Cytochrome P450, nicotinamide adenine dinucleotide-cytochrome P450 reductase and application thereof

A technology of nicotinamide adenine and cytochrome, applied in the fields of biotechnology and microbiology, can solve the problems of high cost and limited yield of Ganoderma lucidum triterpenes.

Active Publication Date: 2018-11-23
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of ganoderma triterpenoids is very high and the output is limited

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Cytochrome P450, nicotinamide adenine dinucleotide-cytochrome P450 reductase and application thereof
  • Cytochrome P450, nicotinamide adenine dinucleotide-cytochrome P450 reductase and application thereof
  • Cytochrome P450, nicotinamide adenine dinucleotide-cytochrome P450 reductase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1, Cloning of Cytochrome P450 and Nicotinamide Adenine Dinucleotide (NADPH)-cytochrome P450 Reductase

[0125] The six primers synthesized respectively have the nucleotide sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 in the sequence listing.

[0126] Ganoderma lucidum RNA was extracted and reverse-transcribed to obtain ganoderma lucidum cDNA. The cDNA was used as a template for PCR amplification, and the above three pairs of primers SEQ ID NO:4 / 5, SEQ ID NO:6 / 7 and SEQ ID NO:8 / 9 were used for PCR amplification respectively. The high-fidelity PrimeSTAR DNA polymerase from Treasure Bioengineering Co., Ltd. was selected as the DNA polymerase. PCR products were detected by agarose gel electrophoresis ( figure 1 ). Under UV irradiation, the target DNA band is excised. Then, Axygen Gel Extraction Kit (AEYGEN Company) was used to recover DNA from the agarose gel, which was the amplified DNA fragment. Ligate this DNA...

Embodiment 2

[0130] Example 2, Yeast Recombinant Expression Vector Construction of Cytochrome P450 Gene GPO1 and GPO2

[0131]The synthetic primers respectively have SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24 and SEQ ID in the sequence listing Nucleotide sequence of NO:25.

[0132] Using the genome of Saccharomyces cerevisiae CEN.PK113-3C (purchased from Euroscarf) as a template, the Trp expression element dTrp-1 (that is, dTrp, whose 5' and 3' ends are both amplified with primers SEQ ID NO:24 and SEQ ID NO:25) Contains sequences homologous to pESC-His). The PCR product was recovered from the agarose gel with the AxyPrep DNA Gel Extraction Kit from AXYGEN. Plasmid pESC-His (purchased from Agilent, USA) was digested with Nde I and Pst I from Fermentas, and DNA fragments were recovered from the agarose gel with AxyPrep DNA Gel Extraction Kit from XYGEN. The DNA fragment and the digested pESC-His plasmid were transformed into Saccharomy...

Embodiment 3

[0136] Example 3, Expression of Cytochrome P450 Genes GPO1 and GPO2 in Saccharomyces cerevisiae

[0137] Preparation of SCO medium: 0.67% (w / v) parent amino acid-free basic nitrogen source, 2% (w / v) glucose. Inoculate PNGP1 and PNGP1 in SCO medium, culture at 250 rpm at 30°C to reach the stationary phase. Prepare YPD medium: 1% (w / v) yeast extract, 2% (w / v) bacto-peptone, 2% (w / v) glucose, collect the bacteria by centrifugation, resuspend in rich YPD medium, 30 Cultivate at 250rpm for 24h. Glucose was replaced by galactose to induce protein expression. Bacteria were collected by centrifugation, cells were lysed, and microsomes were obtained by ultracentrifugation. Take an appropriate amount of microsomes for SDS-PAGE electrophoresis detection. Compared with the recombinants transfected with the pdTrp-NCP1 control vector, PNGP1 and PNGP2 recombinants have no obvious band characteristics, such as image 3 with Figure 5 . Use anti-6×His TagWestern Blot to detect expression...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention relates to cytochrome P450, a nicotinamide adenine dinucleotide-cytochrome P450 reductase and application thereof. The invention discloses the set of new cytochrome P450 (P450 GP01 and P450 GP02) which performs the effect in the terpenoid hydroxylation catalysis and synthesis of Ganoderma triterpenes, and the nicotinamide adenine dinucleotide-cytochrome P450 reductase (GLCPR) which completes hydroxylation catalysis by cooperating with the cytochrome P450. In specific, the cytochrome P450 GP01 can specifically and efficiently catalyze the hydroxylation of C-23 site of a tetracyclic triterpene compound substrate. The cytochrome P450 GP02 performs hydroxylation om ganoderic acid DM and ganoderic acid TR to generate various kinds of products. The GLCPR can be paired with the cytochrome P450, and assists the cytochrome P450 to perform the hydroxylation activity.

Description

technical field [0001] The invention belongs to the fields of biotechnology and microbiology; more specifically, the invention relates to novel cytochrome P450, nicotinamide adenine dinucleotide-cytochrome P450 reductase and application thereof. Background technique [0002] Ganoderma lucidum triterpenoids are highly oxidized lanosterol derivatives isolated from Ganoderma lucidum and fungi of the same genus (such as Zizhi, Tongue, etc.), belonging to tetracyclic triterpenoids, and one of the main medicinal components of Ganoderma lucidum. At present, at least 150 triterpenoids have been isolated from Ganoderma lucidum, some of which have been proven to have a wide range of physiological functions and potential medicinal value: including anti-tumor, anti-virus, liver protection, and cholesterol-lowering effects. [0003] Structurally, Ganoderma lucidum triterpenes are bioactive molecules formed by lanosterol through a series of oxidation, reduction and acetylation reactions. ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/06C12N9/02C12N15/53
CPCC12N9/0042C12N9/0081C12P33/06C12Y106/02004C12Y114/15006Y02P20/55
Inventor 周志华杨成帅严兴肖友利李伟超李晨
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap