate androst-4-ene-3, 17-dione (4-AD) into the crude enzyme solution, and reacting to obtain the C1, 2-dehydrosteroid compound.

In the above method, further comprising: before the reaction, adding electron acceptor menadione into the crude enzyme solution, wherein the mass concentration of the electron acceptor menadione in the reaction solution is less than or equal to 1%.

In the above method, further comprising: adding catalase into the crude enzyme solution before reaction, wherein the concentration of the catalase in the reaction solution is less than or equal to 100U/L.

In the above method, wherein the recombinant KstD enzyme has Maltose Binding Protein (MBP) bound to the N-terminus or C-terminus.

The invention obtains a recombinant KstD fusion enzyme by modifying the KstD211 enzyme of mycobacterium HGMS 2; the KstD fusion enzyme is prepared by utilizing the KstD fusion enzyme efficiently expressed by recombinant escherichia coli and adopting high-density fermentation, and the specific enzyme activity reaches 31.6U/mg.

The soluble MBP/KstD fusion protein is formed by fusing MBP protein at the N terminal or C terminal of the KstD211, so as to enhance the solubility of the KstD protein, thereby solving the problem of the solubility of the enzyme. MBP can be placed either N-or C-terminal to the KstD, without altering KstD725 activity.

Through optimizing the type and the dosage of an electron acceptor in the KstD fusion enzyme reaction, the coenzyme is subjected to low-cost and high-efficiency regeneration circulation, and a process for converting androstenedione (4-AD) into 1, 4-Androstenedione (ADD) by the fusion enzyme is established. In the shake flask process, 10g/L of 4-AD is used as a raw material, the KstD fusion enzyme is used for converting the 4-AD, and the conversion rate is more than 98%. By amplifying and optimizing a KstD fusion enzyme catalytic system, 80 g/L4-AD is taken as a raw material in a 15L reaction kettle, the conversion rate is over 97 percent, and the purity after refining can reach over 99.5 percent. In a 15-ton reaction kettle, large-scale high-efficiency conversion from 4-AD to ADD is realized, and the concentration of a substrate is as high as 80-100 g L^-1The conversion rate reaches 98%, and the product is single without other impurities. The enzymatic conversion of the invention has the unique advantages of high efficiency, specificity, mildness and the like. The fusion enzyme of the invention is also suitable for producing other steroid drug intermediates with high value by high-efficiency conversion.

Drawings

FIG. 1 shows that 3-ketosteroid-. DELTA.1-dehydrogenase catalyzes the dehydrogenation of 4-AD to ADD.

FIG. 2 shows SDS-PAGE analysis comparing expression of soluble KstD enzyme. a) Unfused KstD expression, 1,2 are supernatant and pellet induced for 10 h; 3. 4 is the supernatant and pellet induced for 20 h. b) The fusion protein expresses MBP-KstD, 5 and 6 are supernatant and sediment which are induced for 10 hours; 7.8 is the supernatant and pellet induced for 20 h.

FIG. 3 shows a process curve for fed-batch fermentation of recombinant E.coli, a t, T: OD600 nm; completed before another branch: dissolving oxygen; ● - - ●: (ii) temperature; solid- -. diamond solid: the pH value; ■ - - ■: the rotational speed.

FIG. 4 shows the preparation of the KstD enzyme by high density fermentation. M: a protein Marker; 1, crude enzyme liquid supernatant is carried out for-10 hours; 2: precipitating the crude enzyme solution for-10 h; 3: supernatant of the crude enzyme solution is-12 h; 4: precipitating the crude enzyme solution for-12 h; 5: supernatant of the crude enzyme solution is-20 h; 6: the crude enzyme solution is precipitated for-20 h.

FIG. 5 shows a schematic representation of the coupled C1, 2-dehydrogenase coenzyme regeneration system.

FIG. 6 shows the effect of different coenzyme regeneration systems on conversion.

