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3-sterone-1,2-dehydrogenase and application thereof

A technology of dehydrogenase and steroid, applied in the field of biochemistry, can solve the problems of uncloning, amino acid sequence and enzyme activity and application reports, low enzyme activity and so on

Inactive Publication Date: 2020-07-28
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the activity of these enzymes is low
[0008] 3-sterone-1,2-dehydrogenase (KsdD211) is from Mycobacterium sp.HGMS2, ​​there is no related clone, amino acid sequence and enzyme activity and application report

Method used

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  • 3-sterone-1,2-dehydrogenase and application thereof
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  • 3-sterone-1,2-dehydrogenase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: PCR amplification KsdD211 gene

[0035] From the Mycobacterium strain HGMS2GL genome, the target gene fragment size of KsdD211 was amplified by PCR with a size of 1692bp. The PCR primers were: pre-primer KsdD211-F: 5'-GTAGGATCCATGACTGAACAGGACTAC-3' and post-primer KsdD211-R: 5'- For GCAGAATTCTCAGGCCTTTCCAGCGAG-3', temperature gradient PCR was performed at 50°C, 52°C, 54°C, 56°C, and 60°C. The results of running agarose gel analysis are as follows: figure 1 shown.

[0036] The PCR products at 56°C and 60°C were selected for enzyme digestion and ligation. Enzyme digestion reaction system is 50μL: PCR product: 1ug; BamHI 2U; EcoR Ⅰ2U; 10×Kbuffer 5ul; H 2 O 1ul; 37°C, time: 3h. The reaction product was purified with a PCR purification kit (Tiangen Biotechnology Co., Ltd., Beijing) and used for purification of the pRSV vector. The pRSV vector was digested and purified using the same method and system as above. After digestion, the vector pRSV and the targ...

Embodiment 2

[0042] Example 2: KsdD211 gene sequencing

[0043] Sequencing was carried out at Wuhan Boshang Sequencing Company, and the analysis of the sequencing results was correct. See SEQ ID NO.1 for the KsdD211 gene. The amino acid sequence of KstD211 is shown in SEQ ID NO.2.

Embodiment 3

[0044] Embodiment 3: His-KsdD211 protein expression optimization

[0045] Transform pRSV-KsdD211 into Escherichia coli BL21 competent cells, culture 100mL pRSV-KsdD211-BL21 to OD 600 =0.8-10, divided into 15 test tubes, 5mL per tube, divided into three groups, each group has 5 test tubes, corresponding to three temperature gradients: 18°C, 25°C, 37°C, each temperature corresponds to 5 isotropic The concentrations of propylthiogalactoside (IPTG) were: 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM; in different time periods: 5h, 9h, 24h, respectively, 1mL of each test tube was sampled, and the OD was corrected. 600 =1.0, 4000rpm, 10min, 4°C to collect the bacterial cells. Centrifuge to remove the supernatant, add 100 μL of 50 mM Tris-HCl (10% glycerol, pH 8.0), break the cells with an ultrasonic cell disruptor, centrifuge at 13000 rpm, 10 min, 4 °C, take 40 μL of the supernatant in a 1.5 mL EP tube, add 10 μL of 5 ×loading buffer and vortex to mix; add 50μL 1×loading buffer to the precipi...

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Abstract

The invention provides 3-sterone-1,2-dehydrogenase and an application thereof. An amino acid sequence of the 3-sterone-1,2-dehydrogenase is as shown in SEQ ID.2. The KsdD211 of the invention can catalyze a reaction of androsta-4-ene-3,17-dione (4-AD) to produce androst-1,4-diene-3,17-dione (ADD, 1,4-androstenedione), and the TLC analysis verifies the high enzymatic activity of the KsdD211. The HPLC analysis of the KsdD211catalytic reaction shows that the KsdD211 completely converts 4-AD into ADD within 18 h.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to 3-sterone-1,2-dehydrogenase and application thereof. Background technique [0002] When the double bond is introduced into the steroidal compound, for example, after the double bond is introduced into the C1,2 position of the mother nucleus of the anti-inflammatory steroid hormone drug, its anti-inflammatory effect can be multiplied, such as the C1,2 position of cortisone acetate The anti-inflammatory effect of dehydrocortisone acetate formed after the introduction of double bonds has been increased by 3-4 times, and the side effects caused by sodium retention have been reduced; there are also many important clinically used steroid compounds produced , especially the production of most anti-inflammatory adrenocortical hormones involves the dehydrogenation reaction of microbial C1,2 positions, including prednisolone (prednisolone), dexamethasone (dexamethasone), paramethasone (parameth...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12P33/04
CPCC12N9/0026C12Y105/05001C12N15/70C12P33/04
Inventor 苏正定成细瑶宋士奎王宏伟黄永棋
Owner HUBEI UNIV OF TECH
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