3-sterone-delta1Dehydrogenase and coding gene and application thereof
阅读说明：本技术 一种3-甾酮-δ1-脱氢酶及其编码基因和应用 (3-sterone-delta1Dehydrogenase and coding gene and application thereof ) 是由 张瑞 王玉 冯进辉 陈曦 姚培圆 吴洽庆 朱敦明 马延和 于 2021-09-17 设计创作，主要内容包括：本发明提供一种3-甾酮-Δ～(1)-脱氢酶及其编码基因和应用,提供了含有其基因的表达载体和含有该表达载体的基因工程重组菌株。本发明提供的3-甾酮-Δ～(1)-脱氢酶为一种新的3-甾酮-Δ～(1)-脱氢酶,该酶具有宽广的底物谱,对大多数3-酮基甾体化合物具有高的活性。本发明提供的重组菌株可用于转化3-酮基甾体化合物,以高底物投料浓度,获得高收率的目标产物,无副产物,转化率不低于94％,而且转化时间短,所用生物催化剂用量少,制备方法简单方便、条件温和、环境友好,具有很好的产业化前景。(The invention provides 3-sterone-delta 1 Dehydrogenase, its coding gene and application, and provides an expression vector containing its gene and a genetically engineered recombinant strain containing the expression vector. The 3-sterone-delta provided by the invention 1 The dehydrogenase is a novel 3-sterone-delta 1 Dehydrogenases, which have a broad substrate spectrum and have a high activity on most 3-ketosteroids. The recombinant strain provided by the invention can be used for transformationThe 3-keto steroid compound is prepared by using high substrate feeding concentration to obtain a target product with high yield, no by-product, conversion rate not lower than 94%, short conversion time, small using amount of the used biocatalyst, simple and convenient preparation method, mild condition, environmental protection and good industrialization prospect.)
1. 3-sterone-delta1A dehydrogenase which is a protein of the following (1) or (2):
(1) the amino acid sequence is shown as SEQ ID NO: 1;
(2) 3-sterone-delta having at least 99% homology with the protein of (1) and having the protein of (1)1A protein catalytically active by a dehydrogenase and derived fromSaccharopolyspora kobensis。
2. A polynucleotide encoding the 3-sterone- Δ of claim 11A dehydrogenase.
3. The polynucleotide of claim 2, having the sequence set forth in SEQ ID NO: 2, respectively.
4. A genetically engineered expression vector comprising the polynucleotide of claim 2 or 3.
5. The expression vector of claim 4, wherein the coding sequence isThe polynucleotide is operably linked to a multiple cloning site in a vector to direct mRNA synthesis for expression of 3-sterone-delta1Dehydrogenase, or for homologous recombination.
6. The expression vector of claim 4 or 5, wherein the starting vector is pET-21 a.
7. A genetically engineered expression strain comprising a genetically engineered expression vector according to any one of claims 4 to 6, or a polynucleotide according to claim 2 or 3, which is exogenous and integrated into its genome.
8. The strain of claim 7, wherein the starting strain is E.coli.
9. 3-sterone- Δ according to claim 11-dehydrogenase, or the polynucleotide of claim 2 or 3, or the genetically engineered expression strain of any one of claims 7 to 8, at a Δ of a 3-ketosteroid1-use in dehydrogenation reactions.
10. The use according to claim 9, wherein 3-keto steroid is used as a substrate, an electron acceptor is added, said electron acceptor is phenazine methosulfate or 1, 4-naphthoquinone, and conversion reaction is carried out to obtain C1, 2-dehydrosteroid; the 3-ketosteroid compound is one of the following compounds: androst-4-ene-3, 17-dione; androst-4-ene-3, 11, 17-trione; androsta-4, 9(11) -diene-3, 17-dione; 17 β -hydroxyandrost-4-ene-3, 17-dione; pregn-4-ene-3, 20-dione; 4-pregnene-3, 11, 20-trione; 17 α -hydroxypregn-4-ene-3, 20-dione; 17 α, 21-bishydroxypregn-4-ene-3, 20-dione; 17 α, 21-bishydroxypregn-4-ene-3, 11, 20-trione; 11 β,17 α, 21-trihydroxypregn-4-ene-3, 20-dione; 11 beta, 17 alpha, 21-trihydroxypregn-4-ene-3, 20-dione-21-acetate; 21-hydroxy-pregna-4, 9(11), 16-triene-3, 20-dione-21-acetate.