FIG. 7 shows the ADD reaction generated by conversion of 4-AD by the recombinant KstD enzyme by Thin Layer Chromatography (TLC) analysis. 1:4-AD standard; 2.3, 4 and 5 represent products of enzyme reactions for 6h, 12h, 18h and 24h respectively.

Detailed Description

The following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the invention in any way.

The present application carried out whole genome sequencing (Genbank ID: CP031414.1) on 4-AD industrial production strain Mycobacterium HGMS2, and identified unique KstD enzyme gene from HGMS2 strain, named KstD211(SEQ ID NO.1) and amino acid sequence (SEQ ID NO. 2).

The invention clones, expresses, analyzes and transforms the KstD enzyme gene of the mycobacterium HGMS 2. The invention constructs an expression vector of escherichia coli, introduces the cloned KstD gene into the escherichia coli through a pRSV expression vector for induction expression, and characterizes the enzyme specificity and selectivity.

Previous studies have shown that the highest activity of recombinant KstD in E.coli expression systems is only 6U/mg. By comparing the amino acid sequences of the KstD211 and other mycobacteria KstD, the invention carries out sequence modification and optimal design on the KstD211 to obtain the KstD725 mutant with high activity. The KstD725 mutant has the amino acid sequence of SEQ ID NO.3. the DNA sequence coding the KstD725 mutant is optimized by the preferred codon of Escherichia coli, is suitable for efficient expression of Escherichia coli, and has the DNA sequence of SEQ ID.4.

KstD belongs to a membrane-bound enzyme protein and has low solubility. Previous studies have shown that the KstD gene obtained from rhodococcus sp SQ1 can be expressed in e.coli expression systems, but KstD has low solubility, only a small fraction of which is soluble and active, mainly in the form of inclusion bodies. In order to overcome the problem of low solubility of membrane proteins, the invention adopts a fusion expression strategy to fuse MBP protein at the N terminal or C terminal of KstD to form soluble MBP/KstD fusion protein, and the solubility of the KstD protein can be enhanced in the expression process, thereby solving the problem of enzyme solubility. MBP can be placed either N-terminal or C-terminal to KstD725, without altering KstD725 activity. This is also necessary for large scale enzymatic reactions. Then, it is overexpressed.

The invention utilizes the MBP/KstD fusion protein efficiently expressed by recombinant escherichia coli, prepares the fusion KstD enzyme by high-density fermentation, and the specific activity of the enzyme reaches 31.6U/mg after purification.

The invention optimizes the proportion of electron acceptor, regenerates coenzyme system with low cost and high efficiency, utilizes MBP/KstD725 fusion enzyme in vitro enzyme conversion method in a shake flask, takes 10 g/L4-AD as raw material, and realizes the conversion rate of more than 98 percent.

According to the invention, by amplifying and optimizing an MBP/KstD725 fusion enzyme catalysis system, 80 g/L4-AD is taken as a raw material in a 15L reaction kettle, the conversion rate is more than 97%, and the purity after refining can reach more than 99.5%.

The invention realizes large-scale high-efficiency conversion from 4-AD to ADD in a 15-ton reaction kettle by using an MBP/KstD725 fusion enzyme in-vitro enzyme conversion method, wherein the concentration of a substrate is as high as 80-100 g L^-1The conversion rate reaches 98%, and the product is single without other impurities. The enzymatic conversion of the invention has the unique advantages of high efficiency, specificity, mildness and the like, and is a new technology in steroid biotransformation. The MBP/KstD725 fusion enzyme is also suitable for efficiently converting and producing other steroid drug intermediates with high value, and indicates a new direction for the development of steroid drug industry, and the enzyme conversion process of the invention reaches the requirements of industrialized production. The present invention utilizes a form of fusion expression to increase the solubility of KstD as well as the stability of expression. A soluble protein MBP, i.e. MBP-KstD, is linked to the N-terminus of KstD.

Therefore, the research provides an economic and environment-friendly method and a technical process for the biosynthesis of the steroid drug C1,2 dehydrogenation.

