WO2019010971A1

WO2019010971A1 - Preparation method for dehydroepiandrosterone, and enzyme for preparation thereof

Info

Publication number: WO2019010971A1
Application number: PCT/CN2018/075822
Authority: WO
Inventors: 傅荣昭; 刘立辉; 陈小春; 曹磊; 刘滔滔
Original assignee: 邦泰生物工程(深圳)有限公司
Priority date: 2018-02-08
Filing date: 2018-02-08
Publication date: 2019-01-17
Also published as: CN109312382A

Abstract

A preparation method for dehydroepiandrosterone, comprising: in a protective atmosphere, adding potassium tert-butoxide to tert-butanol, stirring evenly, adding 4-androstenedione to obtain a mixture, and adding the mixture dropwise to a sodium ascorbate-containing acetic acid solution for a reaction to obtain 5-androstenedione; dissolving the 5-androstenedione in an organic solvent, adding a ketone reductase, a glucose dehydrogenase, glucose and a redox coenzyme to obtain a mixture, controlling the pH of the mixture to be 6.0-6.3, stirring and reacting for 1-6 hours at 22-26°C to obtain a reaction solution, and performing separation and purification on the reaction solution to obtain dehydroepiandrosterone, the ketone reductase and the glucose dehydrogenase being coexpressed by a microbial strain and added in the form of a crude enzyme solution. The synthesis process of the preparation method has few steps, simple operations, high yields and low costs, and may be widely applied to industrial scale production. Also provided is an enzyme for preparation.

Description

Preparation method of dehydroepiandrosterone and preparation enzyme thereof

Technical field

The invention relates to the technical field of biomedicine, in particular to a preparation method of dehydroepiandrosterone and an enzyme for preparation thereof.

Background technique

Dehypoepiandrosterone (DHEA) is a C19 steroid carrier compound with the chemical name 3β-hydroxyandrost-5-en-17-one, the molecular formula C ₁₉ H ₂₈ O ₂ . DHEA is a large number of adrenal steroidal compounds coexisting in human plasma. It has anti-inflammatory, anti-proliferative and anti-depressant activities. Its derivatives can promote the immune response of animals in experiments and have a good neuroprotective effect. In addition, DHEA is an important intermediate for the synthesis of steroids. In recent years, DHEA has gradually developed into one of the most popular research objects due to its important research value.

The synthesis of traditional DHEA is mostly carried out by chemical methods, for example, using pregnane dienolone or gestational dienolone acetate as a raw material, and after deuteration, rearrangement and hydrolysis, a product is obtained. However, the synthesis method is completed in three or more chemical reactions, the raw material price is high, and the solvent used in the process such as benzene, alkyl halide or pyridine is harmful to the environment. In addition, the products in the traditional process have low purity, low efficiency, and complicated purification operations in the later stage.

Therefore, it is of great significance to develop a DHEA synthesis method with few steps, low cost, high yield and green environmental protection.

Summary of the invention

In order to solve the above technical problems, the present invention provides a preparation method of dehydroepiandrosterone and an enzyme for preparation thereof, and the preparation method of the dehydroepiandrosterone involves a two-step reaction, which greatly reduces the preparation process of the traditional method. The synthesis process of the present invention provides the synthesis process of the dehydroepiandrosterone with high yield, low cost and green environmental protection.

In a first aspect, the present invention provides a method for preparing dehydroepiandrosterone, comprising:

(1) Adding potassium t-butoxide to t-butanol under a protective atmosphere, stirring uniformly, adding 4-androstenedione to obtain a mixture, and adding the mixture to an acetic acid solution containing sodium ascorbate to obtain a mixture 5-androstenedione;

(2) dissolving the 5-androstenedione in the step (1) in an organic solvent, adding a ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme to obtain a mixture, and controlling the pH of the mixture. For 6.0-6.3, the reaction is stirred at 22-26 ° C for 1-6 hours to obtain a reaction solution, and the reaction solution is separated and purified to obtain dehydroepiandrosterone, and the ketoreductase and the glucose dehydrogenase are The microbial strain is co-expressed and added as a crude enzyme solution derived from one or more of the genus Burkholderia, Rhodococcus and Sphingomonas.

In the present invention, the specific process route of the preparation method of the dehydroepiandrosterone is as follows:

Wherein, the process comprises a two-step reaction, the first step is a chemical method, the second step is a biological enzymatic method, and the 4-androstenenedione (4-Androstene-3, 17-dione, 4-AD) is as As shown in the formula (I), the 5-androstenone (5-Androstene-3, 17-dione, 5-AD) is represented by the formula (II), and the dehydroepiandrosterone is as defined in the formula (III). ) shown.

Optionally, in the step (1), the protective atmosphere refers to the environment filled with nitrogen or an inert gas. The inert gas includes one or more of helium (He), neon (Ne), argon (Ar), krypton (Kr), and xenon (Xe).

Optionally, in the step (1), the specific preparation process of the mixture comprises: adding potassium t-butoxide to t-butanol at room temperature, then raising the temperature to 55-65 ° C, and stirring for 0.3-0.8 hours. After the potassium t-butoxide was completely dissolved, the temperature was maintained at 25 to 30 ° C, and 4-androstenedione was added thereto for stirring for 0.3 to 0.8 hours, followed by cooling to obtain the mixture. In the present invention, after the addition of potassium t-butoxide, the temperature is controlled at 55-65 ° C, the dissolution time of potassium t-butoxide can be greatly shortened, and the dissolution time of the conventional 3 hours or more can be shortened to about 0.5 hour; Maintaining at 25-30 ° C can promote the efficient forward reaction of the 4-androstenedione. If the temperature is too high, it is easy to reversely react to form 4-androstenedione. If the temperature is too low, the reaction takes a long time. , production efficiency is low.