11. The use according to claim 10, wherein the reaction temperature is 25-35 ℃ and the reaction time is 6-24 h.
Technical Field
The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to 3-sterone-delta1Dehydrogenases and their use.
Background
Steroid drugs have strong pharmacological actions such as anti-infection, anti-allergy, anti-virus and anti-shock, and have become the second main class of drugs second to antibiotics. Classification of steroid hormones drugs: adrenocortical hormones including hydrocortisone, prednisone, etc., for treating Addison's disease, anti-inflammatory, antiallergic, antishock, etc.; the protein assimilation hormone has the main physiological functions of inhibiting protein dissimilation and promoting protein synthesis, and is mainly used for treating diseases caused by protein increase and synthesis deficiency; sex hormones, including estrogen, androgen and progestin.
The anti-inflammatory activity can be increased by times after a double bond is introduced into the C1, 2-position of the A ring of the steroid drug parent nucleus, at present, the production of a plurality of clinically important steroid compounds is commonly used, particularly, the production of most of adrenocortical hormones with anti-inflammatory capability relates to dehydrogenation reaction of C1, 2-position, and comprises prednisolone (prednisolone), dexamethasone (dexamethasone), paramethasone (paramethasone), betamethasone (betamethasone), fluocortolone (fluocortolone), fluocinolone (fluocinolone), triamcinolone (triamcinolone), methylprednisolone (medrol) and the like. Methods for the 1, 2-position dehydrogenation of steroids generally include chemical methods and microbial fermentation methods. The chemical dehydrogenation generally needs to use SeO2, has low yield, has great environmental pollution caused by SeO2, and is gradually replaced by a biological method at present. The microorganism fermentation dehydrogenation method, which is currently applied to a large number of strains, is arthrobacter (CN 101760495, CN 200710060202), and although the defects of the chemical method are avoided, the method has the defects of low substrate concentration, long conversion time, low conversion rate and yield, difficult separation and purification and the like.
3-sterone-delta1Dehydrogenase (3-ketosteroid-. DELTA.1-dehydrogenases, KsdD), a flavoprotein-dependent dehydrogenase, which can catalyze the dehydrogenation of carbon-carbon single bond (C-C) at 1-and 2-positions of a ring a of 3-ketosteroid mother nucleus to carbon-carbon double bond (C ═ C) by taking Flavin Adenine Dinucleotide (FAD) as a cofactor in the catalytic reaction process, and has a broad application prospect, as shown in the following figure.
3-sterone-delta1The dehydrogenase is widely existed in microorganisms, and 3-sterone-delta is continuously prepared and obtained from different microorganism cells1Dehydrogenase, 1990 by Itagaki et alNocardia corallinaKsdD is obtained by purification, the activity of the enzyme to various substrates is measured, and the enzyme activity is reduced after C11 position of C19 type compound androst-4-ene-3, 17-diketone (4-AD) is replaced by carbonyl, and the activity is basically not high after C11 position is replaced by hydroxyl; for the C21 compound 17 alpha, 21-bishydroxypregn-4-ene-3, 20-dione (cortixolone), the enzyme activity was increased to some extent when the C11 position was substituted with a carbonyl group, while the C11 position was substituted with a hydroxyl group and was substantially inactive [ Itagaki E, Hatta T, Wakabayashi T& Suzuki K. Spectral properties of 3-ketosteroid-Δ1-dehydrogenase from Nocardia corallina. Biochim Biophys Acta1990, 1040:281-286 ]. Subsequently, in 1995, Choi et al willArthrobacter simplexIn (1)kstDThe genes construct shuttle plasmidsStreptomyces lividansIn the expression of heterologous genes, and the enzymatic properties and substrate profilesThe research is carried out, and the experimental result shows that the catalytic activity of the enzyme is greatly reduced after the C11 position of the steroid parent nucleus is replaced by hydroxyl group [ Choi KP, Moln a r T, Yamashita M& Murooka Y. Purification and characterization of the 3-ketosteroid-Δ1-dehydrogenase of Arthrobacter simplex produced in Streptomyces liuidans. J Biochem1995, 117: 1043-. In 2007, the Lufuping subject group willArthrobacter simplexIn (1)kstDThe gene is expressed in the bacillus subtilis through a vector pWB980, and the intracellular and extracellular enzyme activities are respectively 0.11U/mg and 0.015U/mg by taking 4-AD as a substrate. 