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A kind of method and application of in vitro enzyme reaction synthesis phloroglucinol

A phloroglucinol, in vitro enzyme technology, applied in the field of bioengineering, can solve problems such as increasing the cost of the production process

Active Publication Date: 2018-08-17
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, in the process of producing phloroglucinol by engineering bacteria based on traditional metabolic engineering ideas, inducers and antibiotics need to be used, which increases the cost of the production process.

Method used

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  • A kind of method and application of in vitro enzyme reaction synthesis phloroglucinol
  • A kind of method and application of in vitro enzyme reaction synthesis phloroglucinol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The in vitro reaction of enzymes related to phloroglucinol synthesis and metabolism, the specific steps are as follows:

[0034](1) Take 100ml of Escherichia coli culture fluid expressing acetyl-CoA synthetase, 4 subunits of acetyl-CoA carboxylase, and polyketide synthase at 6000rpm, centrifuge at 4°C for 5min, discard the supernatant, and wash with 50mM, pH 7.4 phosphoric acid Wash twice with salt buffer, and finally use 5ml phosphate buffer to resuspend; then use ultrasonic breaker (20kHz, 60w, 20min), centrifuge at 13000rpm4°C for 20min after breaking, separate the supernatant from the precipitate, keep the supernatant, and use test Protein content was determined by the Mas Brilliant Blue binding assay kit. The enzyme liquid was purified by nickel column from Novagen, the target protein was collected, and the protein content was determined. The mass ratio of the four subunits of the purified acetyl-CoA carboxylase is compounded according to the ratio of accA:accB:ac...

Embodiment 2

[0038] Using the in vitro reaction of enzymes to determine the key enzymes in the synthesis and metabolism of phloroglucinol, the specific steps are as follows:

[0039] (1) Take 100ml of Escherichia coli culture fluid expressing acetyl-CoA synthetase, 4 subunits of acetyl-CoA carboxylase, and polyketide synthase at 6000rpm, centrifuge at 4°C for 5min, discard the supernatant, and wash with 50mM, pH 7.4 phosphoric acid Wash twice with salt buffer, and finally use 5ml phosphate buffer to resuspend; then use ultrasonic breaker (20kHz, 60w), centrifuge at 13000rpm4°C for 20min after breaking, separate the supernatant from the precipitate, keep the supernatant, and use Coomassie brilliant blue Binding assay kits were used to determine protein content. The enzyme liquid was purified by nickel column from Novagen, the target protein was collected, and the protein content was determined. The mass ratio of the four subunits of the purified acetyl-CoA carboxylase is compounded accordi...

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Abstract

The invention discloses a method for synthesizing phloroglucinol by virtue of in vitro enzyme reaction and an application of the method, and belongs to the technical field of biological engineering. The method comprises the following steps: respectively constructing recombinant vectors containing an acetyl coenzyme A synthetase ACS gene, an acetyl coenzyme A carboxylase subunit gene accA, an acetyl coenzyme A carboxylase subunit gene accB, an acetyl coenzyme A carboxylase subunit gene accC, an acetyl coenzyme A carboxylase subunit gene accD and a polyketone synthetase gene phlD; respectively converting the recombinant vectors into host bacteria, and then performing over-expression; crushing the bacteria by using ultrasonic waves; performing separation and purification to obtain an enzyme solution; and mixing the enzyme solution with reaction substrates in proportion and then performing in vitro enzyme reaction to synthesize phloroglucinol. By using the method disclosed by the invention, the phloroglucinol is synthesized by virtue of in vitro enzyme reaction, and the used amounts of each enzyme and accessory factors can be accurately adjusted and controlled.

Description

technical field [0001] The invention relates to a method for synthesizing phloroglucinol by in vitro enzyme reaction and its application, belonging to the technical field of bioengineering. Background technique [0002] Phloroglucinol, also known as 1,3,5-trihydroxybenzene, is an important fine chemical product and an intermediate in the synthesis of flavonoids and isoflavones. Flavonoids have a wide range of anti-cancer and anti-cardiovascular disease effects, and are increasingly valued by pharmacologists. A new drug against immunodeficiency virus synthesized from phloroglucinol, which belongs to the second generation of non-nucleoside reverse transcriptase inhibitors; Euglobals analogs synthesized from phloroglucinol can effectively inhibit Epstein-Barr virus and have significant anticancer effects . At present, the methods for industrial production of phloroglucinol are all chemical synthesis, but in the chemical synthesis method, due to the large amount of by-products...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P7/22C12R1/19
Inventor 咸漠张汝兵
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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