Optionally, in the step (1), the reaction mixture is added to an acetic acid solution containing sodium ascorbate to obtain a 5-androstenedione, and the reaction temperature is 25-30 ° C, and the reaction is carried out. The time is 0.3-0.8 hours. Further preferably, the reaction temperature may be 26 to 30 ° C, and the reaction time is 0.5 to 0.8 hours. For example, the reaction temperature is 30 °C.

Optionally, in the step (1), the molar ratio of the 4-androstenedione and the potassium t-butoxide is 1: (0.75-1.2). Further optionally, the molar ratio of the 4-androstenedione and the potassium t-butoxide is 1: (0.8-1.2).

Optionally, in the step (2), the organic solvent comprises a hydrophobic solvent. Further optionally, the organic solvent comprises ethyl acetate. The preferred ethyl acetate solvent in step (2) of the present invention is effective to increase the yield of said dehydroepiandrosterone.

Optionally, in the step (1), the molar ratio of the 4-androstenedione, the acetic acid and the sodium ascorbate is 1: (0.5-1): (0.04-0.4). Further optionally, the molar ratio of the 4-androstenedione, the acetic acid and the sodium ascorbate is 1: (0.6-1): (0.05-0.2).

Optionally, in the step (2), the ketoreductase, glucose dehydrogenase, glucose and redox coenzyme are an aqueous solution or a buffer solution. Further optionally, the buffer solution comprises a phosphate buffer, a Tris-HCl buffer or other buffer solution having a pH of 6.0-7.0. For example, the buffer solution is a sodium phosphate buffer. Optionally, the buffer solution has a concentration of 50-120 mmol/L. Further optionally, the buffer solution has a concentration of 60-100 mmol/L. Preferably, the buffer solution has a concentration of 80-120 mmol/L. For example, the concentration of the buffer solution is 60 mmol/L, or 70 mmol/L, or 90 mmol/L, or 100 mmol/L, or 120 mmol/L.

Alternatively, the four components of the ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme may be added after mixing four kinds of ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme. Adding simultaneously; or separately adding the ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme, wherein, when added alone, the ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme are sequentially added in order. And the order of addition is not limited. The ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme may be added in the form of one or more of the ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme, and The remaining components of the ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme are mixed and added to the organic solvent containing 5-androstenedione. For example, the ketoreductase in the ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme and the glucose dehydrogenase are first mixed together, and then in a mixed group of ketoreductase and glucose dehydrogenase. Adding the remaining components of glucose and redox coenzyme to the fraction, and obtaining a mixed component containing ketoreductase, glucose dehydrogenase, glucose and redox coenzyme, and adding it to the 5-containing A mixed solution is obtained in an organic solvent of androstenedione.

Optionally, in the step (2), the concentration of the glucose is 0.04-0.12 mg/mL. Further optionally, the concentration of the glucose is from 0.08 to 0.1 mg/mL. For example, the concentration of the glucose in the buffer solution is 0.1 mg/mL, or 0.12 mg/mL.

Optionally, in the step (2), the microbial strain comprises one or both of Rosetta (DE3) E. coli and BL21 (DE3) E. coli. Further optionally, the microbial strain comprises Rosetta (DE3) E. coli.

Optionally, in the step (2), the specific process of co-expressing the microbial strain to produce the ketoreductase and the glucose dehydrogenase comprises: constructing a recombinant expression plasmid, transfecting the recombinant expression plasmid After the microorganism strain is introduced, the microorganism strain containing the recombinant expression plasmid is induced to express the ketoreductase and the glucose dehydrogenase in a microorganism culture solution, wherein the recombinant expression plasmid includes the ketoreductase and The gene coding sequence of the glucose dehydrogenase. The ketoreductase and the glucose dehydrogenase may be produced intracellularly in the microbial strain, or may be released from the microbial culture solution after intracellular production of the microbial strain. The crude enzyme solution is obtained by collecting, washing, and granulating the microorganism strain capable of expressing a ketoreductase and a glucose dehydrogenase, and the crude enzyme solution contains the ketoreductase and the glucose Dehydrogenase.

In the present invention, the ketoreductase and the glucose dehydrogenase are two independent protein molecules, and are obtained by the above-mentioned co-expression, so that the operation can save production cost and simplify the experimental process. The recombinant expression plasmid may simultaneously contain the gene coding sequence of the ketoreductase and the glucose dehydrogenase, and simultaneously express the ketoreductase and the glucose dehydrogenase protein molecules. In the present invention, the reaction in the step (2) is carried out in the form of a crude enzyme solution, for example, the crude enzyme solution may be an expression of the ketoreductase and the glucose dehydrogenase induced by Escherichia coli Rosetta (DE3). After the Escherichia coli Rosetta (DE3) is collected by centrifugation and washing, after the cells are suspended in a buffer solution and subjected to ultrasonic disruption, the ketoreductase and the glucose dehydrogenase are released to a buffer solution. The crude enzyme solution is obtained. The invention adopts the co-expression form to obtain the ketoreductase and the glucose dehydrogenase crude enzyme solution to participate in the preparation of DHEA, not only streamlining the preparation process, but also improving the yield, saving cost and being green. Further, the present invention can also purify the ketoreductase and glucose dehydrogenase and participate in the synthesis reaction.

Alternatively, the gene coding sequence of the ketoreductase includes any one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. Alternatively, the gene coding sequence of the glucose dehydrogenase comprises the nucleotide sequence as shown in SEQ ID NO: 4. The present invention preferably has the above ketoreductase comprising any one of the nucleotide sequences shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, which can be efficiently expressed in the microorganism strain The crude enzyme solution has a higher concentration of the ketoreductase.