The following description will be given with reference to specific examples.

Example 1 construction and shake flask expression of recombinant KstD fusion enzyme plasmids:

coli BL21(DE3) cells containing the recombinant plasmid were cultured in LB medium containing 35. mu.g/ml kanamycin and spun at 37 ℃ at 200 rpm. When OD600nm reached 0.8, 0.4mM isopropyl-. beta. -D-thiogalactopyranoside (final concentration) was added, the incubation temperature was lowered to 20 ℃ and incubation was continued for 18-20 hours. The expression level of the soluble target enzyme protein was significantly increased by SDS-PAGE (FIG. 2).

Example 2, high density fermentation preparation of KstD fusion enzyme:

in order to obtain a high-density cell culture with a large amount of enzymes, a high-density medium was optimized, cells were cultured in a 15L fermenter, and some modifications were made, such as induction by glycerol instead of glucose, yeast powder as a nitrogen source, lactose, and the like. At the beginning of the fermentation, the temperature of the fermentor was controlled at 37 ℃ to allow rapid cell growth. In the fed-batch phase, dissolved oxygen is controlled between 10% and 30% and pH is controlled between 6.2 and 7.8. Prior to the induction phase, the cell culture temperature was lowered to 20 ℃ and held for a period of time, then lactose mother liquor was added until the final concentration reached 0.4mM, the induction time was 18h-20 h. Protein expression is induced. After fermentation, the E.coli cells were collected by centrifugation at 4 ℃ and resuspended in 50mM Tris-HCl buffer (pH 8.0), and the suspension was disrupted by a high-pressure homogenizer under conditions: the crude enzyme solution can be obtained after 3 cycles at the temperature below 5 ℃ and the pressure of 1200 bar. The crude enzyme solution was centrifuged at 6000r/min at 4 ℃ for 20 minutes to remove solid impurities such as cell debris. At this time, the enzyme solution is used for the enzyme-catalyzed reaction and is temporarily stored at a low temperature.

By optimizing the high-density fermentation formula, the invention adopts a fed-batch fermentation mode to perform high-density fermentation on recombinant escherichia coli in a 15L fermentation tank. The liquid loading amount is 10L culture medium, and the culture medium is sterilized at 121 ℃ for 30 min. When the temperature is reduced to 37 ℃, inoculation is carried out with the inoculation amount of 5 percent, and fermentation is started. The changes in temperature, dissolved oxygen and pH throughout the fermentation are shown in figure 3. As can be seen from the graph, feeding was started while the dissolved oxygen was reduced to the minimum 5 hours after inoculation and thereafter the dissolved oxygen was increased back. The dissolved oxygen is maintained in a certain fluctuation range (20-30%) during the feeding process. And (5) fermenting for 16h, reducing the temperature, and supplementing an inducing solution at a low temperature. During the induction phase, the feeding rate is correspondingly reduced, and the growth rate of the thallus is kept lower so as to ensure the normal expression of the protein. The pH fluctuation was not so great throughout the fermentation, and no acid or alkali was added during the period from 7.2 at the beginning of the fermentation to 6.7 at the end of the fermentation to adjust the pH. The whole feeding process adopts an intermittent feeding process, namely the system automatically feeds at a constant speed (the feeding rate of every several seconds). The pH of the feed solution used in the present invention is not adjusted and is overall slightly acidic (pH is between about 5.0 and 6.0). Adjustment of feed rate before induction: feeding is started when the dissolved oxygen returns, feeding is stopped when the dissolved oxygen starts to decrease, and the dissolved oxygen is maintained in a certain fluctuation range (20% -30%), wherein the pH change in the process is as follows: during feeding, the pH value begins to decrease, and after feeding is stopped, the pH value slowly increases, so that the pH value at the stage is maintained to fluctuate around 7.0. Feed rate adjustment after induction: the growth of the thalli is slow at low temperature, the feeding rate is properly reduced, the consumption of oxygen is reduced in the process of the stage, the dissolved oxygen is always kept in a high state (80% -90%), based on the pH value, feeding is started when the pH value rises again, feeding is stopped when the pH value begins to decrease, and the pH value in the process of the stage is maintained to fluctuate around 7.0. The intermittent feeding process adopted by the invention ensures that the pH is not reduced due to excessive feeding and acid production of thalli in the whole fermentation process, and the pH is not increased due to autolysis of thalli caused by insufficient feeding.

