Mutant of short-chain dehydrogenase, encoding gene, encoding gene obtaining method and application of mutant

A technology of short-chain dehydrogenase and coding gene, applied in the direction of microorganism-based methods, applications, genetic engineering, etc., can solve the problems of low conversion rate, low concentration of prednisolone substrate, high substrate conversion rate, etc., to achieve High conversion rate, reduced production cost, and high yield

Pending Publication Date: 2022-08-09
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems of low substrate concentration and low conversion rate of prednisolone prepared by existing biological methods, the object of the present invention is to provide a mutant of short-chain dehydrogenase, which ca

Method used

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  • Mutant of short-chain dehydrogenase, encoding gene, encoding gene obtaining method and application of mutant
  • Mutant of short-chain dehydrogenase, encoding gene, encoding gene obtaining method and application of mutant
  • Mutant of short-chain dehydrogenase, encoding gene, encoding gene obtaining method and application of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Construction of engineering bacteria that can express each mutant

[0092] According to the amino acid sequence of wild-type short-chain dehydrogenase (the amino acid sequence is shown in SEQ ID NO. 1), the encoding gene (nucleotide sequence shown in SEQ ID NO. 8) is synthesized, and then the plasmid pET28a is used as the vector , the recombinant plasmid pET28a containing the encoding gene was obtained through conventional preparation operations, and the recombinant plasmid was transferred into Escherichia coli BL21 to obtain an engineering recombinant strain of wild-type short-chain dehydrogenase. Activated on the LB plate, cultured at 37°C for 18h, picked a single colony into a 50mL LB conical flask that also contained 1‰Kan resistance, cultured at 37°C and 220rpm until the OD600 was about 0.6, and extracted the plasmid according to the instructions of the plasmid mini-extraction kit. . Then, using the extracted plasmid as a template, a point mutation kit w...

Embodiment 2

[0103] Example 2 Induced expression of each mutant

[0104] The engineered bacteria expressing the wild enzyme constructed in Example 1 and the engineered bacteria expressing each mutant were inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C overnight, and the prepared bacteria were kept for storage, and then added with 1% of the inoculum amount (v / v) was inoculated into 50 mL LB medium containing 50 μg / mL kanamycin, cultivated at 37°C and 200 rpm until the bacterial concentration OD600 was about 0.6, and IPTG with a final concentration of 0.1 mmol / L was added. After 16 hours of induction and culture at 25°C, the wet cells were collected by centrifugation at 4°C and 8000 rpm for 10 min to obtain the wet cells of the engineered bacteria expressing wild enzymes and the engineered bacteria expressing each mutant, and stored at -80°C for later use.

Embodiment 3

[0105] Embodiment 3 (prednisolone prednisone acetate concentration 60g / L prepared by mutant I158A)

[0106] Mix 0.06g prednisone acetate, 200mg hydrolase (purchased from Azure Bio), 200mg GDH, 0.03g glucose, 200mg mutant I158A wet cells (from Example 2), add 1mL containing 10% (v / v) K of N,N-dimethylformamide 2 HPO 4 -KH 2 PO 4 Buffer (100mM, pH 6.5), placed at 37°C, under the condition of 220rpm constant temperature shaker for 24h. After the reaction, it was extracted with N,N-dimethylformamide and centrifuged, and 2 times the volume of N,N-dimethylformamide was added with water, vacuum rotary-evaporated at 60°C for 3 h, followed by acetonitrile filtration, high performance liquid chromatography (HPLC) ) analysis to determine its conversion rate and yield, the liquid chromatogram of prednisolone standard is as follows figure 1 As shown, the liquid chromatogram of the material system after this implementation reaction is as follows figure 2 As shown, the substrate conve...

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Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to a mutant of short-chain dehydrogenase, and the mutant is a mutant L104A, a mutant A150Y, a mutant Y155A, a mutant I158A, a mutant I202A or a mutant T205A. The amino acid sequence of the short-chain dehydrogenase is as shown in SEQ ID NO. 1. Compared with wild type short-chain dehydrogenase, the short-chain dehydrogenase mutant has higher enzyme activity, prednisolone can be prepared from prednisone or prednisone acetate matched with hydrolase as a substrate, the conversion rate of the substrate is high, the yield of the product is high, no by-product is generated, the reaction can be carried out at a higher substrate concentration level, and the method is suitable for industrial production. The method is suitable for large-scale production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a short-chain dehydrogenase mutant, an encoding gene, a method for obtaining the encoding gene, and application of the mutant Background technique [0002] Prednisolone is an adrenal cortical hormone drug, which is mainly used to treat various acute severe bacterial infections, severe allergic diseases, collagen diseases, rheumatism, rheumatoid arthritis, severe bronchial asthma, etc. It is an important steroid compound and can also be used as a precursor of nedide drugs such as dessonide and budesonide, and has a huge market potential. [0003] At present, the preparation of prednisolone mainly includes chemical method and biological fermentation method. The chemical method mainly takes steroid mother nucleus or hydrocortisone as raw material to obtain prednisolone through multi-step chemical synthesis steps. The method generally has shortcomings such as lon...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/11C12N15/70C12N1/21C12P33/08C12R1/19
CPCC12N9/0006C12N15/70C12P33/08C12Y101/01184
Inventor 于洪巍李东炎夏娜娜
Owner ZHEJIANG UNIV
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