Optionally, in the step (2), the molar ratio of the ketoreductase, the glucose dehydrogenase, and the 4-androstenedione is (0.015-0.04): (0.01-0.04):1. .

In the step (2), the redox coenzyme includes one or more of NAD ⁺ , NADP ⁺ , NADH, and NADPH. Further optionally, in the step (2), the redox coenzyme comprises NAD ⁺ . Optionally, in the step (2), the concentration of the redox coenzyme is 0.04-0.4 mg/mL. Further optionally, the concentration of the redox coenzyme is from 0.08 to 0.2 mg/mL. For example, the concentration of the redox coenzyme in the buffer solution is 0.1 mg/mL, or 0.15 mg/mL, or 0.2 mg/mL.

Wherein, the NAD ⁺ is Nicotinamide adenine dinucleotide (NAD ⁺ ), also known as oxidized coenzyme I; the NADH is a reduced state of nicotinamide adenine dinucleotide, also called Is a reduced coenzyme I; the NADP ⁺ is nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), also known as oxidized coenzyme II; the NADPH is nicotinamide adenine dinucleotide The reduced state of phosphoric acid, also known as reduced coenzyme II. In the presence of oxygen (O ₂ ), a redox coenzyme, glucose dehydrogenase and glucose form a coenzyme regeneration system, which is an electronic cycle system, which can effectively make 5 under the action of ketoreductase - androstenedione is reduced to form dehydroepiandrosterone, which increases the yield of dehydroepiandrosterone. System of the present invention, DHEA has very high yields can only use the NAD ^{^+,} NADP ^+, NADH and NADPH in NAD ⁺ can be implemented with cycles and the.

Optionally, the process of separating and purifying in the step (2) comprises a sequential filtration, washing and extraction process. Further optionally, the step (2) further comprises recrystallizing the dehydroepiandrosterone.

Optionally, the specific process of separating and purifying in the step (2) comprises: adding the reaction liquid to diatomaceous earth to obtain a filtrate and a filter cake, and washing the filter cake with ethyl acetate to obtain a washing liquid, and collecting And mixing the washing liquid and the filtrate to obtain an aqueous phase and an organic phase, and extracting the aqueous phase with ethyl acetate for two or three times to obtain an extract, and mixing the extract with the organic phase and concentrating Dehydroepiandrosterone is obtained by filtration, washing, washing and drying.

The method for preparing dehydroepiandrosterone provided by the first aspect of the present invention, the preparation method is simple, time-saving, high-efficiency, low-cost, green and environmentally friendly, and the preparation method is applicable to large-scale industrial production; The resulting dehydroepiandrosterone has an extremely high yield.

In a second aspect, the present invention provides a ketoreductase comprising ketoreductase KRED3 (abbreviated as KRED3), ketoreductase KRED4 (abbreviated as KRED4) and ketoreductase KRED5 (abbreviated as KRED5) One or more of the gene coding sequences of the ketoreductase KRED3 include the nucleotide sequence set forth in SEQ ID NO: 1, and the gene coding sequence of the ketoreductase KRED4 includes as set forth in SEQ ID NO: The nucleotide sequence shown, the gene coding sequence of the ketoreductase KRED5 includes the nucleotide sequence shown in SEQ ID NO: 3.

Optionally, the gene coding sequence of KRED3 further comprises a nucleotide sequence of at least 95% homology of the nucleotide sequence shown in SEQ ID NO: 1, and the gene coding sequence of KRED4 further comprises SEQ ID NO: a nucleotide sequence of at least 95% homology of the nucleotide sequence shown by 2, the gene coding sequence of KRED5 further comprising at least 95% homology of the nucleotide sequence as shown in SEQ ID NO: Nucleotide sequence.

Wherein the amino acid sequence of KRED3 comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence of KRED4 comprises the amino acid sequence set forth in SEQ ID NO: 6, and the amino acid sequence of KRED5 comprises SEQ ID NO: NO: The amino acid sequence shown in 7. Alternatively, the amino acid sequence of the glucose dehydrogenase comprises the amino acid sequence set forth in SEQ ID NO: 8.

Alternatively, the gene of the ketoreductase KRED3 of the present invention is derived from Burkholderia territorii, the gene of the ketoreductase KRED4 is derived from Rhodococcus, the ketoreductase The gene for KRED5 is derived from Sphingomonas sp. The gene for the glucose dehydrogenase is derived from Bacillus megaterium. Preferably, the ketoreductase of the present invention has strong chemical selectivity and regioselectivity, and under certain conditions, the 5-androstenedione can be efficiently converted into the dehydroepiandrosterone.

Alternatively, the gene fragment of any one of the ketoreductases KRED3, KRED4 and KRED5 further includes a His tag. Further optionally, the gene fragment of any one of the ketoreductases KRED3, KRED4 and KRED5 further comprises an 8×His tag. The gene fragment of the glucose dehydrogenase further includes a 6 x His tag. As the number of His tags of the His tag increases, the binding efficiency of the protein increases, and the purification yield increases; the present invention can connect different numbers of His on the ketoreductase and the glucose dehydrogenase. The His tag is separately purified.

The ketoreductases KRED3, KRED4 and KRED5 provided by the second aspect of the present invention are one of the enzymes for preparing the method for preparing dehydroepiandrosterone according to the first aspect of the present invention, and a His tag is added to the gene fragment (group) The nucleotide sequence of the amino acid tag enables the expressed protein to be tagged with His tag. The His tag facilitates the isolation and purification of the expressed protein, and is analyzed and traced in experiments, such as analysis for immunoblot experiments. .

