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Method for generating ADD by converting AD through recombined Escherichia coli whole cells

A recombinant vector and chemical transformation technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of inactivity, undetectable KSDD enzyme activity, and low enzyme activity level, and achieves simple process, environmental protection, and raw material utilization rate. high effect

Inactive Publication Date: 2017-05-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When KSDD from different sources is expressed in the E. coli system, it mainly exists in the form of inactive inclusion bodies, and the enzyme activity of KSDD cannot be detected or the enzyme activity level is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Embodiment 1: Construction of recombinant Escherichia coli BL21 bacterial strain

[0034] Take the lab with 3-sterone-Δ 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 pET28a vector. After successful verification, the model strain Escherichia coli BL21 was transformed. Positive transformants were screened on the resistance plate, inoculated in shake flasks for fermentation, and the product ADD in the fermentation liquid was detected by 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 Escherichia coli BL21 strain with transformed AD...

Embodiment 2

[0035] Embodiment 2: the enzyme activity assay of recombinant bacterial strain

[0036] The strain was cultured overnight in LB medium, centrifuged at 8000rpm for 10min, washed 3 times with 50mM PBS buffer solution of pH 7.5, suspended in the buffer solution, and subjected to ultrasonic crushing to prepare crude enzyme solution. 3ml reaction mixture consists of 100μL crude enzyme solution, 50mM PBS (pH7.5), 40μM 2,6 dichlorophenol indophenol, 1.5mM phenazine methyl sulfate, 500μM AD (dissolved in 2% methanol) , to detect the change of absorbance value at 600nm. The enzyme activity unit is defined as: 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 1.75U / mg.

Embodiment 3

[0037] Example 3: Detection of whole cell transformation performance of recombinant strains

[0038] Recombinant Escherichia coli BL21 was inoculated in 100ml LB medium with 1% inoculum, cultivated for 24h, centrifuged to recover the thalli, washed twice, redissolved with an appropriate 50mM PBS (pH7.5) buffer, and added 0.1% ( w / v) 4AD and 0.1% (v / v) Tween 80, continue culturing at 30° C., 160 rpm for 10 h. After detection and analysis by HPLC, the molar conversion rate of the final substrate reaches 76.5%, which is about 25 times higher than that of the starting bacteria.

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Abstract

The invention discloses a method for generating ADD by converting AD through recombined Escherichia coli whole cells, and belongs to the field of gene engineering and enzyme engineering. The method comprises the following steps: firstly, performing gene amplification on 3-sterone-delta1-dehydrogenase (KSDD) in a new golden mycobacterium; realizing over expression of a gene in a type strain, namely, escherichia coli BL21 by using a pET28a plasmid. An escherichia coli engineering strain capable of producing ADD at a high yield is constructed by taking 4-AD as a substrate through a whole cell conversion method. As proved by researches on the enzyme activity and fermentation performance of the strain, the activity of the 3-sterone-delta1-dehydrogenase is remarkably increased compared with an original strain; 0.1 percent (w / v) 4-AD is taken as the substrate, and whole cell conversion is performed for 10 hours, so that the molar conversion rate of the substrate is up to 76.5 percent, being 25 times the relative conversion rate of the original strain. By adopting the method, beneficial guidance is provided for the industrial production of the ADD through a one-step fermentation method of microorganisms.

Description

technical field [0001] The invention relates to a method for transforming AD into ADD by using recombinant Escherichia coli whole cells, belonging to the fields of genetic engineering and enzyme engineering. [0002] technical background [0003] Steroidal drugs can be obtained through total synthesis or degradation of natural steroidal compounds and transformation of their functional groups. Steroidal drugs have strong pharmacological effects such as anti-infection, just 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. In 2000, the sales of steroid drugs in the global drug market have exceeded 20 billion US dollars, accounting for about 66% of the world's total pharmaceutical sales. %. [0004] Classification of steroid hormone drugs: adrenocortical hormones, including hydrocortisone, prednisone, etc. It can treat Addison's disease, anti-inflammation, anti-allerg...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P33/02C12R1/19
CPCC12N9/0073C12N15/70C12N2800/101C12P33/02C12Y114/13054
Inventor 饶志明邵明龙沙宗焱张显杨套伟徐美娟许正宏
Owner JIANGNAN UNIV