Example 3 KstD fusion enzyme Activity

An appropriate amount of 2.3 centrifuged supernatant was taken and Mbp-KstD2 was measured at 30 ℃ using Phenazine Methosulfate (PMS) and 2, 6-dichlorophenol-benzenediol (dccip) on a Nano Drop 2000 spectrophotometer (Thermo Scientific) at 600nm ([ xi ] 600nm ═ 18.7 × 103 cm-1M-1). The reaction mixture (1ml) consisted of 50mM Tris-HCl buffer (pH 7.0), 150mM PMS, 8mM DCPIP, a suitable concentration of supernatant or cell extract and 100mM AD methanol. Activity is expressed in units per mg protein; 1U is defined as a 1 μmolmin-1DCPIP reduction. No activity was detected in the reaction mixture without 4-androstenone-3, 17-dione (AD). The remaining supernatant was analyzed by SDS-PAGE using 8% separation gel and 5% stacking gel to identify whether the protein was expressed correctly. Protein expression in the fermentor is shown in FIG. 4.

Example 4 enzymatic regeneration of circulating KstD fusion enzyme cofactor

As shown in FIG. 5, normally, in the KstD enzymatic Δ 1-dehydrogenation reaction, KstD, after removing two hydrogens from the substrate, passes to molecular oxygen, which is used as a hydrogen acceptor, producing hydrogen peroxide (H)₂O₂) The coenzyme regeneration thus forms a cyclic process. After dissolved oxygen in the reaction system is exhausted, the reaction is immediately terminated, and the generated hydrogen peroxide can also influence the enzyme activity.

In the invention, the enzyme conversion system is optimized and adjusted, and we find for the first time that the conversion rate of a substrate can be improved greatly by adding a small amount of catalase into the enzyme conversion system. The catalase can promote hydrogen peroxide to be decomposed to generate water and oxygen, so that the toxic effect of the hydrogen peroxide is reduced, the dissolved oxygen in the system is increased, and meanwhile, the dissolved oxygen is supplemented in the enzyme catalysis reaction system to prevent the reaction from slowing down or stopping due to insufficient dissolved oxygen.

Example 5 Shake flask KstD fusion enzyme transformation 4-AD reaction Process

Androst-4-ene-3, 17-dione (4-AD) powder was put into 50mM ofTris-HCl buffer (pH 8.0) to prepare a 10% concentration, emulsified with a homogenizer for 2 hours (preventing generation of a large amount of bubbles, and a small amount of antifoaming agent was added) to reduce 4-AD particles, and uniformly dispersed in a buffer to prepare a substrate emulsion. The enzyme activity test was carried out as described above for the enzyme-catalyzed reaction. The high-density fermented cells were resuspended in Tris-HCl buffer (Ph 8.0), and disrupted by a high-pressure homogenizer to obtain a crude enzyme solution.

The volume of the enzyme catalysis is 100ml, and the reaction is carried out according to 1 percent of feeding amount. 50ml of crude enzyme solution was weighed into a 250ml shake flask, and 25ml of 4-AD (about 1g) emulsion and 25ml of Tris-HCl buffer solution were added to conduct a coenzyme factor regeneration system experiment, and the different systems are shown in Table 1.