In a third aspect, the present invention also provides a use of a ketoreductase for catalyzing the conversion of a ketone or an aldehyde to a chiral alcohol, the gene coding sequence of the ketoreductase comprising SEQ ID NO: 1, SEQ ID NO: 2 And any one of the nucleotide sequences shown in SEQ ID NO: 3. That is, the ketoreductase of the present invention can efficiently and selectively catalyze the reduction of a carbonyl group on an aldehyde or a ketone to a chiral hydroxyl group.

Alternatively, the use of the catalytic ketone or aldehyde to convert to a chiral alcohol includes the use of catalyzing the conversion of 5-androstenedione to dehydroepiandrosterone. Preferably, the ketoreductase of the present invention has strong chemical selectivity and regioselectivity, and under certain conditions, the 5-androstenedione can be efficiently and selectively converted to the dehydroepiandrosterone. .

In a fourth aspect, the present invention also provides a recombinant plasmid comprising a gene coding sequence of a ketoreductase, wherein the gene coding sequence of the ketoreductase comprises a gene coding sequence of KRED3, a gene coding sequence of KRED4, and KRED5 One or more of the gene coding sequences, the gene coding sequence of KRED3 comprising the nucleotide sequence shown in SEQ ID NO: 1, the gene coding sequence of KRED4 comprising as shown in SEQ ID NO: The nucleotide sequence of the KRED5 gene coding sequence includes the nucleotide sequence shown in SEQ ID NO: 3.

Optionally, the recombinant plasmid further comprises a gene coding sequence of glucose dehydrogenase, and an RBS sequence is inserted between the gene coding sequence of the glucose dehydrogenase and the gene coding sequence of the ketoreductase, and the glucose is removed. The gene coding sequence of the hydrogenase includes a nucleotide sequence as shown in SEQ ID NO: 4, and the RBS sequence includes the nucleotide sequence as shown in SEQ ID NO: 17. Alternatively, the recombinant plasmid includes a gene coding sequence for a ketoreductase ligated from the 5' end to the 3' end, a RBS sequence, and a gene coding sequence for glucose dehydrogenase. The RBS sequence of the present invention is a ribosome binding site (RBS) sequence, which can effectively promote the independent transcription and translation of two homologous genes (the ketoreductase gene and the glucose dehydrogenase gene).

The recombinant plasmid of the fourth aspect of the invention can be used for co-expressing ketone reductase and glucose dehydrogenase, and the ketoreductase and glucose dehydrogenase have excellent activity and can be widely used in the fields of biopharmaceuticals, protein production and the like.

In a fifth aspect, the present invention also provides a method for preparing a recombinant plasmid, comprising:

(1) providing or preparing a gene template comprising a ketoreductase and a glucose dehydrogenase;

(2) preparing an upstream primer and a downstream primer of the ketoreductase and the gene of the glucose dehydrogenase, and amplifying the ketoreductase and the gene fragment of the glucose dehydrogenase, the gene of the ketoreductase The coding sequence includes any one of the nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the genetic coding sequence of the glucose dehydrogenase includes SEQ ID NO: a nucleotide sequence as shown in 4;

(3) taking the pET22b(+) vector plasmid, inserting the ketoreductase and the glucose dehydrogenase gene fragment amplified by the step (2) into the pET22b(+) vector plasmid, and purifying and recycling The recombinant plasmid was obtained.

Alternatively, the gene for the ketoreductase is inserted between the Nde I and EcoR I cleavage sites in the pET22b(+) vector plasmid. When the gene of the ketoreductase is inserted into the pET22b(+) vector plasmid, the 3' end of the gene coding sequence of the ketoreductase can be added to the stop codon (such as TAA) and the pET22b (+) vector plasmid in the EcoRI digestion. The sites are connected.

The gene for the glucose dehydrogenase was inserted between the EcoR I and Xho I restriction sites in the pET22b(+) vector plasmid. When the gene of the glucose dehydrogenase is inserted into the pET22b(+) vector plasmid, the 3' end of the gene coding sequence of the glucose dehydrogenase may be added to a stop codon (such as TAA) and a pET22b (+) vector plasmid. I cleavage sites are linked.

Optionally, inserting an RBS sequence between the ketoreductase and the glucose dehydrogenase gene coding sequence, the RBS sequence comprising the nucleotide sequence set forth in SEQ ID NO: 17 optionally The gene coding sequence of the ketoreductase includes any one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the gene coding sequence of the glucose dehydrogenase includes A nucleotide sequence as shown in SEQ ID NO: 4. The sequence of the ketoreductase and the glucose dehydrogenase gene coding sequence in the pET22b(+) vector plasmid may be that the 3' end of the ketoreductase gene is located 5' of the glucose dehydrogenase gene Before the end, or the 5' end of the ketoreductase gene is located after the 3' end of the glucose dehydrogenase gene.

Further optionally, the RBS sequence is inserted before the 5' end of the glucose dehydrogenase gene sequence. A linker may be disposed between the 3' end of the RBS sequence and the 5' end of the glucose dehydrogenase gene sequence to facilitate translation recognition of the glucose dehydrogenase, which is beneficial to ketoreductase and glucose dehydrogenase. Translational expression can be performed separately in the same vector. Preferably, the sequence of the linker may be "ATATACAT".

Alternatively, in the step (3), the cleavage sites used for the pET22b(+) vector plasmid include Nde I endonuclease and Xho I endonuclease.

