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Cytochrome P450 119 and mutant and purpose thereof

A CYP119, 1.CYP119 technology, applied in the field of CYP119 enzymes and their mutants, can solve the problems of catalyzing thioether substrates that have not been reported, and achieve excellent economic benefits, high catalytic efficiency, and wide application prospects

Active Publication Date: 2019-09-17
SOUTHWEST MEDICAL UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing CYP119 enzymes and their mutants have not been reported to catalyze thioether substrates

Method used

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  • Cytochrome P450 119 and mutant and purpose thereof
  • Cytochrome P450 119 and mutant and purpose thereof
  • Cytochrome P450 119 and mutant and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040]Example 1 Construction and expression of CYP119 mutant enzyme

[0041] 1. Construction of CYP119 enzyme mutants: according to the pET30a-CYP119 vector, the Quickchange Lighting Site-directed Mutagenesis Kit (Agilent Technologies) was used for point mutation. The PCR product was double digested and ligated to the pET30a vector to construct the pET30a-CYP119 mutant plasmid. This plasmid was transformed into Escherichia coli BL21(DE3)plysS. Positive clones were selected and sequenced to verify the correctness of the mutant plasmid.

[0042] 2. Expression of wild-type and T213G+F1533G mutants: Inoculate the positive plasmid in 2 mL double-antibody LB liquid medium, and culture overnight at 37°C with shaking. Take 100 μL of the overnight cultured bacterial solution in 50 mL of double-antibody TB medium. Add 250 μL / LTrace Element, culture at 37°C with shaking until OD 0.6. Add 0.4mmol·L -1 IPTG, 32°C, induced for 24h. 12000rpm, centrifuge at 4°C for 10min, discard the su...

Embodiment 2

[0044] Example 2 Screening of reaction conditions for CYP119 wild-type and mutant enzymes to catalyze the oxidation of thioanisole

[0045] In this embodiment, screening is carried out for the concentration of TBHP, and the screening conditions are shown in Table 1.

[0046] Table 1 The results of CYP119 wild-type enzyme catalyzing the reaction of thioanisole at different TBHP concentrations at 35°C and pH 7.5

[0047]

[0048] This embodiment explores the concentration of the nutrient TBHP, and finally determines the optimal condition: the amount of TBHP is 10 times that of the substrate concentration (in this embodiment, the substrate concentration is 2mmol L -1 ), 35°C, pH 7.5. GC detection results of CYP119 wild-type enzyme catalyzed thioanisole oxidation reaction Figures 1A-1E .

Embodiment 3

[0049] Example 3 CYP119 wild-type enzyme catalyzes the oxidation reaction of thioanisole and the effect verification of thioanisole respectively

[0050] Reaction conditions: temperature 35°C, substrate sulfide anisole 2mmol·L -1 , Enzyme 7 μmol L -1 . The reaction buffer system is 50mmol·L -1 bis-Tris buffer, pH 7.5, total system 200μL. After preheating at 35°C for 10 min, add TBHP (20 mmol L -1 ) to initiate the reaction. See Figures 1A-1E .

[0051] After 1 hour, with 200 μL CH 2 Cl 2 Quench the reaction, add 1.5 μL of 0.25 mol of acetophenone, and extract three times. The conversion of the substrate was checked by GC. GC-MS detection conditions: Thermo Scientific ITQ900, DB-1MS column (30m×0.25mm), 90°C for 2min; 90-210°C, 10°C / min to 210°C; 210°C for 1min. Under this condition, the wild-type CYP119 enzyme catalyzes the oxidation of thioanisole to benzyl sulfoxide and benzyl sulfone, and the yields are 59.3% for benzyl sulfoxide and 30.3% for benzyl sulfone.

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Abstract

The invention provides a cytochrome P450 119 and a mutant and purpose thereof. The mutant of the cytochrome P450 119 provided by the invention is T213G+F153G. The cytochrome P450 119 and the mutant thereof provided by the invention can efficiently catalyze the oxidization of thioether type substrates into sulfoxide and sulphone at normal temperature; and besides, n-caprylic acid is added to a reaction system, the catalysis capacity of the mutant for performing enzyme catalysis on an oxidation reaction of thioanisole is improved by 2.38 times than that of a wild type mutant. The invention also finds that the mutation enzyme can increase the chemical selectivity of a reaction, namely increasing the generation of sulfoxide and reducing the generation of the sulphone. The invention provides the new purpose of the cytochrome P450 119, and also provides the new purpose of the mutant of the cytochrome P450 119 to improvement of the catalysis capacity of the thioether type oxidation reaction and improvement of the chemical selectivity of the reaction, excellent economic benefits can be obtained, and the cytochrome P450 119 has great application prospects.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a CYP119 enzyme, mutants and applications thereof. Background technique [0002] The CYP119 enzyme (cytochrome P450 119) comes from the acidophilic and thermophilic Sulfolobus solfataricus isolated from the crater of Yellowstone Park, so the enzyme has the functions of acid resistance and high temperature resistance. Current literature reports that CYP119 enzyme can catalyze the hydroxylation of lauric acid and the epoxidation of styrene and its similar substrates under the conditions of hydrogen peroxide or pseudomonas redoxin-reductase (Pd / PdR) and coenzyme NADPH. Response to green environmental protection has a high application prospect. Since the natural substrate of this enzyme has not been identified, the catalytic efficiency of epoxidation reaction using styrene as the substrate of CYP119 enzyme is low. Zhang et al. conducted a series...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12N15/53C12P11/00
CPCC12N9/0065C12P11/00C12Y111/01007
Inventor 王钦张春魏潇遥杜羲
Owner SOUTHWEST MEDICAL UNIVERISTY
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