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Novel 17 beta-hydroxyl steroid dehydrogenase, gene and application thereof

A technology of hydroxysteroid and dehydrogenase, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of less in-depth research, and achieve the effect of less consumption, less by-products, and environmental friendliness

Active Publication Date: 2019-02-05
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Since the 1930s, it has been confirmed that there are constitutive or inducible 17β-hydroxysteroid dehydrogenases in different microorganisms, but there are still few in-depth studies on this type of enzymes. At present, 17β-hydroxysteroid dehydrogenases that are more studied Hydroxysteroid dehydrogenase is derived from Cochliobolus lunatus 17β-HSDcl ( M B, Stojan J, T L. Journal of Steroid Biochemistry and Molecular Biology, 2012(129):92-98, Kristan K, Stojan J, G, et al.Molecular and Cellular Endocrinology, 2005(241):80-87), however, the use of this enzyme as a biocatalyst in the conversion of substrate 17-carbonyl steroids to synthesize 17β-hydroxy steroids has not been seen. to report

Method used

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  • Novel 17 beta-hydroxyl steroid dehydrogenase, gene and application thereof
  • Novel 17 beta-hydroxyl steroid dehydrogenase, gene and application thereof
  • Novel 17 beta-hydroxyl steroid dehydrogenase, gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: Cloning of 17β-hydroxysteroid dehydrogenase (17β-HSDcab) gene and construction of genetically engineered bacteria

[0021] 1.1 PCR amplification of 17β-hydroxysteroid dehydrogenase gene

[0022] With Candida albicans as the limited strain, the hypothetical 17β-hydroxysteroid dehydrogenase gene sequence was searched in the NCBI database with 17β-hydroxysteroid dehydrogenase as the search term, and the following two primers were designed according to the sequence:

[0023] Forward primer: 5′-GGAATTC CATATG TCAGAACGTCAAAAAGGTTA-3′ (Nde Ⅰ restriction site is underlined)

[0024] Reverse primer: 5′-CCG CTCGAG CTAATTAACTTTCTCTTTCAGATAC-3′ (Xho Ⅰ restriction site is underlined)

[0025] Using the genomic DNA of Candida albicans 7103 as a template, the target gene fragment was amplified.

[0026] The PCR conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 50°C for 30 s, extension at 68°C for 1 min, and 30 cycles; f...

Embodiment 2

[0039] Example 2: Induced expression and purification of 17β-hydroxysteroid dehydrogenase (17β-HSDcab)

[0040] Prepare 50 mL of seed liquid, and the medium is LB liquid medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L), pick a single colony of genetically engineered bacteria with an inoculation loop and insert it into the medium, 37°C, 200rpm Incubate overnight. The overnight cultured seed solution was transferred to the fermentation medium (LB medium) at an inoculum size of 1%, and cultivated to A at 37°C and 200rpm 600 0.6-1.0, add 0.25mM IPTG, and place at 37°C, 200rpm to induce for 10-12h. 4°C, centrifuged at 6000rpm to collect the bacteria, washed twice with sodium phosphate buffer (50mM, pH 7.5), crushed with a high-pressure homogenizer, centrifuged at 13000rpm to get the supernatant, and then used metal affinity chromatography (Ni The target protein was purified and recovered by column) method, and the target protein was dialyzed to remove imidazole to obtain 17...

Embodiment 3

[0041] Example 3: Substrate spectrum of 17β-hydroxysteroid dehydrogenase (17β-HSDcab)

[0042] Enzyme activity assay system: total reaction volume 0.2mL, pH 7.0-7.5, sodium phosphate buffer solution with a concentration of 100mM, 0.5mM NADPH and 0.8mM different substrates, add an appropriate amount of pure enzyme solution and start detection at 30°C, measure 340nm changes in absorbance.

[0043] The results are shown in Table 1. 17β-hydroxysteroid dehydrogenase (17β-HSDcab) is active on a series of 17-carbonyl steroid compounds, among which the substrate androst-4-ene-3,17-di Ketones and androst-4,9(11)-diene-3,17-dione had the highest activity.

[0044] Table 1 The substrate spectrum of 17β-hydroxysteroid dehydrogenase (17β-HSDcab)

[0045]

[0046]

[0047] Notes: ① The activity of the enzyme on androst-4-ene-3,17-dione was set as 100% (4.4U / mg). ②Enzyme activity definition: the amount of enzyme required to catalyze the oxidation of 1 μmol NADPH per minute is 1 enzy...

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Abstract

The invention discloses a novel 17 beta-hydroxyl steroid dehydrogenase (17 beta-HSDcab) derived from candida albicans 7103 and a coding gene thereof; the enzyme is utilized as a biocatalyst to catalyse a substrate 17-carbonyl steroid compound to synthesize a 17 beta-hydroxyl steroid compound. The enzyme has a wide substrate spectrum, and especially has very high activity for androstane-4-alkene-3,17-diketone, androstane-4,9(11)-diene-3,17-diketone. A recombinant bacterium resting cell is taken as a biocatalyst, androstane-4-alkene-3,17-diketone is taken as a substrate, so that a target producttestosterone with a high yield is obtained with relatively high substrate feeding concentration, by-products are less, and the yield is not lower than 95%; and moreover, the conversion time is short,the used biocatalyst is less in dosage, and the space time yield is far higher than the current reported level.

Description

technical field [0001] The present invention relates to a new gene and its protein product, in particular to a novel 17β-hydroxysteroid dehydrogenase and its gene derived from Candida albicans (Candida albicans 7103), which is used to biocatalyze the synthesis of 17β-hydroxy A steroid compound belongs to the field of applied microorganism and enzyme engineering. Background technique [0002] Steroidal drugs have strong pharmacological effects such as anti-infection, anti-allergy, anti-virus and anti-shock. With the continuous development of the times, steroid drugs have become the second largest class of drugs after antibiotics. Classification of steroid hormone drugs: adrenocortical hormones, including hydrocortisone, prednisone, etc., which can treat Addison’s disease, anti-inflammatory, anti-allergic, anti-shock, etc.; anabolic hormones, whose main physiological functions are Inhibit protein dissimilation and promote protein synthesis, mainly used to treat diseases caus...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12P33/06
CPCC12N9/0006C12P33/06
Inventor 王玉陈曦刘媛媛冯进辉吴洽庆朱敦明马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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