In the present invention, the preparation method can prepare a recombinant plasmid containing only a ketoreductase or a glucose dehydrogenase, and can also prepare a recombinant plasmid containing both a ketoreductase and a glucose dehydrogenase. Alternatively, one or both of the ketoreductase and glucose dehydrogenase of the present invention can be obtained by transfecting the recombinant plasmid into a microbial strain to induce expression.

The beneficial effects of the present invention include the following aspects:

1. The preparation method of dehydroepiandrosterone according to the present invention comprises a two-step reaction, using 4-androstenedione as a starting material, and adopting a biological enzyme such as ketoreductase, which has low production cost, simple process and time saving. High efficiency, mild reaction conditions, greatly improving the yield of the product;

2. The preparation method of dehydroepiandrosterone according to the invention is green and environmentally friendly, avoids environmental pollution problems from the root causes, further reduces costs, and can be widely applied to industrial scale production;

3. Dehydroepiandrosterone prepared by the method of the present invention has high purity and can be widely used in the pharmaceutical field or biomedical field.

DRAWINGS

1 is a plasmid map of a recombinant plasmid pET22b-KRED3-GDH according to an embodiment of the present invention;

2 is a plasmid map of a recombinant plasmid pET22b-KRED4-GDH according to an embodiment of the present invention;

3 is a plasmid map of a recombinant plasmid pET22b-KRED5-GDH according to an embodiment of the present invention;

4 is a gel electrophoresis diagram of a KRED3-GDH crude enzyme solution according to an embodiment of the present invention;

5 is a gel electrophoresis diagram of a KRED4-GDH crude enzyme solution according to an embodiment of the present invention;

6 is a gel electrophoresis diagram of a KRED5-GDH crude enzyme solution according to an embodiment of the present invention;

7 is a high performance liquid chromatogram of 4-androstenedione according to an embodiment of the present invention;

8 is a high performance liquid chromatogram of 5-androstenedione according to an embodiment of the present invention;

9 is a high performance liquid chromatogram of a dehydroepiandrosterone standard according to an embodiment of the present invention;

Figure 10 is a high performance liquid chromatogram of a crude product of dehydroepiandrosterone according to an embodiment of the present invention.

Detailed ways

The following are the preferred embodiments of the embodiments of the present invention, and it should be noted that those skilled in the art can make some improvements and refinements without departing from the principles of the embodiments of the present invention. And retouching is also considered to be the scope of protection of the embodiments of the present invention.

Unless otherwise stated, the raw materials and other chemical reagents used in the examples of the present invention are all commercially available.

The present invention provides a preparation method of a ketoreductase and a glucose dehydrogenase, wherein the ketoreductase and the glucose dehydrogenase are enzymes for preparation in the preparation method of the dehydroepiandrosterone, and the specific experiment The steps include:

(1) Construction of pET22b-KRED3/4/5-GDH recombinant plasmid

The upstream primer and the downstream primer were provided, and the gene coding sequences of ketoreductase KRED3, ketoreductase KRED4, ketoreductase KRED5 and glucose dehydrogenase were obtained by PCR amplification experiments. Wherein the gene coding sequence of the ketoreductase KRED3 is as shown in SEQ ID NO: 1, the gene coding sequence of the ketoreductase KRED4 is as shown in SEQ ID NO: 2, and the coding sequence of the ketoreductase KRED5 is encoded. As shown in SEQ ID NO: 3, the gene coding sequence of the glucose dehydrogenase is shown in SEQ ID NO: 4. The base sequence of the upstream primer corresponding to the ketoreductase KRED3 is shown in SEQ ID NO: 9, and the base sequence of the downstream primer is shown in SEQ ID NO: 10. The base sequence of the upstream primer corresponding to the gene of the ketoreductase KRED3 is shown in SEQ ID NO: 9, and the base sequence of the downstream primer is shown in SEQ ID NO: 10. The base sequence of the upstream primer corresponding to the gene of the ketoreductase KRED4 is shown in SEQ ID NO: 11, and the base sequence of the downstream primer is shown in SEQ ID NO: 12. The base sequence of the upstream primer corresponding to the gene of the ketoreductase KRED5 is shown in SEQ ID NO: 13, and the base sequence of the downstream primer is shown in SEQ ID NO: 14. The base sequence of the upstream primer corresponding to the gene of the glucose dehydrogenase is shown in SEQ ID NO: 15, and the base sequence of the downstream primer is shown in SEQ ID NO: 16. The RBS sequence is inserted before the 5' end of the gene of the glucose dehydrogenase, and can be inserted into the plasmid pET22b(+) by the upstream and downstream primers as shown in SEQ ID NO: 17.

The PCR amplification system was carried out by the corresponding upstream primer and downstream primer, respectively, and the amplification system was configured as follows:

The PCR amplification procedure was: predenaturation at 98 ° C for 2 min; denaturation at 98 ° C for 10 s; annealing at 60 ° C for 30 s; extension at 72 ° C for 1 min; after 30 cycles, extension at 72 ° C for 10 min. The PCR products were purified by gel recovery kit and digested with restriction endonucleases Nde I and EcoR I, EcoR I and Xho I respectively. After digestion, they were ligated with T4 ligase and digested with Nde I and Xho I. Plasmid pET22b(+). The ligation product was transferred into E. coli DH5α, and after screening with ampicillin resistance (Amp ⁺ ), the colony was picked and sequenced. After successful sequencing, the recombinant plasmid pET22b-KRED3/4/5 co-expressed by ketoreductase and glucose dehydrogenase was obtained. - GDH (including pET22b-KRED3-GDH, pET22b-KRED4-GDH or pET22b-KRED5-GDH). The plasmid map of the recombinant plasmid pET22b-KRED3-GDH is shown in Figure 1; the plasmid map of the recombinant plasmid pET22b-KRED4-GDH is shown in Figure 2; the plasmid map of the recombinant plasmid pET22b-KRED5-GDH is shown in Figure 3 is shown. The DNA templates described in the present embodiment are plasmids pET22b-KRED3, pET22b-KRED4, pET22b-KRED5, and pET22b-GDH, respectively.

