WO2004099398A1 - Verfahren zur herstellung eines hydroxylierungskatalysators und seine verwendung - Google Patents
Verfahren zur herstellung eines hydroxylierungskatalysators und seine verwendung Download PDFInfo
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- WO2004099398A1 WO2004099398A1 PCT/EP2004/004748 EP2004004748W WO2004099398A1 WO 2004099398 A1 WO2004099398 A1 WO 2004099398A1 EP 2004004748 W EP2004004748 W EP 2004004748W WO 2004099398 A1 WO2004099398 A1 WO 2004099398A1
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- sol
- cyp
- hydroxylation
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- embedding
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
Definitions
- the present invention relates to a process for the preparation of a hydroxylation catalyst based on a cytochrome P450 monooxygenase, and to processes for the hydroxylation of organic substrates by means of these hydroxylation catalysts.
- Cytochrome P450 monooxygenases catalyze the hydroxylation of a number of hydrophobic substrates on activated and non-activated carbon atoms.
- One oxygen atom of O 2 is built into the substrate, while the other oxygen atom is reduced to H 2 O with simultaneous oxidation of nicotine adenine dinucleotide (phosphate) (N ⁇ D (P) H).
- N ⁇ D (P) H nicotine adenine dinucleotide
- this hydroxylation occurs regionally and stereospecifically.
- cytochrome P450 monooxygenase is the gene originally cloned from Bacillus megaterium, hereinafter called CYP BM-3. It is a 119 kDa natural fusion protein which is composed of a heme-containing monooxygenase domain and a FAD and FMN-containing reductase domain (LP.Wen, AJ Fulco, J. Biol. Chem. 1987, 262, 6676-6682; AJFuico, RTRuettinger, Life Sei. 1987, 40, 1769-1775).
- the natural substrates for CYP BM-3 are long-chain fatty acids (C12-C20), which are exclusively hydroxylated at the subterminal positions co-1, ⁇ -2, ⁇ -3, sometimes with high enantioselectivity.
- Variants of the CYP BM-3 also show activity against non-natural substrates such as short-chain fatty acids (Ost et al. FEBS Lett 2000, 486, 173-177; Li et al. Biochim. Biophys. Acta 2001, 1545, 114-121), indoles (Li et al. Chemistry 2000, 6, 1531-1536), polycyclic aromatic hydrocarbons (Li et al. Appl. Environ.
- the invention relates to a method for producing a hydroxylation catalyst by i) embedding a cytochrome P450 monooxygenase in a sol-gel matrix, ii) embedding an enzymatic NADPH regeneration system in a sol-gel matrix, and both components i) and ii ) merges, unless they already exist before the
- Cytochrome P450 monooxygenases and their use in biotransformations are known to the person skilled in the art, for example, from E.T. Farinas et al. Adv. Synth. Catal. 2001, 343, 601-606 or V. Urlacher and R.D. Schmid Curr. Opin. Biotechnol. 2002, 13, 557-564.
- those CYPs which are originally isolated from microorganisms are particularly suitable for the method according to the invention.
- a particularly suitable cytochrome P450 monooxygenase is the protein originally cloned from Baciilus megaterium, hereinafter called CYP BM-3. It is a 119 kDa natural fusion protein made from a heme-containing monooxygenase. Domain and a reductase domain containing FAD and FMN (LP.Wen, AJ Fulco, J. Biol. Chem. 1987, 262, 6676-6682; AJFuIco, RTRuettinger, Life Sei. 1987, 40, 1769-1775).
- muteins can be produced by recombinant DNA techniques which carry changes in certain amino acid positions compared to the wild-type CYP BM-3.
- Muteins particularly suitable for the process according to the invention are those which carry an amino acid other than Ala at position 74 and / or carry an amino acid other than Phe at position 87 and / or carry a different amino acid than Leu at position 188 and / or carry an amino acid other than Phe at position 386.
- the changes to the listed positions can either be introduced as single mutations or cumulatively as multiple mutations.
- Such a mutein which is particularly suitable because of its large substrate range, is the one which, compared to the wild-type CYP BM-3, has the following three amino acid substituents. tion has: Position 74 Ala replaced by Gly, Position 87 Phe replaced by Val, Pos 188 Leu replaced by Gin. This mutein can be used in particular for the hydroxylation of alkanes and aromatics.