1 g/L of substrate 4-AD is transformed by using the bacillus subtilis recombinant cells, and the maximum transformation rate reached by 40 h of transformation is only 45.5% [ Li Y, Lu F, Sun T& Du L. Expression of ksdD gene encoding 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex in Bacillus subtilis. Letters in Applied Microbiology2007, 44:563-568 ]. In 2013, the group of questions related to the symptomatology willMycobacterium neoaurumInksdDThe gene is expressed in the bacillus subtilis, and the intracellular and extracellular enzyme activities are respectively 1.75U/mg and 0.08U/mg by taking 4-AD as a substrate. 1 g/L substrate 4-AD is transformed by using the bacillus subtilis recombinant cells, and the maximum transformation rate reached by 10H of transformation is only 65.7% [ Zhang WQ, Shao ML, Rao ZM, Xu MJ, Zhang X, Yang TW, Li H& Xu ZH. Bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione by recombinant Bacillus subtilis expressing ksdd gene encoding 3-ketosteroid-∆1-dehydrogenase from Mycobacterium neoaurum JC-12. J Steroid Biochem Mol Biol, 2013, 135:36-42】。
In summary, the current 3-sterone-delta1The dehydrogenase has low activity on steroid drug intermediates, particularly on intermediates substituted by related bond groups at C11 on steroid parent nucleus, even has no activity, so that the dehydrogenase still has the problems of low substrate feeding concentration, low conversion rate and the like when being applied to the synthesis of steroid C1 and 2 dehydrogenation drug intermediates.
Therefore, KsdD enzyme gene resources are fully excavated, and one with high activity to most steroid substrates is screened3-sterone-delta1Dehydrogenase and application in the synthesis of steroid C1, 2-position dehydrogenation drug intermediate, which is a key requirement for the industrialization of steroid enzyme process.
Disclosure of Invention
In order to overcome the problems of the prior art, the invention aims to provide a novel 3-sterone-delta1Dehydrogenase and its coding gene and application to solve the problem of 3-sterone-delta1The dehydrogenase has lower activity to steroid drug intermediate, the substrate feeding concentration is small when in use, the conversion rate is low and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a 3-sterone- Δ1A dehydrogenase, said 3-sterone-Delta1The dehydrogenase is a protein of the following (1) or (2); (1) the amino acid sequence is shown as SEQ ID NO: 1; (2) a protein having at least 80%, at least 90%, at least 95%, at least 99% homology with the protein of (1) and having the function of the protein of (1).
Most preferably, the 3-sterone- Δ1The amino acid sequence of the dehydrogenase is as set forth in SEQ ID NO: 1 is shown.
In a second aspect of the present invention, there is provided a polynucleotide encoding the aforementioned 3-sterone- Δ1A dehydrogenase. Preferably, the encoding is the aforementioned 3-sterone-delta1The nucleotide sequence of the polynucleotide of the dehydrogenase is as set forth in SEQ ID NO: 2, respectively.
In a third aspect of the invention, there is provided a genetically engineered expression vector comprising the polynucleotide as described above. Methods well known to those skilled in the art can be used to construct the recombinant expression vectors. These methods include recombinant DNA techniques, DNA synthesis techniques and the like. Can encode the 3-sterone-delta1The DNA of the dehydrogenase is operatively linked to a multiple cloning site in the vector to direct the synthesis of mRNA for the expression of 3-sterone-Delta1Dehydrogenase, or for homologous recombination. In a preferred embodiment of the present invention, pET-21a is used as the expression vector.
In a fourth aspect of the invention, a genetically engineered expression strain is provided, which comprises the recombinant expression vector or a polynucleotide having an exogenous sequence integrated into its genome.
In a preferred embodiment of the invention, the genetically engineered expression strain is escherichia coli. The Escherichia coli is preferably BL21(DE 3).
