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Omega-transaminase mutant capable of catalyzing sitafloxacin five-membered key intermediate

A mutant, transaminase technology, applied in the field of genetic engineering and enzyme engineering, can solve the problems of inability to apply ω-transaminase, reduce development and application, etc.

Active Publication Date: 2019-03-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although ω-transaminase has great application and research value, the ω-transaminase screened from wild bacteria cannot specifically catalyze the key intermediate of the five-membered ring of sitafloxacin, making it impossible to further apply the ω-transaminase to industry In production, the development and application are greatly reduced

Method used

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  • Omega-transaminase mutant capable of catalyzing sitafloxacin five-membered key intermediate
  • Omega-transaminase mutant capable of catalyzing sitafloxacin five-membered key intermediate
  • Omega-transaminase mutant capable of catalyzing sitafloxacin five-membered key intermediate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Preparation and construction of ω-transaminase site-directed mutants

[0028] One site-directed mutant Y32L / S190A / L212M / I215M of ω-transaminase derived from Bacillus pumilus W3:

[0029] In the present invention, the most similar thermophilic archaea transaminase crystal structure (PDB ID: 5E25) is used as a template to construct the three-dimensional simulation structure of Bacillus pumilus W3 ω-transaminase (ω-BPAT) through the Swiss-Model online server ( figure 1 ). Through amino acid primary sequence alignment, it was found that the similarity between thermophilic archaea aminotransferase and ω-BPAT reached 51.21%, which was in line with the parameters of homology modeling. Therefore, it can be considered that ω-BPAT has a similar three-dimensional structure to thermophilic archaea aminotransferase. According to the results of software analysis and prediction, mutant Y32L / S190A / L212M / I215M was constructed by PCR-mediated site-directed mutagenesis.

[00...

Embodiment 2

[0041] Example 2: Expression and purification of native ω-transaminase and site-directed mutants thereof

[0042] Pick the positive single clones transferred into the expression host Escherichia coli BL21 (DE3) and grow them in LB liquid medium (containing 30 μg / mL ampicillin) for 8-10 hours, and transfer the seed fermentation broth to LB liquid medium ( containing 30 μg / mL ampicillin); Escherichia coli was cultured on a shaker at 37°C for 2 hours until OD 600 = about 0.6, the mutant Y32L / S190A / L212M / I215M recombinant strain was added with 0.05mM final concentration of IPTG to induce extracellular expression, and continued to culture and ferment on a shaker at 15°C for 24h, then centrifuged the fermentation broth at 4°C and 8000g for 10min to remove Bacteria, and the centrifuged fermentation supernatant was collected. Slowly add 60% (NH 4 ) 2 SO 4 , placed at 4°C for salting out overnight. Centrifuge at 10,000 g for 20 min at 4°C to collect the precipitate. After redisso...

Embodiment 3

[0043] Embodiment 3: enzyme activity analysis method

[0044] The determination method of ω-transaminase activity refers to Gao, S. (Gao, S., Su, Y., Zhao, L., Li, G., Zheng, G., 2017.Characterization of a(R)-selective amine transaminase from Fusariumoxysporum. Process. Biochem. 63, 130-136.).

[0045] Take an appropriate amount of bacterial supernatant (or purified and diluted enzyme solution), add 500 μL sodium dihydrogen phosphate / disodium hydrogen phosphate buffer (100 mM, pH7.0), which contains 20 mM (R)-α-phenethylamine ( or (S)-α-phenethylamine), 20mM sodium pyruvate, 0.1mM pyridoxal 5'-phosphate (PLP), mix well, react at 45°C for 15min respectively, and then add the same amount of ethyl acetate to terminate the reaction. The absorbance of the solution at 254 nm was measured before and after the reaction.

[0046] Under the above conditions, the amount of enzyme required to catalyze 1 μmol of related ketones in 1 minute is defined as one enzyme activity unit (U / ml). ...

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Abstract

The invention discloses an omega-transaminase mutant capable of catalyzing a sitafloxacin five-membered key intermediate, and belongs to the technical fields of gene engineering and enzyme engineering. According to the omega-transaminase mutant disclosed by the invention, amino acid with higher B factors in an omega-transaminase crystal structure is subjected to site-directed mutation, so that Y positioned at the 32nd point of the omega-transaminase is mutated into L, S positioned at the 190th point is mutated into A, L positioned at the 212th point is mutated into M, and I positioned at the 215th point is mutated into M, so that the omega-transaminase mutant capable of catalyzing a sitafloxacin five-membered key intermediate is obtained. The catalytic efficiency Kcat / Km of the mutant Y32L / S190A / L212M / I215M disclosed by the invention is 0.64min<-1>.mM<-1>; and when (S)-5-benzyl-5-azaspiro[2.4]heptan-7-amine is catalyzed to generate 5-benzyl-5-azaspiro[2.4]heptan-7-one, the maximum conversion ratio of the 5-benzyl-5-azaspiro[2.4]heptan-7-one of the mutant is 79.02 percent. The mutant disclosed by the invention is suitable for being applied to industrial chiral synthesis of the sitafloxacin five-membered key intermediate.

Description

technical field [0001] The invention relates to an omega-transaminase mutant capable of catalyzing the key intermediate of the five-membered ring of sitafloxacin, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Sitafloxacin Hydrate, a broad-spectrum quinolone antibacterial drug developed by Daiichi Pharmaceutical Sankyo Co., Ltd. in 2008, has a good bactericidal effect on many common clinical fluoroquinolone-resistant strains and is used to treat severe refractory infections. disease. Compared with other quinolone drugs, this product has low daily treatment cost, good therapeutic effect and safety. However, the key technical difficulty in its synthesis process is the asymmetric synthesis of the key intermediate of the five-membered ring of sitafloxacin. The instability of the process, high cost, and technical limitations that are difficult to scale up production severely limit the production of sitafloxacin. C...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P13/00C12P13/04C12P17/10
CPCC12N9/1096C12P13/001C12P13/005C12P13/04C12P17/10C12Y206/01
Inventor 廖祥儒翟李欣赖英杰殷方荣蔡宇杰管政兵
Owner JIANGNAN UNIV
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