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3-sterone-1, 2-dehydrogenase as well as gene sequence and application thereof

A gene sequence and dehydrogenase technology, which is applied to 3-sterone-1,2-dehydrogenase and its gene sequence and application fields, can solve the problems of low enzyme activity and achieve the effects of high enzyme activity and increased solubility

Active Publication Date: 2020-08-07
武汉艾默佳华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of these enzymes is low

Method used

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  • 3-sterone-1, 2-dehydrogenase as well as gene sequence and application thereof
  • 3-sterone-1, 2-dehydrogenase as well as gene sequence and application thereof
  • 3-sterone-1, 2-dehydrogenase as well as gene sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: PCR amplification KsdD211 gene

[0038] From the Mycobacterium strain HGMS2GL genome, the KsdD211 target gene fragment was amplified by PCR with a size of 1692bp. The PCR primers were: the front primer KsdD211-F and the back primer KsdD211-R. ℃, 54℃, 56℃, 60℃. The results of running agarose gel analysis are as follows: figure 1 shown.

[0039] The PCR products at 56°C and 60°C were selected for enzyme digestion and ligation. The digestion reaction system is 50 μL: PCR product: 1ug; BamHI 2U; EcoRI 2U; 10×K buffer 5ul; H 2 O 1ul; 37°C, time: 3h. The reaction product was purified with a PCR purification kit (Tiangen Biotechnology, 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 target fragment KsdD211 were ligated by T4 ligase reaction, and the 20 μL reaction system was ligated overnight at 4°C. Table 1 shows the pRSV-Ks...

Embodiment 2

[0044] Example 2: KsdD211 gene sequencing

[0045] 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 KsdD211 is shown in SEQ ID NO.2.

Embodiment 3

[0046] Example 3: Optimization of KsdD211 enzyme structure based on comparison of amino acid sequence and three-dimensional protein structure

[0047] Compared with other KsdD amino acid sequences, it is found that the amino acid sequence of KsdD211 is very different, especially in the active center, different KsdD enzymes have different biases, the main difference is that Leu136 and Val364 are hydrophobic amino acids ( image 3 ), which is beneficial to substrate binding, but unfavorable for dehydrogenation reaction. Therefore, changing to carry out amino acid, changing Leu136 to His, Thr, Ser, His, Glu or Asp amino acid is beneficial for dehydrogenation reaction. Similarly, the change of Val364 to His, Thr, Ser, His, Glu or Asp amino acid is also beneficial to the dehydrogenation reaction. The three-dimensional structure of KsdD211 shows that there are five redundant loop regions in the protein conformation, namely Asp161-S188, Pro253-Glu257, Arg340-Pro344, Gly377-G382 and A...

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Abstract

The invention provides 3-sterone-1, 2-dehydrogenase as well as a gene sequence and application thereof. The invention discloses a gene sequence for expressing 3-sterone-1, 2-dehydrogenase, the gene sequence is shown as SEQ ID. 4, and the gene sequence is used for expression in escherichia coli. The 3-ketosterone-1, 2-dehydrogenase disclosed by the invention can be used for efficiently catalyzing androstane-4-ene-3, 17-dione (4-AD) reaction to generate androstane-1, 4-diene-3, 17-dione (ADD), and has high enzymatic activity. In addition, the soluble 3-sterone-1, 2-dehydrogenase is obtained, andthe solubility of the enzyme is improved.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to 3-sterone-1,2-dehydrogenase and its gene sequence and application. 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), parametha...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12P33/02
CPCC12N9/001C12N15/70C12P33/02
Inventor 苏正定成细瑶宋士奎周曦
Owner 武汉艾默佳华生物科技有限公司
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