The present invention also provides the aforementioned 3-sterone-Delta1-dehydrogenase or 3-sterone-delta1-dehydrogenase gene engineering expression of strains in delta of 3-ketosteroids1-use in dehydrogenation reactions.
The application is that 3-keto steroid compound is used as a substrate, an electron acceptor is added, preferably the electron acceptor is phenazine methyl sulfate or 1, 4-naphthoquinone, and conversion reaction is carried out to obtain C1, 2-dehydrosteroid compound; the 3-ketosteroid compound is one of the following compounds: androst-4-ene-3, 17-dione; androst-4-ene-3, 11, 17-trione; androsta-4, 9(11) -diene-3, 17-dione; 17 β -hydroxyandrost-4-ene-3, 17-dione; pregn-4-ene-3, 20-dione; 4-pregnene-3, 11, 20-trione; 17 α -hydroxypregn-4-ene-3, 20-dione; 17 α, 21-bishydroxypregn-4-ene-3, 20-dione; 17 α, 21-bishydroxypregn-4-ene-3, 11, 20-trione; 11 β,17 α, 21-trihydroxypregn-4-ene-3, 20-dione; 11 beta, 17 alpha, 21-trihydroxypregn-4-ene-3, 20-dione-21-acetate; 21-hydroxy-pregna-4, 9(11), 16-triene-3, 20-dione-21-acetate.
The invention also provides a method for preparing the C1, 2-dehydrogenation steroid compound, which comprises the following steps: the 3-sterone-delta of the first aspect of the invention1The dehydrogenase or the gene engineering expression strain of the fourth aspect acts on the 3-ketosteroid compound, and simultaneously an electron acceptor is added, the conversion is carried out at 25-35 ℃, preferably 30 ℃, and the C1, 2-dehydrosteroid compound is obtained after the conversion time is 6-24 hours.
In a preferred embodiment of the present invention, the 3-ketosteroid compound is one of the following compounds: androst-4-ene-3, 17-dione; androst-4-ene-3, 11, 17-trione; androsta-4, 9(11) -diene-3, 17-dione; 17 β -hydroxyandrost-4-ene-3, 17-dione; pregn-4-ene-3, 20-dione; 4-pregnene-3, 11, 20-trione; 17 α -hydroxypregn-4-ene-3, 20-dione; 17 α, 21-bishydroxypregn-4-ene-3, 20-dione; 17 α, 21-bishydroxypregn-4-ene-3, 11, 20-trione; 11 β,17 α, 21-trihydroxypregn-4-ene-3, 20-dione; 11 beta, 17 alpha, 21-trihydroxypregn-4-ene-3, 20-dione-21-acetate; 21-hydroxy-pregna-4, 9(11), 16-triene-3, 20-dione-21-acetate.
Compared with the prior art, the invention has the following beneficial effects: the inventor screens out 3-ketosteroid-delta with wide substrate spectrum and high activity to most steroid substrates by gene excavation1Dehydrogenase, with other 3-sterone-delta presently reported in the literature1The similarity of dehydrogenase is lower than 60 percent, has obvious difference and is a novel 3-sterone-delta1Dehydrogenase, providing conditions for further research on the evolutionary relationship of the enzyme family, and constructing the highly effective 3-sterone-delta by using genetic engineering technology1The dehydrogenase gene engineering bacteria provide more gene resources. In particular, the invention uses genetic engineering means to transform the 3-sterone-delta1Heterologous over-expression of dehydrogenase gene in colibacillus to obtain high-efficiency expression 3-sterone-delta1Genetically engineered bacteria of dehydrogenases. The established biotransformation process of the genetically engineered bacteria for the series of 3-keto steroid compounds obtains a target product with high yield by high substrate feeding concentration, has no by-product, the transformation rate is not lower than 94 percent, the transformation time is short, the used biocatalyst is less in dosage, the preparation method is simple and convenient, the conditions are mild, the environment is friendly, and the industrial prospect is good.
Drawings
FIG. 1 shows the HPLC chart of the genetically engineered strain BL21-Sat in example 4.1 for transformation of androst-4-ene-3, 17-dione. Wherein, a is a standard product of the product androstane-1, 4-alkene-3, 17-diketone, and b is a conversion sample of the genetic engineering strain BL21-Sat for converting androstane-4-alkene-3, 17-diketone.
