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A kind of monascus monooxygenase mutant and its application

A monooxygenase and mutant technology, which is applied in the direction of application, oxidoreductase, and introduction of foreign genetic material by using a carrier, can solve the problems of low catalytic activity and hinder application, and achieve simple separation, high yield, and high purity. Effect

Active Publication Date: 2022-03-25
HUAZHONG AGRI UNIV
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Problems solved by technology

However, the low catalytic activity of the enzyme hinders related applications

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  • A kind of monascus monooxygenase mutant and its application

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Embodiment

[0023] 1. Construct E. coli expression strains of wild-type monooxygenase and its mutants.

[0024] After culturing Monascus in PDB medium for 10 days, the mycelium was obtained by filtration. Weigh 200 mg of mycelia and place in liquid nitrogen for precooling. Then put the mycelia into a mortar, add liquid nitrogen to grind quickly, then add 1mL TRIZOL solution and shake immediately to extract total mRNA. The total cDNA was then obtained by reverse transcriptase.

[0025] The DNA fragment encoding the monooxygenase is amplified by PCR technology, as shown in SEQ ID NO.3, and the primers used are shown in SEQ ID NO.4 and SEQ ID NO.5. The monooxygenase DNA fragment and the empty pET30a(+) DNA fragment were subjected to double digestion with restriction endonucleases (HindIII and EcoRI) at 37°C, respectively. The two DNA fragments were then purified and recovered with a DNA fragment recovery kit, and under the action of T4 DNA ligase, the two DNA fragments were enzyme-ligated...

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Abstract

The invention belongs to the field of monooxygenase, and in particular relates to a monascus monooxygenase mutant and application thereof. The specific technical solution is: a monooxygenase mutant whose amino acid sequence is shown in SEQ ID NO.2. Compared with the wild-type monooxygenase, the monooxygenase mutant provided by the present invention can catalyze and generate the product azone with higher efficiency. The synthesis efficiency of the reaction product azone was increased from 44.2% to 92.8%, and the content of azone after separation and purification was higher than 99%. The azone produced by the monooxygenase catalyzed by the invention has higher yield, higher purity and simpler separation.

Description

technical field [0001] The invention belongs to the field of monooxygenase, and in particular relates to a monascus monooxygenase mutant and application thereof. Background technique [0002] Azotropes are a class of compounds with a pyranoquinone bicyclic structure, which are mainly produced by microorganisms or plants. In the molecular structure of azotropin, the oxygen atom is easily replaced by the nitrogen atom in the organic nitrogen compound, hence the name azotropin. These compounds have been found to have various medical functions such as anticancer, antibacterial, anti-inflammatory, weight loss, lowering blood pressure and lowering blood fat. [0003] At present, the most commonly used method for preparing azone compounds is to extract them from plants. However, this method is affected by factors such as planting area, plant variety and climate change, resulting in unstable yield and quality of azone. However, if microorganisms are used to produce azone, the abo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P17/06C12R1/19
CPCC12N9/0004C12N15/70C12P17/06
Inventor 李牧刘佳玮段雅丽易志强
Owner HUAZHONG AGRI UNIV
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