Method for preparing catechol compounds by biotransformation

A catechol and biotransformation technology, which is applied in the biological high field, can solve the problems of poor applicability of the biotransformation method, and achieve the effect of wide applicability

Inactive Publication Date: 2018-09-04
南京乐夫朗生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the problem of poor applicability of the biotransformation method, the present invention provides

Method used

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  • Method for preparing catechol compounds by biotransformation

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Effect test

Embodiment 1

[0016] The construction of embodiment 1 aniline oxidase expression bacterial strain

[0017] The gene cluster atdAlA2A3A4A5 encoding ATD was synthesized with reference to the sequence in GenBank (accession number AB008831.1), and then the synthetic DNA fragment was connected to the NdeI and EcoRI sites of the expression vector pET28a (purchased from Treasure Bioengineering Co., Ltd.) to obtain the expression vector pET-atd. Similarly, the gene cluster adoQTA1A2B encoding ADO was synthesized with reference to the sequence in GenBank (accession number CPO10956.1), and then the synthetic DNA fragment was connected to the NdeI and XhoI sites of the expression vector pET28a to obtain the expression vector pET-ado. Transform pET-atd and pET-ado into Escherichia coli BL21(DE3) (purchased from Treasure Bioengineering Co., Ltd.), respectively, to obtain expression strains BL21-ATD and BL21-ADO. The transcription of atdAlA2A3A4A5 and adoQTA1A2B is controlled by the T7 promoter.

Embodiment 2

[0018] The cultivation of embodiment 2 bacterial cells

[0019] The fermentation medium of bacterial strain BL21-ATD and BL21-ADO is glucose 1%, (NH 4 ) 2 SO 4 0.4%, K 2 HPO 4 0.22%, KH 2 PO 4 0.3%, MgSO 4 0.01%, NaCl 0.01%, fish meal 0.2%, yeast extract 0.01%, 1 times trace element mother solution, pH 7.0. The culture temperature was 37°C. In the middle and late stages of logarithmic growth, 0.1% lactose was added to induce the synthesis of ATD or ADO, and the induction time was 6 hours. After induction, the bacterial cells were collected by centrifugation and washed 3 times with pure water.

Embodiment 3

[0020] Embodiment 3 biotransformation method produces 4,5-dichlorocatechol

[0021] Using pure water as a solvent, the amount of Escherichia coli cells that synthesized aniline oxidase ADO was 10 9 CFU / ml, the amount of 3,4-dichloroaniline added is 0.4g / L, the reaction temperature is 30°C, the rotation speed is 150 rpm, the reaction time is 12 hours, the bacteria are removed by centrifugation, and the supernatant is concentrated by distillation The product 4,5-dichlorocatechol was obtained with a conversion rate of 95%. As shown in Figure 2, only raw material 3,4-dichloroaniline ( figure 1 A), after the reaction, only product 4,5-dichlorocatechol ( figure 1 B). The bacterial cells removed by centrifugation were harmlessly treated by steam sterilization.

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Abstract

The invention belongs to the field of biological high technology, and discloses a method for preparing catechol compounds by biotransformation. The method comprises the steps that two kinds of anilineoxidase ATD and ADO are efficiently synthesized by escherichia coli; under normal circumstances, escherichia coli cells containing the ATD and ADO can efficiently convert aniline compounds to producea plurality of catechol compounds which include catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol, 4,5-dichlorocatechol, p-tert-butylcatechol and isopropyl catechol, and the conversion rate is greater than 95%. The waste produced in the scheme is mainly bacterial cells, the bacterial cells can be killed by high temperature, and the environmental burden is almost notproduced.

Description

technical field [0001] The invention belongs to the field of biological high technology, and discloses a biotransformation preparation method of catechol compounds. Background technique [0002] Catechol compounds, such as catechol, p-methylcatechol, p-tert-butylcatechol, 3-chlorocatechol, 4-chlorocatechol and 4,5-dichlorocatechol Catechol, etc., are widely used in chemical industry and scientific research. Therefore, the development of green and efficient synthetic methods of catechol compounds has important application value. [0003] The synthesis of catechol compounds mainly has the following methods. First, the traditional synthetic method uses phenolic compounds as raw materials, undergoes formylation, Dakin oxidation, or oxidation, reduction and other processes. The disadvantage of this method is that the selectivity is low, a variety of products are produced, and the separation of the target product is increased. cost. The second is the transition metal-catalyzed...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P7/22
CPCC12N15/70C12P7/22
Inventor 张磊朱叶清
Owner 南京乐夫朗生物科技有限公司
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