CN111500549A - Enzyme for preparing C1, 2-dehydrogenation steroid compound and application thereof - Google Patents

Abstract

The invention provides an enzyme for preparing a C1, 2-dehydrogenation steroid compound and application thereof. The amino acid sequence of the enzyme for preparing the C1, 2-dehydrosteroid compound is SEQ ID.3. The enzyme may have Maltose Binding Protein (MBP) bound to the N-or C-terminus. The invention obtains the enzyme with high activity by modifying the enzyme. In addition, the solubility problem of the enzyme is solved by forming soluble MBP/KstD fusion proteins to enhance the solubility of the KstD protein.

Description

Enzyme for preparing C1, 2-dehydrogenation steroid compound and application thereof

Technical Field

The invention relates to the field of biochemistry, in particular to an enzyme for preparing a C1, 2-dehydrogenation steroid compound and application thereof.

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) (as shown in 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 chemical dehydrogenation processes have been used in the steroid pharmaceutical industry, enzymatic processes are highly efficient, produce no byproduct build-up, and produce high product purity, as compared to multi-step chemical synthesis of hormone synthesis, and thus the development of "green" highly efficient enzymatic methods for the preparation of steroid intermediates by heterologous expression of KstD has now begun to be of interest. M. smegmatis mc²155 MstKstD 1 can lead to a cortisol yield of 90% in 3 hours at a concentration of 6 g/L KstD2 of M.neoformans DSM1381 can lead to almost complete conversion of 30 g/L4-AD, but the low substrate concentrations and low conversion rates mentioned above limit the industrial 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 problems, the invention provides an enzyme for preparing C1, 2-dehydrosteroid compounds, wherein the amino acid sequence of the enzyme is SEQ ID.3.

In the above enzyme, wherein Maltose Binding Protein (MBP) is bound to the N-terminus or C-terminus of the enzyme.

The invention also provides a gene sequence for expressing the enzyme, and the gene sequence is SEQ ID.4.

The invention also provides an expression vector, wherein the expression vector is used for expressing the enzyme.

The invention also provides an expression system, wherein the expression system comprises the gene sequence.

The invention also provides application of the enzyme in preparation of C1, 2-dehydrogenation steroid compounds.

The invention obtains the KstD725 mutant with high activity by carrying out sequence modification and optimal design on the KstD 211. The KstD725 mutant has the amino acid sequence of SEQ ID NO.3. the DNA sequence coding the KstD725 mutant amino acid sequence is optimized by escherichia coli preferred codons and is suitable for efficient expression of escherichia coli, and one DNA sequence is SEQ ID NO. 4.

The invention obtains a soluble KstD fusion enzyme by modifying the KstD211 enzyme of mycobacterium HGMS 2; 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.

The KstD fusion enzyme efficiently expressed by recombinant escherichia coli is used, high-density fermentation is adopted to prepare the fusion KstD fusion enzyme, the enzyme specific activity reaches 31.6U/mg, the KstD fusion enzyme reaction electron acceptor type and dosage are optimized, low-cost and high-efficiency regeneration circulation is carried out on coenzyme, and a process for converting androstenedione (4-AD) into 1, 4-Androstenedione (ADD) by the fusion enzyme is established^-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 electrophorograms to analytically compare the expression of soluble KstD enzyme. Unfused KstD expression: 1. 2 is supernatant and pellet induced for 10 h; 3. 4 is the supernatant and pellet induced for 20 h. The fusion protein expresses MBP-KstD: 5. 6 is supernatant and pellet induced for 10 h; 7.8 is the supernatant and pellet induced for 20 h.

FIG. 3 shows the curves of the process of fed-batch fermentation of recombinant E.coli and induction of expression, wherein, xxx is OD600nm, ★ - ★ is dissolved oxygen, ● - ● is temperature, ◆ - ◆ is pH, ■ - ■ is rotation speed.

FIG. 4 shows an SDS-PAGE pattern of high density fermentatively produced KstD enzymes. 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 T L C (thin layer chromatography) analysis of the reaction of converting recombinant KstD enzyme into 4-AD for 1: 6 hours and 2: 20 hours.

FIG. 6 shows the conversion of 4-AD into ADD by T L C assay recombinant KstD enzyme 1:4-AD standard, 2, 3, 4, 5 represent the products of 6h, 12h, 18h, 24h enzyme reactions, respectively.

FIG. 7 shows the reaction of HP L C in the analysis of the activity of recombinant KstD enzyme converting 4-AD to produce ADD.a) the analysis of 4-AD standard HP L C, b) the analysis of ADD standard HP L C, and C) the analysis of KstD catalytic reaction solution HP L C.