FIG. 2 shows the HPLC chart of the sample transformed by genetically engineered strain BL21-Sat in example 4.2 to transform androsta-4, 9(11) -diene-3, 17-dione.
FIG. 3 is an HPLC chart showing a sample of the transformation of pregna-4-ene-3, 20-dione by the genetically engineered strain BL21-Sat in example 4.3.
FIG. 4 is a HPLC chart showing a sample of the transformation of 17 α -hydroxypregn-4-ene-3, 20-dione by the genetically engineered strain BL21-Sat in example 4.4.
FIG. 5 shows an HPLC chart of hydrocortisone transformed by the genetically engineered strain BL21-Sat in example 4.5. Wherein, a is a standard product of the product prednisolone, and b is a transformation sample of hydrocortisone transformed by the genetic engineering strain BL 21-Sat.
FIG. 6 shows the HPLC chart of the genetically engineered strain BL21-Sat in example 4.6 transformed into 21-hydroxy-pregna-4, 9(11), 16-triene-3, 20-dione-21-acetate. Wherein, a is a standard substance of a substrate 21-hydroxy-pregna-4, 9(11), 16-triene-3, 20-diketone-21-acetate, and b is a conversion sample of a genetic engineering strain BL21-Sat for converting 21-hydroxy-pregna-4, 9(11), 16-triene-3, 20-diketone-21-acetate.
FIG. 7 shows an SDS-PAGE pattern after purification of the Sat protein. Wherein, M is a standard protein; 1-breaking the supernatant after induced expression; purifying by using a 2-nickel column.
Detailed Description
The following further illustrates the invention by way of specific embodiments, but should not be construed as limiting the invention.
Example 1: 3-sterone-delta1Synthesis of dehydrogenase (Sat) Gene and construction of genetically engineered bacterium
1.3-sterone-. DELTA.1Synthesis of dehydrogenase Gene
Searching for possible 3-sterone-delta from NCBI databases by gene mining techniques1Dehydrogenases, with a protein sequence identity of between 30% and 80%, with a very conserved FAD binding region at the N-terminus by sequence analysis (GSGX)5- 6AX2AX3GLX5E) And conserved amino acid residues exist and are mined fromSaccharopolyspora kobensisPossible 3-sterone-delta1-a dehydrogenase sequence having the nucleotide sequence set forth in SEQ ID NO: 2, and the coding amino acid sequence is shown as SEQ ID NO: 1 is shown. The nucleotide sequence was synthesized by general biosystems (Anhui) Ltd and ligated to pET21a vector to obtain 3-sterone-. DELTA.1Recombinant plasmids of dehydrogenases.
2. Transformation of recombinant plasmids
Preparing competent Escherichia coli cells by calcium chloride method.
(1) mu.L of the recombinant plasmid was placed in 50. mu.L of E.coli BL21(DE3) competent cells and ice-cooled for 30 min.
(2) And (3) carrying out heat shock on the mixture in a water bath at 42 ℃ for 45 s, and quickly placing the mixture on ice for 1-2 min.
(3) Adding 600 mu L of fresh LB liquid culture medium, and carrying out shake culture at 37 ℃ for 45-60 min.
(4) And (3) coating 200 mu L of bacterial liquid on the surface of LB solid medium containing ampicillin, and culturing at 37 ℃ for 12-16 h until single colonies appear.
Example 2: 3-sterone-delta1Inducible expression and purification of the dehydrogenase (Sat)
Preparing 50 mL of seed solution, wherein the culture medium is LB liquid culture medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L), picking a single colony of the genetic engineering strain BL21-Sat by using an inoculating loop, inoculating into the culture medium, and culturing at 37 ℃ and 200rpm overnight. Transferring the seed liquid cultured overnight to a fermentation medium (LB medium) with the inoculation amount of 1%, culturing at 37 ℃ and 200rpm until the A is 6000.6-1.0, adding 0.05 mM IPTG, and inducing at 25 ℃ and 200rpm for 10-12 h. Centrifuging at 4 deg.C and 6000rpm to collect thallus, washing with Tris-HCl buffer solution (50mM, pH 8.0) twice, crushing with high pressure homogenizer, centrifuging at 13000rpm to collect supernatant, purifying and recovering target protein by metal affinity chromatography (nickel column), dialyzing to remove imidazole to obtain pure enzyme solution. SDS-PAGE shows that the protein band obtained by purification is single, and the electrophoretic purity is achieved (see figure 7).