TABLE 1

The reaction mixture was placed in a shaker and reacted at 30 ℃ with shaking at 200 rpm. Samples were taken at reaction 20, extracted with ethyl acetate and centrifuged. After evaporating ethyl acetate from the extraction reaction solution, 100. mu.L of methanol was added: the reaction was dissolved in a 6:4 mobile phase solution and analyzed by HPLC, the mobile phase being: methanol: water 6: 4. The results of the analysis calculated the 4-AD conversion. As shown in FIG. 6, different coenzyme factor regeneration systems have different influences on the conversion rate, and the conversion rates of the systems 2,6, 7 and 8 reach more than 97 percent.

Example 6 reaction Process for conversion of Pilot-plant KstD fusion enzyme to 4-AD

The scale-up experiment was continued for the enzyme conversion test of kilogram grade substrate, and the experiment was performed in a 15L reactor. The total reaction volume was 10L, again at 10% substrate concentration (i.e.1 Kg input). The reaction conditions are the same as those of the shake flask, and the coenzyme factor regeneration system adopts a system 8. Samples were taken at 6h, 12h, 18h, and 24h of reaction, extracted with ethyl acetate, centrifuged, and the supernatant was dipped in ethyl acetate by a capillary and spotted. The silica gel plate was placed in a developing solvent (V petroleum ether/V ethyl acetate ═ 5:3) and after the solvent climbed to two thirds of the silica gel plate, the solvent was taken out and blown dry. The silica gel plate was then exposed to 254nm UV light, and the results are shown in FIG. 7.

EXAMPLE 7 Large Scale KstD fusion enzyme conversion 4-AD reaction Process

And carrying out a high-concentration substrate enzyme conversion production test according to the optimized enzyme catalytic system. The experiment was carried out in a 15 ton autoclave. The total reaction volume was 10 tons, again at 10% substrate concentration (i.e.1 ton input). The reaction conditions were the same as those of the pilot plant. Sampling at the reaction time of 24h, extracting with ethyl acetate, centrifuging, dipping the supernatant ethyl acetate by a capillary tube, performing TLC spot plate detection on the reaction progress, and further performing HPLC detection to confirm that the conversion rate reaches more than 97 percent, so that the refining process can be performed. And after the reaction is carried out for 20-24 hours, the conversion rate reaches more than 97, and the reaction is stopped.

Those skilled in the art will appreciate that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the application.

Sequence listing

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<120> method for preparing C1, 2-dehydrosteroid compound

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Glu Ala Cys His His Met Tyr Gly Gly Gln Tyr Gly Gln Glu Asn Val

325 330 335

Pro Ala Trp Met Val Phe Asp Gln Gln Tyr Arg Asp Arg Tyr Ile Phe

340 345 350

Ala Gly Leu Gln Pro Gly Gln Arg Ile Pro Lys Lys Trp Met Glu Ser

355 360 365

Gly Val Ile Val Lys Ala Asp Ser Val Ala Glu Leu Ala Glu Lys Thr

370 375 380

Gly Leu Ala Pro Asp Ala Leu Thr Ala Thr Ile Glu Arg Phe Asn Gly

385 390 395 400

Phe Ala Arg Ser Gly Val Asp Glu Asp Phe His Arg Gly Glu Ser Ala

405 410 415

Tyr Asp Arg Tyr Tyr Gly Asp Pro Thr Asn Lys Pro Asn Pro Asn Leu

420 425 430

Gly Glu Ile Lys Asn Gly Pro Phe Tyr Ala Ala Lys Met Val Pro Gly

435 440 445

Asp Leu Gly Thr Lys Gly Gly Ile Arg Thr Asp Val His Gly Arg Ala

450 455 460

Leu Arg Asp Asp Asn Ser Val Ile Glu Gly Leu Tyr Ala Ala Gly Asn

465 470 475 480

Val Ser Ser Pro Val Met Gly His Thr Tyr Pro Gly Pro Gly Gly Thr

485 490 495

Ile Gly Pro Ala Met Thr Phe Gly Tyr Leu Ala Ala Leu His Leu Ala

500 505 510

Gly Lys Ala

515

<210> 4

<211> 1548

<212> DNA

<213> Artificial sequence ()