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 invention carries out whole genome sequencing (GenbankID: CP031414.1) on 4-AD industrial production strain mycobacterium HGMS2, and identifies a unique KstD enzyme gene from HGMS2 strain, which is named as KstD211(SEQ ID NO.1) and an 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 amino acid sequence is optimized by escherichia coli preferred codons and is suitable for efficient expression of escherichia coli, and one DNA sequence is SEQ ID NO. 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 form soluble MBP/KstD fusion protein by fusing MBP protein (maltose-binding protein) at the N terminal or C terminal of KstD, 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 realizes the conversion rate of more than 98 percent by optimizing the proportion of the electron acceptor, regenerating the coenzyme system with low cost and high efficiency, utilizing the MBP/KstD725 fusion enzyme in vitro enzyme conversion method in a shake flask and taking 10 g/L4-AD as the raw material.

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. 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 is given in conjunction with specific examples to better understand the present invention.

EXAMPLE one construction and Shake flask expression of recombinant KstD fusion enzyme plasmids

Coli B L21 (DE3) cells containing the recombinant plasmid were cultured in L B medium containing 35. mu.g/ml kanamycin and rotated at 200rpm at 37 ℃ when OD600nm reached 0.8, 0.4mM isopropyl- β -D-thiogalactopyranoside (final concentration) was added and the culture temperature was decreased to 20 ℃ and the culture was continued for 18-20 hours, and the expression of soluble target enzyme protein was significantly increased as shown in FIG. 2 by SDS-PAGE gel.

EXAMPLE two preparation of KstD fusion enzymes by high Density fermentation

In order to obtain a high-density cell culture with a large amount of enzyme, a high-density medium is optimized, cells are cultured in a 15L fermenter, and some modifications are made, such as replacing glucose by glycerol, yeast powder as a nitrogen source, inducing by lactose, etc., at the start of fermentation, the temperature of the fermenter is controlled at 37 ℃ to allow rapid growth of the cells, in a fed-batch phase, dissolved oxygen is controlled at 10% to 30%, pH is controlled at 6.2 to 7.8, before the induction phase, the cell culture temperature is reduced to 20 ℃ and maintained for a certain period of time, then lactose mother liquor is added until the final concentration reaches 0.4mM, induction time is 18h to 20h to induce protein expression, after fermentation, E.coli cells are collected by centrifugation at 4 ℃, resuspended in 50mM Tris-HCl buffer (pH8.0), the suspension is disrupted by a high pressure homogenizer, disruption conditions of 5 ℃ or less and 1200bar, 3 cycles are performed to obtain a crude enzyme solution, the crude enzyme solution is retained for 20 minutes after 4 ℃ and 6000r/min, and the cell debris is removed by centrifugation, and then the enzyme is used as a solid reverse enzyme.

The method comprises the steps of optimizing a high-density fermentation formula, performing high-density fermentation on recombinant escherichia coli in a fermentation tank of 15L by adopting a fed-batch fermentation mode, wherein the liquid loading amount is 10L culture medium, performing high-temperature sterilization at 121 ℃, performing inoculation for 30min when the temperature is reduced to 37 ℃, and starting fermentation by adopting an inoculation amount of 5 percent, wherein the temperature, dissolved oxygen and pH change in the whole fermentation process is shown in figure 3. As can be seen from figure 3, the dissolved oxygen is reduced to the minimum in 5h after inoculation, and then the dissolved oxygen is increased back, the feeding is started, the dissolved oxygen is maintained in a certain fluctuation range (20-30 percent) in the feeding process, the fermentation is started to reduce the temperature in 16h, and an inducing solution is supplemented at a low temperature, the feeding speed is correspondingly reduced during an inducing phase, the lower growth speed of the thalli is maintained, so that the normal expression of the protein is ensured, the fluctuation of the pH is not large in the whole fermentation process, the pH fluctuation is not increased from 7.2 at the beginning of the fermentation to 6.7 after the fermentation is finished, the feeding is probably because the acid or alkali is not added, the acid is properly controlled, the feeding is not added, and the pH is increased, so that the acid is increased, and the pH is increased.