(2) expression of ketoreductase and glucose dehydrogenase

Transfer one or more of the constructed recombinant plasmids pET22b-KRED3-GDH, pET22b-KRED4-GDH and pET22b-KRED5-GDH into E. coli Rosetta (DE3) to obtain ketone reductase and glucose dehydrogenase E. Rosetta (DE3). Escherichia coli containing the recombinant plasmid pET22b-KRED3-GDH, pET22b-KRED4-GDH or pET22b-KRED5-GDH was inoculated to a 50 mL containing 10 mL of LB medium (100 μg/mL ampicillin (Amp)) at a 1% inoculation amount. In a triangular flask, maintain a constant shaking temperature of 37 ° C and 200 rpm. After overnight culture, transfer the bacterial solution to a 2 L flask containing 1 L of LB medium (100 μg/mL Amp) at 1% inoculation. The OD600 value of the medium cultured at 37 ° C was adjusted to about 0.6, and isopropyl-β-D-thiogalactoside (IPTG) inducer (systemic concentration: 0.5 mM) was added, and cultured at 37 ° C for 8 hours. The cells were collected by centrifugation. The cells were resuspended in 4 times the mass of 100 mM potassium phosphate buffer (pH=6.5) and sonicated for 20 min to obtain KRED3-GDH crude enzyme solution, KRED4-GDH crude enzyme solution or KRED5-GDH crude enzyme solution. The KRED3-GDH crude enzyme solution contains a ketoreductase KRED3 and a glucose dehydrogenase. The KRED4-GDH crude enzyme solution contains a ketoreductase KRED4 and a glucose dehydrogenase. The KRED5-GDH crude enzyme solution contains a ketoreductase KRED5 and a glucose dehydrogenase.

The obtained KRED3-GDH crude enzyme solution, KRED4-GDH crude enzyme solution or KRED5-GDH crude enzyme solution was identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 4 is a gel electrophoresis diagram of the KRED3-GDH crude enzyme solution, wherein the lane M is the protein Marker (Thermo Scientific PageRuler), the lane 1 is the total protein after the cell disruption, and the lane 2 is the supernatant after the cell disruption. The solution wherein KRED3 has a molecular weight of about 28 kDa and GDH has a molecular weight of 26 kDa. Figure 5 is a gel electrophoresis diagram of the KRED4-GDH crude enzyme solution, wherein the lane M is the protein Marker (Thermo Scientific PageRuler), the lane 1 is the supernatant after the cell disruption, and the lane 2 is the total cell after the cell is broken. The protein, wherein KRED4 has a molecular weight of 29 kDa and the GDH has a molecular weight of 26 kDa. Figure 6 is a gel electrophoresis diagram of the KRED5-GDH crude enzyme solution, wherein the lane M is the protein Marker (Thermo Scientific PageRuler), the lane 1 is the supernatant after the cell disruption, and the lane 2 is the total cell after the cell is broken. The protein, wherein KRED5 has a molecular weight of 28 kDa and the GDH has a molecular weight of 26 kDa. The ketoreductases KRED3, KRED4 and KRED5, as well as the molecules of GDH, are similar in theoretical calculations for the corresponding proteins.

Embodiment 1

A method for preparing dehydroepiandrosterone comprises the following steps:

(1) Under nitrogen protection, add 82.5g of potassium t-butoxide to 388mL of tert-butanol, then raise the temperature to 60 ° C, stir for 0.5h to completely dissolve potassium t-butoxide, cool down to 30 ° C, continue to add nitrogen protection 100 g of 4-AD was continuously stirred for 0.5 h and then cooled to room temperature to obtain a mixture. Under a nitrogen atmosphere, 6.0 g of sodium ascorbate was added to 205.3 g of a 26.8% aqueous acetic acid solution to obtain an acetic acid solution containing sodium ascorbate. Under nitrogen protection, the mixture was slowly added dropwise to the acetic acid solution containing sodium ascorbate at 30 ° C for 30 min, maintained at a temperature of 30 ° C for 0.5 h, and cooled at room temperature to obtain a reaction containing 5-AD. liquid.

(2) 500 mL of ethyl acetate was added to the reaction solution of the 5-AD, and the mixture was stirred uniformly, and 400 mL of a 1.8 g/L KRED3 and GDH crude enzyme solution, 300 mL of 100 mmol/L sodium phosphate buffer (pH 6.5) was added. 100 g of glucose, 200 mg of NAD ⁺ , stirred at 25 ° C until completely dissolved, adding a reaction solution containing 5-AD, controlling pH to 6.3, temperature 26 ° C, stirring at 200 rpm for 3 h to obtain an enzyme reaction solution; adding the enzyme reaction solution After stirring to diatomaceous earth, the filter cake was collected, and the filter cake was washed twice with ethyl acetate. The washing liquid and the filtrate were mixed, the aqueous phase and the organic phase were separated, and the aqueous phase was extracted twice with ethyl acetate. The liquid was mixed with the organic phase, and the organic phase was concentrated under reduced pressure. 600 g of water was added, and the mixture was thoroughly stirred and then filtered. The filter cake was washed twice with 120 g of water, and the washed cake was vacuum dried to obtain a crude DHEA product. The crude DHEA product was transferred to a clean container, and 234 g of acetone was added to dissolve, and then 200 g of water was added, and the above operation was repeated to carry out recrystallization to obtain 78.02 g of DHEA final product. In the present example, the yield of DHEA was 78.02%.