<400> 4

atgaccgaac aggattactc ggttttcgac gtcgtggtcg tcggatctgg agcggcgggc 60

atggtggcag cgctgacggc tgcgcaccag gggttatcta cagtggtagt cgagaaagca 120

cctcactatg gcggttcgac cgcgcgttca ggtggggggg tatggatacc gaacaacgag 180

gtattacagc gggacggtgt gaaagatacg cctgctgagg cccgtaaata tttgcatgcc 240

atcattggcg atgttgttcc agcagaaaag atagatacgt atctggatcg cagtccagag 300

atgttgtctt ttgtactgaa aaactcgccg ttaaaactgt gctgggtccc cgggtacagt 360

gactattatc ctgaaacacc gggtggtaaa gctactggtc gcagcgtgga gccgaaaccc 420

ttcaatgcca aaaagttagg gccggatgag aaaggcttgg aacctccata cgggaaagtc 480

gtatgggcga atgctactgg caaaaattta gtcggcatgg gccgtgcgtt gattgctcct 540

ttacgtatag ggctgcagaa agcaggagta cccgtccttc ttaacactgc attaacagat 600

ttatatcttg aagacggcgt cgttcgtggc atctatgttc gggaagctgg agcgcctaag 660

ctgatacgtg cgcgcaaggg cgttatcctg ggcagtggcg gtttcgagca caaccaggaa 720

atgcggacca aataccagcg gcaacccatc accacagaat ggacggtcgg cgccgtagct 780

aacacgggag atggtattgt tgccgcggag aagttaggag ctgcgcttga gttgatggaa 840

gatgcgtggt ggggtcccac agtacctctg gtgggcgcac cgtggttcgc tctttctgag 900

ggtagtatca ttgttaatat gaacggaaag agatttatga acgaatctat gccttatagc 960

gaagcatgtc atcacatgta cggtggacag tatggtcagg agaatgttcc cgcttggatg 1020

gtttttgatc agcagtaccg ggaccggtac atatttgctg ggctgcaacc cgggcaacgg 1080

ataccgaaga agtggatgga gagtggggtg atcgtgaagg ccgattccgt tgctgagtta 1140

gccgaaaaga ccggtctggc cccggacgcc ttaacggcca cgatcgaacg cttcaatggt 1200

ttcgcaagaa gcggagtgga cgaggatttt cacagaggcg aatctgctta tgatcgctac 1260

tatggagatc caacgaataa acccaaccct aatctgggcg aaatcaaaaa tggaccattc 1320

tatgcggcta aaatggtgcc aggtgacctt gggaccaagg gcggaattag aacagatgtt 1380

catggaagag cactgcgcga cgacaacagc gtaatcgagg gattatacgc tgcagggaac 1440

gtcagctcac cggtgatggg acacacgtat ccgggaccag gaggcacgat aggacccgca 1500

atgacgtttg gctacttagc cgctctgcac ttagctggca aggcctga 1548

Claims (2)

1. A process for the preparation of a C1, 2-dehydrosteroid comprising:

constructing an expression vector for expressing the recombinant KstD enzyme, wherein the amino acid sequence of the recombinant KstD enzyme is SEQ ID NO.3, and Maltose Binding Protein (MBP) is combined at the N end of the recombinant KstD enzyme;

expressing the recombinant KstD enzyme by using the expression vector and a cell culture mode to obtain a crude enzyme solution;

adding electron acceptor menadione into the crude enzyme solution, wherein the mass concentration of the electron acceptor menadione in the reaction solution is less than or equal to 1%;

adding a substrate androst-4-ene-3, 17-dione (4-AD) into the crude enzyme solution, and reacting to obtain the C1, 2-dehydrosteroid compound.

2. The method of claim 1, further comprising:

adding catalase into the crude enzyme solution before reaction, wherein the concentration of the catalase in the reaction solution is less than or equal to 100U/L.

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