- Another mutein particularly suitable for the hydroxylation of ⁇ -ionone is that which has the following three amino acid substitutions compared to the wild-type CYP BM-3: position 74 Ala replaced by Glu, position 87 Phe replaced by Val, pos
- Sol-gel matrices which are particularly suitable for the process according to the invention are based on silica.
- Particularly suitable sol-gel matrices can be produced from alkoxysilanes, in particular from tetraalkoxysilanes, especially tetraethoxysilanes (TEOS) and tetramethoxysilanes (TMOS).
- TEOS tetraethoxysilanes
- TMOS tetramethoxysilanes
- NADPH -RS The enzymatic NADPH regeneration system
- NADPH -RS consists of an NAD + or NADP + -dependent enzyme that oxidizes a substrate to a product while simultaneously reducing the NAD + to NADH or the NADP + to NADPH.
- NAD + or NADP + -dependent dehydrogenases in particular microbial dehydrogenases and in particular formate dehydrogenases, are suitable for this.
- a preferred embodiment of the NADPH-RS is the NADP + -dependent formate dehydrogenase (EC 1.2.1.2), hereinafter abbreviated to FDH from Pseudomonas sp. 101.
- FDH catalyzes the NAD + dependent oxidation of formate to CO 2 .
- the advantages of this system are the low substrate costs (formate) and the easy removal of the resulting product (CO 2 ).
- a mutated form of FDH has a high activity with NADP + and can therefore be used particularly well as a NADPH regenerating enzyme in combination with CYP. This particularly suitable mutant form of FDH has been described by Tishkov et al. Biotechnol. Bioeng.
- CYP and NADPH-RS can be embedded in a sol-gel matrix simultaneously or in separate batches. Simultaneous embedding means that first a preparation of CYP, preferably a solution of CYP, is combined with a preparation of NADPH-RS, preferably a solution of NADPH-RS, and this combined preparation is subsequently combined in the sol-gel matrix is embedded.
- a separate embedding is to be understood to mean that a preparation of CYP, preferably a solution of CYP, is embedded in a sol-gel matrix and in a second approach a preparation of NADPH-RS, preferably a solution of NADPH-RS, in a Sol-Gel matrix is embedded and then these two approaches are combined.
- Separate embedding is preferably used for the method according to the invention, since the stoichiometric ratio of CYP to NADPH-RS can also be flexibly adjusted afterwards.
- the invention further relates to preparations which contain a CYP and a NADPH-RS embedded in a sol-gel matrix.
- preparations which contain a CYP and a NADPH-RS embedded in a sol-gel matrix.
- Such preparations can be produced by the processes described above. These preparations have the advantage that it is possible to produce hydroxylation catalysts in a stable, readily storable form and to prepare them for use in specific hydroxylation reactions.
- the invention further relates to processes for the enzymatic hydroxylation of a substrate using one of the hydroxylation catalysts which can be prepared according to the invention.
- a large number of organic compound classes are suitable as substrates, in particular long-chain fatty acids (C12-C20), which are hydroxylated in particular at the subterminal positions ⁇ -1, ⁇ -2, ⁇ -3, but also short-chain fatty acids (Ost et al. FEBS Lett . 2000, 486, 173-177; Li et al. Bio-chim. Biophys. Acta 2001, 1545, 114 - 121), indoles (Li et al. Chemistry 2000, 6, 1531- 1536), polycyclic aromatic hydrocarbons (Li et al.
- the reaction can be carried out in a wide temperature range between 0 and 70, preferably between 5 and 50 and particularly preferably between 10 and 40 ° C.
- the substrate to be hydroxylated can be dissolved or suspended in an organic or aqueous solvent; in the case of liquid substrates, the addition of solvents can also be dispensed with under certain circumstances.
- DMSO / water mixtures in which DMSO is 1 -10% v / v are particularly suitable.
- the hydroxylation reaction can be carried out batchwise or continuously.
- the reaction is carried out continuously, in addition to the substrate to be hydroxylated, formations of the reaction are also fed continuously and the hydroxylated product and the CO 2 formed are continuously removed from the reaction.
