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Function of a novel cytochrome p450 oxidase

A technology of cytochrome and oxidase, applied in fermentation and other fields

Active Publication Date: 2020-08-28
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At this stage, there are few reports on biotransformation using P450 enzymes of the CYP108 family

Method used

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  • Function of a novel cytochrome p450 oxidase
  • Function of a novel cytochrome p450 oxidase
  • Function of a novel cytochrome p450 oxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 CYP108N7 heterologous expression

[0029] After the plasmid pET28-CYP108N7 was transformed into Escherichia coli BL21 competent, the single clone was extracted and inoculated in LB culture containing 50mg / L kanamycin. Based on 37°C, 180rpm cultured for 16h as seed liquid, transferred to fresh In LB or TB medium (200mL medium in 500mL volumetric shaker flask), shake culture at 37°C for 4h, then add IPTG with a final concentration of 0.5mM to induce the expression of CYP108N7 protein, induce at 20°C for 12h, then incubate at 4°C, 6000rpm Bacteria were harvested by refrigerated centrifugation for 10 min.

Embodiment 2

[0030] Example 2 Separation and purification of CYP108N7 protein

[0031] The bacteria obtained in Example 1 were resuspended in binding buffer (100 mM, pH7.0 sodium phosphate buffer, containing 300 mM NaCl, 5 mM imidazole), crushed by a high-pressure homogenizer, centrifuged at 12000 rpm for 15 min, and the supernatant was mixed with the above-mentioned After incubation with the Ni affinity chromatography resin equilibrated with the binding solution, rinse with a washing buffer (100 mM, pH7.0 sodium phosphate buffer containing 300 mM NaCl, 10 mM imidazole) until there is basically no foreign protein, and then wash with an elution buffer ( 100mM, pH7.0 sodium phosphate buffer, containing 300mMNaCl, 250mM imidazole) to elute and collect the target protein, after electrophoresis to identify the purity, combine the target protein and dialyze with dialysis buffer (100mM, pH7.0 potassium phosphate buffer) for 48h, After ultrafiltration and concentration, the protein concentration w...

Embodiment 3

[0032] Example 3 Construction of CYP108N7 in vitro catalytic system

[0033] Reaction system 1mL, including sodium phosphate buffer (0.1M, pH8.0), CYP108N7 pure enzyme 1μM, a pair of electron transfer proteins 10μM each, 5U glucose dehydrogenase (GDH), 2mM substrate, 0.2mM oxidized coenzyme NADP + , 10 mM glucose. After reacting for 12 hours at 30° C. in a shaking reactor at 200 rpm, an equal volume of ethyl acetate was added to terminate the reaction and extracted, and an appropriate amount of anhydrous sodium sulfate was added to dry it for HPLC detection and analysis.

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Abstract

The invention relates to the catalytic function of a novel cytochrome P450 enzyme CYP108N7, which belongs to the field of applied microorganisms and enzyme engineering. This enzyme belongs to the CYP108 family, which can be used to perform biocatalytic reactions such as epoxidation, hydroxylation, heteroatom oxidation, and C-H bond breaking on different substrates, especially in the asymmetric oxidation reaction of aromatic sulfide compounds. The selectivity is excellent, and it has the application value of preparing optically pure sulfoxide.

Description

technical field [0001] The invention relates to the catalytic function of a cytochrome oxidase CYP108N7, which belongs to the CYP108 family, and can be used to perform biocatalytic reactions such as epoxidation, hydroxylation, heteroatom oxidation, and C-H bond breaking on different substrates. The invention belongs to the field of applied microorganism and enzyme engineering. Background technique [0002] Cytochrome P450 (cytochrome P450, P450) belongs to the heme oxidase superfamily, and is named cytochrome P450 enzyme because the reduced heme oxidase combines with CO to form a complex with a maximum absorption peak near 450nm. Since it was first reported in 1962, more than 35,000 P450 genes have been annotated so far. [0003] P450 enzymes are widely distributed in nature and have diverse catalytic functions. In view of its important role in the body's metabolic activities, P450 enzymes play a prominent role in the synthesis of drugs and drug metabolites. In recent yea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P11/00C12P17/12C12P17/02C12P7/22
CPCC12P7/22C12P11/00C12P17/02C12P17/12
Inventor 吴中柳郭超刘艳裴小琼
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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