The two-step reaction was sampled and tested. The detection conditions were: Ferenbach Phenyl-Hexyl 250×4.6 mm column, wavelength UV210 nm, mobile phase 50% acetonitrile aqueous solution, flow rate 1.0 mL/min, temperature 25 °C. Among them, from the liquid chromatogram of 4-AD (see Figure 7) and the liquid chromatogram of 5-AD synthesis (see Figure 8), it was found that 4-AD can be efficiently converted to produce 5-AD; DHEA The liquid chromatogram of the crude product (Figure 10) is compared to the liquid chromatogram of the DHEA standard (see Figure 9), which contains the higher purity DHEA.

Embodiment 2

A method for preparing dehydroepiandrosterone comprises the following steps:

(1) Under nitrogen protection, add 80.0 g of potassium t-butoxide to 388 mL of tert-butanol, then raise the temperature to 55 ° C, stir for 0.8 h until the potassium tert-butoxide is completely dissolved, cool to 30 ° C, and continue to add nitrogen gas. 100 g of 4-AD was stirred for 0.6 h and then cooled to room temperature to obtain a mixture. Under a nitrogen atmosphere, 6.0 g of sodium ascorbate was added to 200.0 g of a 26.8% aqueous acetic acid solution to obtain an acetic acid solution containing sodium ascorbate. Under nitrogen protection, the mixture was slowly added dropwise to the acetic acid solution containing sodium ascorbate at 30 ° C for 30 min, maintained at a temperature of 30 ° C for 0.5 h, and cooled at room temperature to obtain a reaction containing 5-AD. liquid.

(2) 500 mL of ethyl acetate was added to the reaction solution of the 5-AD, and the mixture was stirred uniformly. 400 mL of a crude enzyme solution of 1.5 g/L of KRED4 and GDH, 300 mL of 80 mmol/L sodium phosphate buffer (pH 6.0) was added. 100 g of glucose, 200 mg of NAD ⁺ , stirred at 25 ° C until completely dissolved, adding a reaction solution containing 5-AD, controlling pH to 6.1, temperature 22 ° C, stirring at 180 rpm for 5 h to obtain an enzyme reaction solution; adding the enzyme reaction solution After stirring to diatomaceous earth, the filter cake was collected, and the filter cake was washed twice with ethyl acetate. The washing liquid and the filtrate were mixed, the aqueous phase and the organic phase were separated, and the aqueous phase was extracted twice with ethyl acetate. The liquid was mixed with the organic phase, and the organic phase was concentrated under reduced pressure. 600 g of water was added, and the mixture was thoroughly stirred and then filtered. The filter cake was washed twice with 120 g of water, and the washed cake was vacuum dried to obtain a crude DHEA product. The crude DHEA product was transferred to a clean container, and 234 g of acetone was added to dissolve, and then 200 g of water was added. The above operation was repeated for recrystallization to obtain 75.16 g of DHEA final product. In the present example, the yield of DHEA was 75.16%.

Embodiment 3

A method for preparing dehydroepiandrosterone comprises the following steps:

(1) Under nitrogen protection, add 90.0g of potassium t-butoxide to 400mL of tert-butanol, then raise the temperature to 65 ° C, stir for 0.3h to completely dissolve potassium t-butoxide, cool down to 30 ° C, continue to add nitrogen protection 100 g of 4-AD was continuously stirred for 0.5 h and then cooled to room temperature to obtain a mixture. Under a nitrogen atmosphere, 6.5 g of sodium ascorbate was added to 230 g of a 25% aqueous acetic acid solution to obtain an acetic acid solution containing sodium ascorbate. Under nitrogen protection, the mixture was slowly added dropwise to the acetic acid solution containing sodium ascorbate at 30 ° C for 30 min, maintained at a temperature of 28 ° C for 0.5 h, and cooled at room temperature to obtain a reaction containing 5-AD. liquid.

(2) 500 mL of ethyl acetate was added to the reaction solution of the 5-AD, and the mixture was stirred uniformly, and 400 mL of a 2.0 g/L KRED5 and GDH crude enzyme solution, 300 mL of 100 mmol/L sodium phosphate buffer (pH 7.0) was added. 100 g of glucose, 180 mg of NADH, stirred at 25 ° C until completely dissolved, adding a reaction solution containing 5-AD, controlling pH to 6.2, temperature 26 ° C, stirring at 220 rpm for 2 h to obtain an enzyme reaction solution; adding the enzyme reaction solution to After stirring and filtering in celite, the cake was collected, and the filter cake was washed twice with ethyl acetate, and the washing liquid and the filtrate were mixed, the aqueous phase and the organic phase were separated, and the aqueous phase was extracted twice with ethyl acetate. After mixing with the organic phase, the organic phase was concentrated under reduced pressure, 600 g of water was added, and the mixture was thoroughly stirred and filtered. The filter cake was washed twice with 120 g of water, and the washed cake was vacuum dried to obtain a crude DHEA product. The crude DHEA product was transferred to a clean container, and after adding 250 g of acetone to dissolve, 200 g of water was added, and the above operation was repeated to carry out recrystallization to obtain 77.53 g of DHEA final product. In the present example, the yield of DHEA was 77.53%.