EXAMPLE III KstD fusion enzyme Activity

An appropriate amount of 2.3 centrifuged supernatant was taken, Mbp-KstD2 was assayed at 30 ℃ using a Nano Drop 2000 spectrophotometer (Thermo Scientific) at 600nm (ξ 600nm ═ 18.7 × 103 cm-1M-1) using Phenazine Methosulfate (PMS) and 2, 6-dichlorophenol-benzenediol (dcppi) the reaction mixture (1ml) consisted of 50mM Tris-HCl buffer (pH 7.0), 150mM PMS, 8mM dcppip, an appropriate concentration of supernatant or cell extract and 100mM AD methanol the activity was expressed in units per mg protein, 1U was defined as a 1 μmolmin-1 dcppip reduction, no activity was detected in the reaction mixture without 4-androstenone-3, 17-dione (AD), the remaining supernatant was subjected to PAGE-PAGE electrophoresis, SDS-gel was used with 8% separation gel and 5% concentrated gel, and it was identified whether the protein was expressed correctly as shown in the fermenter 4.

EXAMPLE IV Shake flask KstD fusion enzyme transformation 4-AD reaction Process

Androst-4-ene-3, 17-dione (AD) powder was put into 50mM of Tris-HCl buffer (pH8.0) to make a 10% concentration, emulsified with a homogenizer for 2h (preventing generation of a large amount of air bubbles, a small amount of an antifoaming agent may be added) to make AD particles smaller and uniformly dispersed in a buffer to prepare a substrate emulsion, enzyme activity test was performed according to the enzyme catalysis reaction described above, cells of high density fermentation were taken to be resuspended with Tris-HCl buffer (pH8.0), disrupted with a high pressure homogenizer to obtain a crude enzyme solution, the enzyme catalysis volume was 100ml, reaction was performed according to a 1% loading amount, 50ml of the crude enzyme solution was taken into a 250ml shake flask, 25ml of 4AD (about 1g) emulsion and 25ml of Tris-HCl buffer were added, 1% of an electron acceptor menaquinone was added, the shake flask was placed in a shaker, shaking reaction was performed at 30 ℃ and 200rpm, the reactions were performed for 6h and 20h, extracted with ethyl acetate, centrifuged, the sample was taken out on a capillary tube, the gel plate was taken out, and the sample was taken out of a silica gel filtration system, after the two-gel filtration reaction, the substrate emulsion was performed, the reaction was performed for a two-gel filtration reaction, after the two-ethanol suspension, the gel filtration reaction was performed for 2V reaction, the reaction was performed, the sample was taken out.

EXAMPLE V Pilot-plant KstD fusion enzyme conversion 4-AD reaction Process

The scale-up experiment was continued for the enzymatic conversion of kilogram-scale substrates, and the experiments were carried out in a 15L reaction vessel with a total reaction volume of 10L, again at 1% substrate concentration (i.e. 1Kg input), under the same reaction conditions as in shake flasks, at 6h, 12h, 18h, and 24h respectively, the samples were taken, extracted with ethyl acetate, centrifuged, and the supernatant was dipped in ethyl acetate by capillary tubing for spotting, the silica gel plate was placed in a developing solvent (V petroleum ether/V ethyl acetate 5:3), the solvent was removed and blown dry after the solvent climbed to two thirds of the silica gel plate, and the silica gel plate was exposed to uv light at 254nm, with the results shown in fig. 6.

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

Carrying out enzyme conversion test of tonnage substrate by continuous amplification experiment, carrying out experiment in a 15 ton reaction kettle, wherein the total reaction volume is 10 tons, the same 10% substrate concentration (namely 1 ton feeding amount) is adopted, the reaction conditions are the same as those of a shake flask, sampling is carried out at the reaction time of 6h, 12h, 18h and 24h, ethyl acetate is used for extraction and centrifugation, the ethyl acetate of the supernatant is dipped by a capillary tube for carrying out T L C point plate detection reaction process, HP L C detection is further carried out, the conversion rate is proved to reach more than 97%, the refining process can be carried out, and the conversion rate reaches more than 97% after the reaction is carried out for 20-24 hours as shown in figure 7, 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> enzyme for preparing C1, 2-dehydrosteroid compound and application thereof

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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 (6)

1. An enzyme for preparing C1, 2-dehydrosteroid compounds, wherein the amino acid sequence of the enzyme is SEQ ID.3.

2. The enzyme according to claim 1, wherein Maltose Binding Protein (MBP) is bound to the N-terminus or C-terminus of the enzyme.

3. A gene sequence expressing the enzyme of claim 1, said gene sequence being SEQ id.4.

4. An expression vector for expressing the enzyme according to claim 1 or 2.

5. An expression system, wherein the expression system comprises a gene sequence according to claim 3.

6. Use of an enzyme according to claim 1 or 2 for the preparation of C1, 2-dehydrosteroids.

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