A method for transforming AD into ADD by using recombinant corynebacterium crenatum whole cells

A whole-cell transformation and 4-AD technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems that hinder the optimization of whole-cell transformation conditions and the lack of understanding of enzymatic properties, and achieve environmental protection, easy promotion and application, and raw materials The effect of high utilization

Active Publication Date: 2019-09-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the KSDD enzyme was successfully expressed in Bacillus subtilis and its activity was detected, the active KSDD enzyme was not successfully purified, so the enzymatic properties of the enzyme were poorly understood, which hindered the understanding of AD. Condition optimization for whole-cell transformation to ADD

Method used

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  • A method for transforming AD into ADD by using recombinant corynebacterium crenatum whole cells
  • A method for transforming AD into ADD by using recombinant corynebacterium crenatum whole cells
  • A method for transforming AD into ADD by using recombinant corynebacterium crenatum whole cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Construction of recombinant Corynebacterium crenatum strain

[0039] Take the lab with 3-sterone-△ 1 - The new mycobacterium aureus with dehydrogenase activity is used as the starting strain, and its chromosome is used as a template to obtain the gene of the enzyme by means of PCR, and connect the cloning vector to realize a large amount of amplification of the gene. A large number of amplified KSDD gene fragments were purified and connected to the pXMJ19 vector. After successful verification, the model strain Corynebacterium blunt-toothed SYPA5-5 was transformed. Positive transformants were screened on resistance plates, inoculated in shake flasks for fermentation, and the product ADD in the fermentation broth was detected by TLC and HPLC. The recombinant strains with the ability to transform AD into ADD were successfully constructed if the product ADD was detected. The present invention finally obtains the recombinant corynebacterium bacilli that trans...

Embodiment 2

[0040] Embodiment 2: the enzyme activity assay of recombinant bacterial strain

[0041] The strain was cultured overnight in LBG medium, centrifuged at 8000rpm for 10min, washed 3 times with 50mM Tris-HCL buffer solution of pH 7.0, suspended in the buffer solution, and subjected to sonication to prepare crude enzyme solution. 1ml reaction mixture was composed of 100 μL crude enzyme solution, 50 mM Tris-HCL (pH7.0), 40 μM 2,6-dichlorophenol indophenol, 1.5 mM phenazine methyl sulfate, 500 μM AD (dissolved in 2% methanol) The composition was reacted at 30°C for 50 minutes, and the change of absorbance value at a wavelength of 600nm was detected. The amount of enzyme that can cause a change of 0.01 absorbance unit per minute is defined as a unit U. The measured enzyme activity of the recombinant strain was 2.58U / mg.

[0042] The crude enzyme liquid was purified by Ni-NTA column to obtain pure KSDD. The specific enzyme activity of the purified enzyme liquid was 12.96U / mg, and th...

Embodiment 3

[0055] Example 3: Detection of whole cell transformation performance of recombinant strains

[0056] Inoculate the recombinant Corynebacterium blunt-toothed into 50ml LBG medium with 1% inoculum, cultivate for 24h, recover the thalline by centrifugation, wash twice, redissolve with the appropriate 50mM Tris-HCL (pH7.0) buffer, add 1% (w / v) 4-AD and 3% (w / v) β-cyclodextrin, continue to culture at 30°C, 160rpm for 12h. After detection and analysis by HPLC, the molar conversion rate of the final substrate reaches 83.87%, which is about 23 times higher than that of the starting bacteria.

[0057]

[0058]

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Abstract

A method for realizing whole-cell transformation from AD to ADD by utilizing recombinant corynebacterium crenatum is disclosed and belongs to the field of genetic engineering and enzyme engineering. The method comprises: firstly obtaining gene amplification of 3-ketosteroid-delta 1-dehydrogenase (KSDD) in mycobacterium neoaurum, then utilizing pXMJ19 plasmid to realize overexpression of the gene in a type strain corynebacterium crenatum SYPA5-5, so as to successively purify the active KSDD enzyme coming from mycobacterium aurum for the first time, and researching the enzymatic property of the KSDD enzyme for the first time. Bacillus subtilis engineering strain highly producing ADD is constructed by taking 4-AD as a substrate and performing whole-cell transformation; by researching the enzymatic activity and the fermentation performance of the bacterial strain, 3-ketosteroid-delta 1-dehydrogenase is discovered to have the activity obviously improved compared with the original strain; and by taking 1% (w/v) 4-AD as a substrate and performing whole-cell transformation for 12 h, the molar conversion rate of the substrate reaches 83.87% and is 23 times the relative conversion rate of the original strain.

Description

technical field [0001] The invention discloses a method for transforming AD into ADD by using whole cells of recombinant Corynebacterium crenatum, belonging to the fields of genetic engineering and enzyme engineering. [0002] technical background [0003] At present, although steroid drugs only account for 2.5% of the value of drugs in the international market, the pharmaceutical industry around the world requires more than 2,000 tons of steroid raw materials. Natural steroids are also becoming more and more important. For a long time, sitosterol was considered a waste product in the production of stigmasterol. Since the double bonds at positions 24 and 25 in the stigmasterol structure promote the chemical degradation of the side chains of the steroid structure, stigmasterol is widely used in the synthesis of pregnane derivatives in industry. At the same time, a large amount of sitosterol waste is produced. Now, sitosterol is the cheapest raw material for the synthesis of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/77C12N1/21C12P33/02C12R1/15
Inventor 饶志明许正宏吴丹邵明龙张显徐美娟杨套伟
Owner JIANGNAN UNIV
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