Example 3: 3-sterone-delta1Substrate profile of the dehydrogenase (Sat)
Enzyme activity determination system: the total reaction volume was 0.2 mL, 50mM Tris-HCl pH 8.0, 1.5 mM Phenazine Methosulfate (PMS), 0.12 mM 2, 6-Dichlorophenolindophenol (DCPIP), 0.5 mM of various substrates (obtained by screening the steroid substrate library of the laboratory), and after adding a suitable amount of purified enzyme Sat (8-48 ng), which was purified by the method described in example 2, the change in absorbance at 600nm was measured starting at 30 ℃. The enzyme activity of 1U was defined as the amount of enzyme required to reduce 1. mu. mol DCPIP in 1 min. The results are shown in Table 1.
Example 4: conversion of 3-keto steroid compound by gene engineering strain BL21-Sat
Seed culture: a single colony of the genetically engineered strain BL21-Sat is picked up by an inoculating loop and inoculated into an LB culture medium containing ampicillin, and cultured overnight at 37 ℃ and 200 rpm.
Fermentation induction culture: the seed liquid cultured overnight was transferred to the fermentation medium at an inoculum size of 1%, cultured at 37 ℃ and 200rpm to OD600nmAdding 0.6-1.0 mM IPTG, and inducing at 25 deg.C and 200rpm for 10-12 hr to obtain fermentation liquid OD600nmAbout 10.0.
(4.1): androstane-4-alkene-3, 17-diketone is taken as a substrate
And adding androst-4-ene-3, 17-dione powder into 100 mL of the fermentation liquid, wherein the adding concentration is 100 g/L, adding phenazine methyl sulfate 10g/L, and stirring at 30 ℃ for reaction. And reacting for 24 hours, after the reaction is finished, extracting by ethyl acetate, combining organic phases, drying by anhydrous sodium sulfate, removing the solvent under reduced pressure, and detecting by HPLC that the conversion rate of the reaction reaches 99.0%.
(4.2): androstane-4, 9(11) -diene-3, 17-diketone as substrate
Androstane-4, 9(11) -diene-3, 17-diketone dissolved in DMF (10 mL) is added into 100 mL of the fermentation liquid, the feeding concentration is 50 g/L, then phenazine methyl sulfate is added into the fermentation liquid, 5g/L of phenazine methyl sulfate is added, and the mixture is stirred and reacts at the temperature of 30 ℃. And reacting for 24 hours, after the reaction is finished, extracting by ethyl acetate, combining organic phases, drying by anhydrous sodium sulfate, removing the solvent under reduced pressure, and detecting by HPLC that the conversion rate of the reaction reaches 95.5%.
(4.3): pregna-4-ene-3, 20-dione as substrate
Pregna-4-ene-3, 20-dione powder dissolved in ethylene glycol (30 mL) is added into 100 mL of the fermentation liquid, the feeding concentration is 50 g/L, then 2.5 g/L of 1, 4-naphthoquinone is added, and the mixture is stirred and reacted at 30 ℃. And reacting for 24 hours, after the reaction is finished, extracting by ethyl acetate, combining organic phases, drying by anhydrous sodium sulfate, removing the solvent under reduced pressure, and detecting by HPLC that the conversion rate of the reaction reaches 94.4%.
(4.4): takes 17 alpha-hydroxypregna-4-ene-3, 20-diketone as a substrate
Adding 17 alpha-hydroxypregn-4-ene-3, 20-dione dissolved in isooctanol (40 mL) into 100 mL of the fermentation liquid, wherein the feeding concentration is 50 g/L, adding phenazine methyl sulfate 5g/L, and stirring at 30 ℃ for reaction. And reacting for 24 hours, after the reaction is finished, extracting by ethyl acetate, combining organic phases, drying by anhydrous sodium sulfate, removing the solvent under reduced pressure, and detecting by HPLC that the conversion rate of the reaction reaches 94.8%.