The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

A method for preparing dehydroepiandrosterone, which comprises:

(1) Adding potassium t-butoxide to t-butanol under a protective atmosphere, stirring uniformly, adding 4-androstenedione to obtain a mixture, and adding the mixture to an acetic acid solution containing sodium ascorbate to obtain a mixture 5-androstenedione;

(2) dissolving the 5-androstenedione in the step (1) in an organic solvent, adding a ketoreductase, glucose dehydrogenase, glucose, and redox coenzyme to obtain a mixture, and controlling the pH of the mixture. For 6.0-6.3, the reaction is stirred at 22-26 ° C for 1-6 hours to obtain a reaction solution, and the reaction solution is separated and purified to obtain dehydroepiandrosterone, and the ketoreductase and the glucose dehydrogenase are The microbial strain is co-expressed and added as a crude enzyme solution derived from one or more of the genus Burkholderia, Rhodococcus and Sphingomonas.
The method for preparing dehydroepiandrosterone according to claim 1, wherein in the step (1), the specific preparation process of the mixture comprises: adding potassium t-butoxide to t-butanol at room temperature, Then, the temperature is raised to 55-65 ° C, stirred for 0.3-0.8 hours until the potassium t-butoxide is completely dissolved, the temperature is maintained at 25-30 ° C, 4-androstenedione is added for stirring for 0.3-0.8 hours, and then cooled. The mixture.
The method for preparing dehydroepiandrosterone according to claim 1, wherein in the step (1), the mixture is added dropwise to an acetic acid solution containing sodium ascorbate to obtain 5-androstene. In the course of the ketone, the reaction temperature is 25-30 ° C and the reaction time is 0.3-0.8 hours.
The method for producing dehydroepiandrosterone according to claim 1, wherein in the step (2), the microorganism strain comprises one or both of Rosetta E. coli and BL21 E. coli.
The method for producing dehydroepiandrosterone according to claim 1, wherein in the step (2), the specific process of co-expressing the microbial strain to produce the ketoreductase and the glucose dehydrogenase comprises: Constructing a recombinant expression plasmid, transfecting the recombinant expression plasmid into the microbial strain, and inducing the microbial strain containing the recombinant expression plasmid to express the ketoreductase and the glucose dehydrogenase in a microbial culture solution Wherein the recombinant expression plasmid comprises the ketoreductase and the gene coding sequence of the glucose dehydrogenase.
The method for producing dehydroepiandrosterone according to claim 1 or 5, wherein the gene coding sequence of the ketoreductase comprises as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. Any one of the nucleotide sequences; the gene coding sequence of the glucose dehydrogenase comprises the nucleotide sequence set forth in SEQ ID NO: 4.
The method for producing dehydroepiandrosterone according to claim 1, wherein in the step (2), the redox coenzyme comprises one or more of NAD + , NADP + , NADH and NADPH.
A ketoreductase, wherein the ketoreductase comprises one or more of a ketoreductase KRED3, a ketoreductase KRED4 and a ketoreductase KRED5, and the gene coding sequence of the ketoreductase KRED3 comprises as SEQ ID a nucleotide sequence represented by NO: 1, the gene coding sequence of the ketoreductase KRED4 includes the nucleotide sequence shown in SEQ ID NO: 2, and the gene coding sequence of the ketoreductase KRED5 includes SEQ ID NO: NO: The nucleotide sequence shown by 3.
A ketoreductase for use in catalyzing the conversion of a ketone or an aldehyde to a chiral alcohol, wherein the gene coding sequence of the ketoreductase comprises as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: Any of the nucleotide sequences shown.
The use according to claim 9, wherein the conversion of the catalytic ketone or aldehyde to a chiral alcohol comprises the use of catalyzing the conversion of 5-androstenedione to dehydroepiandrosterone.
A recombinant plasmid, wherein the recombinant plasmid comprises a gene coding sequence of a ketoreductase, and the gene coding sequence of the ketoreductase comprises a gene coding sequence of a ketoreductase KRED3, a gene coding sequence of a ketoreductase KRED4, and a ketone reduction One or more of the gene coding sequences of the enzyme KRED5, the gene coding sequence of the ketoreductase KRED3 includes the nucleotide sequence shown in SEQ ID NO: 1, and the gene coding sequence of the ketoreductase KRED4 includes As the nucleotide sequence shown in SEQ ID NO: 2, the gene coding sequence of the ketoreductase KRED5 includes the nucleotide sequence shown in SEQ ID NO: 3.
The recombinant plasmid according to claim 11, wherein the recombinant plasmid further comprises a gene coding sequence of glucose dehydrogenase, and a gene coding sequence of the glucose dehydrogenase and a gene coding sequence of the ketoreductase are further inserted. There is an RBS sequence, the gene coding sequence of the glucose dehydrogenase comprises a nucleotide sequence as shown in SEQ ID NO: 4, and the RBS sequence comprises the nucleotide sequence shown in SEQ ID NO: 17.
A method for preparing a recombinant plasmid, comprising:

(1) providing or preparing a gene template comprising a ketoreductase and a glucose dehydrogenase;

(2) preparing an upstream primer and a downstream primer of the ketoreductase and the gene of the glucose dehydrogenase, and amplifying the ketoreductase and the gene fragment of the glucose dehydrogenase, the gene of the ketoreductase The coding sequence includes any one of the nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the genetic coding sequence of the glucose dehydrogenase includes SEQ ID NO: a nucleotide sequence as shown in 4;

(3) taking the pET22b(+) vector plasmid, inserting the ketoreductase and the glucose dehydrogenase gene fragment amplified by the step (2) into the pET22b(+) vector plasmid, and purifying and recycling The recombinant plasmid was obtained.
The method for producing a recombinant plasmid according to claim 13, wherein in the step (3), an RBS sequence is inserted between the ketoreductase and the glucose dehydrogenase gene coding sequence, and the RBS sequence includes A nucleotide sequence as shown in SEQ ID NO: 17.