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Method of Preparing Piceatannol Using Bacterial Cytochrome P450 and Composition Therefor

a technology of cytochrome p450 and piceatannol, which is applied in the field of preparation of piceatannol using bacterial cytochrome p450 and composition therefor, can solve the problems of large quantities of metabolites to be produced, inability to meet the requirements of biocatalysis, and difficulty in synthesis of pure metabolites

Inactive Publication Date: 2011-09-29
GLO BIOTECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

"The present invention relates to a method of preparing expensive piceatannol from resveratrol using bacterial cytochrome P450 BM3 (CYP102A1) or mutants thereof. The technical effect of this invention is to provide a cost-effective and efficient way to produce piceatannol, which has various beneficial activities and is a potential anti-cancer agent. The use of bacterial P450s as biocatalysts for the production of fine chemicals has been explored in the past decade, but their low stability, catalytic activity, and availability have limited their use in preparative-scale metabolite synthesis. The invention proposes to use engineered bacterial P450 BM3 or mutants thereof to oxidize resveratrol to produce piceatannol, which has not been previously reported as a substrate of human P450 enzymes."

Problems solved by technology

In spite of the potential use of mammalian P450s in various biotechnology fields, they are not suitable as biocatalysts because of their low stability, catalytic activity, and availability.
However, large quantities of the metabolite need to be produced.
The pure metabolites may be difficult to synthesize.
Hepatic microsomes can be a source of human P450s, but their limited availability make their use in preparative-scale metabolite synthesis impractical.
Metabolite preparation has been demonstrated using human P450s expressed in Escherichia coli and in insect cells (Parikh et al., 1997; Rushmore et al., 2000; Vail et al., 2005), but these systems are costly and have low productivities due to limited stabilities and slow reaction rates (usually −1 (Guengerich et al., 1996)).
However, there has been no research on whether resveratrol can be used as a substrate.
However, a patent application related to a method of preparing piceatannol has not been filed.

Method used

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  • Method of Preparing Piceatannol Using Bacterial Cytochrome P450 and Composition Therefor
  • Method of Preparing Piceatannol Using Bacterial Cytochrome P450 and Composition Therefor
  • Method of Preparing Piceatannol Using Bacterial Cytochrome P450 and Composition Therefor

Examples

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example 1

Construction of P450 BM3 Mutants By Site-Directed Mutagenesis

[0051]Site-directed mutants of CYP102A1 were prepared as disclosed by Kim et al., Drug Metab Dispos, volume 35, p. 2166-2170, 2008. PCR primers used to introduce BamHI / SacI restriction sites and to induce mutation are listed in Table 1. Codons for amino acid substitution are in italics and underlined. The PCR primers were obtained from Genotech (Daejeon, Korea). Genes that encode CYP102A1 mutants were amplified from pCWBM3 by PCR using primers designed to facilitate cloning into an expression vector pCWori (Dr. F. W. Dahlquist, University of California, Santa Barbara, Calif.) or pSE420 (Invitrogen) (Farinas et al., 2001). Oligonucleotide assembly was performed by PCR using the 14 sets of designed primers listed in Table 1. The amplified genes were subsequently cloned into the PCWBM3 BamHI / SacI vector at the BamHI / SacI restriction sites. These plasmids were transformed into Escherichia coli DH5a F′IQ (Invitrogen), and this ...

example 2

Expression And Purification of Wild-Type CYP102A1 And Mutants of CYP102A1

[0052]Plasmids comprising a gene of wild-type CYP102A1 and mutants of CYP102A1 (pCWBM3) were transformed into Escherichia coli DH5α F′-IQ. A single colony was inoculated into 5 ml of a Luria-Bertani medium supplemented with ampicillin (100 g / ml) and cultured at 37° C. This culture was inoculated into 250 ml of a Terrific Broth medium supplemented with ampicillin (100 g / ml). The cells were grown at 37° C. while shaking at 250 rpm to an 0D600 of up to 0.8, at which gene expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM. δ-Aminolevulinic acid (1.0 mM) was also added thereto. Following induction, the cultures were allowed to grow another 36 hours at 30° C. Cells were harvested by centrifugation (15 min, 5000 g, 4° C.). The cell pellet was resuspended in a TES buffer (100 mM Tris-HCl, pH 7.6, 500 mM sucrose, 0.5 mM EDTA) and lysed by sonication (Sonicato...

example 3

Hydroxylation of Trans-Resveratrol By Wild-Type P450 BM3 And Mutants of P450 BM3

[0055]Oxidation of trans-resveratrol, a substrate of human CYP1A2, by CYP102A1 was identified. Typical steady-state reactions for trans-resveratrol hydroxylation included 50 pmol P450 BM3 in 0.25 ml of a 100 mM potassium phosphate buffer (pH 7.4) were performed along with a specified amount of a substrate. To determine the kinetic parameter of several CYP102A1 mutants, 2 to 100 μM of trans-resveratrol was used. An NADPH-generating system was used to initiate reaction solutions (final concentrations: 10 mM glucose 6-phosphate, 0.5 mM NADP+, and 1 IU yeast glucose 6-phosphate per ml). Trans-resveratrol stocks (20 mM) were prepared in DMSO and diluted into the enzyme reactions with the final organic solvent concentration <1% (v / v). Reactions were generally incubated for 10 min at 37° C., and terminated with 105 μl of ice-cold acetic acid / methanol (95 / 5, v / v).

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Abstract

Provided is a method of preparing piceatannol, and more particularly, to a method of preparing piceatannol from resveratrol using bacterial cytochrome P450 BM3 (CYP102A1) or mutants thereof, and a composition and a kit therefor.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of preparing expensive piceatannol from resveratrol using bacterial cytochrome P450 BM3 (CYP102A1) or mutants thereof.[0002]This work was supported in part by the 21C Frontier Microbial Genomics and Application Center Program of the Ministry of Education, Science & Technology of the Republic of Korea [Project No.: MG08-0306-2-0, Title: Development of humanized bacterial monooxygenase for fine chemicals using microbial cytochrome P450 enzyme genomics].BACKGROUND ART[0003]Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin, which is an antitoxic substance produced by a plant tissue in response to external toxicity and found in a wide variety of dietary sources including grapes, plums, and peanuts. It exhibits beneficial effects including anti-oxidant, anti-inflammatory, cardioprotective, and anti-tumor activities (Kundu and Surh, 2008; Pirola and Fröjdö, 2008; Athar, et al., 2007). Currently, numerous preclinical find...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P7/22C12N9/02
CPCC12N9/0071C12Y114/14001C12P7/22C12N15/09
Inventor YUNKIM, DONG HYUN
Owner GLO BIOTECH
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