(4.5): 11 beta, 17 alpha, 21-trihydroxy pregn-4-ene-3, 20-diketone (hydrocortisone) is taken as a substrate
And (2) adding 11 beta, 17 alpha, 21-trihydroxy pregn-4-ene-3, 20-dione powder into 100 mL of the fermentation liquor, wherein the adding concentration is 60 g/L, adding 2.5 g/L phenazine methyl sulfate, and stirring at 30 ℃ for reaction. And (3) reacting for 6-8 h, extracting with ethyl acetate after the reaction is finished, combining organic phases, drying with anhydrous sodium sulfate, removing the solvent under reduced pressure, and detecting by HPLC that the conversion rate of the reaction reaches 96.5%.
(4.6): 21-hydroxy-pregna-4, 9(11), 16-triene-3, 20-diketone-21-acetate is taken as a substrate
Adding 21-hydroxy-pregna-4, 9(11), 16-triene-3, 20-diketone-21-acetate dissolved in butyl acetate (40 mL) into 100 mL of the fermentation liquid, adding phenazine methyl sulfate at a concentration of 50 g/L, and stirring at 30 ℃ for reaction. And reacting for 24 hours, after the reaction is finished, extracting by ethyl acetate, combining organic phases, drying by anhydrous sodium sulfate, removing the solvent under reduced pressure, and detecting by HPLC that the conversion rate of the reaction reaches 99.0%.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> 3-sterone-delta 1-dehydrogenase and coding gene and application thereof
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gtgggcgacc gcacgccccg cgagctgcag gacgcgttct tggacaacgg tgcccgactg 300
gtcgactacc tggaggagga cgacgacttc gagttcatcg tctacccctg gccggactac 360
tacgggtcgg cgccgggagc cagcgcgacc ggcaggcaca tcatgccgat gccgatgcgg 420
ccggagcgga tcggctcgct gcgggagcag ctccggccac cggtggacgt cgaccgggcg 480
ggcgcaccgc tgcccgacct gctcgtcggc ggccaggcgc tgatcggcag gctgctgctc 540
gcgctgtcca agcaggactc cgcccggctg cggcgcggat cggtgtgcga cgagctgctg 600
acctccgacg gcggcgtgat cggcgcggtg gtcgaggagg gcggtgagcg acggcggatc 660
cgcgcgcggc gcggcgtgct gatcgcctcc ggcgggttcg agcgcaacca ggcgatgcgg 720
accgagcacg gggtgccggg cgcggcgcgg gacacgatgg ggccggcgga gaacctgggc 780
aaggcgatcc gggccgggat cgacgtcggc gcgggcaccg acctgatggc ggaggcctgg 840
tggtcgccgg gcatcaccca cccggacggg acctcgacgt tctcgctgtg gttcaccggc 900
ggcatcttcg tcgacggcgc gggcgagcgc ttcgtcaacg agtcctggcc gtacgaccgg 960
atcggccgcg ccgtcctcga ccgcctcggc gcgggccgga tggcgctgcc gttctggatg 1020
atctacgacg accgcgaggg cgagcggccg ccggtgcgct cgacgagcgt cccgatgggg 1080
gagacggctg actacgtcga cgccgggctg tggttcagcg cgggcacgct ggccgaactc 1140
gcggagaaga tcggtgttcc cgcggagaac ctggagcgga ccgtggcccg gttcaacggt 1200
ttcgcgaccg ccgggaccga cgaggacttc caccggggtg acgagcccta cgaccggtcg 1260
ttcgcggatg ggggctcccc gctggtgccg atagagaagg gcccgttcca cgccgcggcc 1320
ttcggcctct ccgatctcgg caccaagggc ggtctgcgca ccgatgcgcg tgcccgggtg 1380
ctcagcacct cgggcgaggt gatccccggc ctgtacgccg cgggcaactc gatggccgcg 1440
gtcagcggga ccacgtatcc gggcgggggc aacccgatcg ggtcctgcat ggtgttcagc 1500
cacctcgccg cgctggacat gctcacccgc tga 1533