- the immobilized enzyme forms a cloudy mixture. Therefore, direct kinetic experiments to determine the activity of the sol-gel-embedded CYP BM-3 were not possible.
- the activity of the sol-gel-embedded CYP BM-3 was therefore determined by incubating old components of the standard pNCA test for a certain period of time. and subsequent centrifugation in order to separate the yellow reaction product from the solid catalyst. The absorption at 410 nm was then determined from the supernatant.
- the enzyme immobilized according to the invention has remarkably high stability even at 25 ° C. At this temperature the half-life is determined to be 29 days.
- Another selective hydroxylation reaction was carried out with the sol-gel-embedded CYP BM-3 on the substrate n-octane. 79% of the starting material was hydroxylated.
- the regioisomers 2-octanol, 3-octanol and 4-octanol were obtained in a molar ratio of 1: 2.1: 1.6 (detected by gas chromatography).
- Co-immobilized enzymes from the first experimental series were placed in the p-NCA test with a ten-fold excess of p-NCA over oxidized NADP + .
- the activity of the separately immobilized FDH in combination with separately immobilized CYP was considerably higher than that of the co-immobilized enzymes.
- the co-immobilized enzymes showed a conversion of up to 28% of the pNCA conversion, while the mixture of separately immobilized enzymes led to a conversion of up to 75%.
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- Organic Chemistry (AREA)
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- Wood Science & Technology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
- Catalysts (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE502004002309T DE502004002309D1 (de) | 2003-05-09 | 2004-05-05 | Verfahren zur herstellung eines hydroxylierungskatalysators und seine verwendung |
JP2006505372A JP5252805B2 (ja) | 2003-05-09 | 2004-05-05 | ヒドロキシ化触媒を製造する方法およびその使用 |
US10/555,719 US7364884B2 (en) | 2003-05-09 | 2004-05-05 | Method for producing a hydroxylation catalyst and the use thereof |
EP04739133A EP1625215B1 (de) | 2003-05-09 | 2004-05-05 | Verfahren zur herstellung eines hydroxylierungskatalysators und seine verwendung |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10321082.2 | 2003-05-09 | ||
DE10321082A DE10321082A1 (de) | 2003-05-09 | 2003-05-09 | Verfahren zur Herstellung eines Hydroxylierungskatalysators und seine Verwendung |
Publications (1)
Publication Number | Publication Date |
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WO2004099398A1 true WO2004099398A1 (de) | 2004-11-18 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2004/004748 WO2004099398A1 (de) | 2003-05-09 | 2004-05-05 | Verfahren zur herstellung eines hydroxylierungskatalysators und seine verwendung |
Country Status (7)
Country | Link |
---|---|
US (1) | US7364884B2 (de) |
EP (1) | EP1625215B1 (de) |
JP (1) | JP5252805B2 (de) |
AT (1) | ATE348150T1 (de) |
DE (2) | DE10321082A1 (de) |
ES (1) | ES2278323T3 (de) |
WO (1) | WO2004099398A1 (de) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013076258A3 (en) * | 2011-11-23 | 2013-08-29 | Dsm Ip Assets B.V. | P450 bm3 mutants and their use for regio- and stereoselective hydroxylation of alpha-and beta-ionone |
WO2021168228A1 (en) * | 2020-02-21 | 2021-08-26 | Dow Silicones Corporation | Method of preparing silanols with selective cytochrome p450 variants and related compounds and compositions |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011055715A1 (ja) * | 2009-11-05 | 2011-05-12 | 国立大学法人神戸大学 | ヒドロキシ脂肪酸類縁体およびそれらの製造方法 |
GB2506880A (en) * | 2012-10-10 | 2014-04-16 | Michael David Fothergill | Method of enzyme conversion using an immobilised composition consisting of at least two enzymes and a co-factor |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
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DE19818665A1 (de) * | 1998-04-27 | 1999-10-28 | Basf Ag | Verfahren zur biotechnologischen Oxidation von substituierten Trimethylcyclohexenylverbindungen |
CA2328614C (en) * | 1999-02-12 | 2012-06-26 | Biostream, Inc. | Matrices for drug delivery and methods for making and using the same |
MY126592A (en) * | 1999-07-27 | 2006-10-31 | Basf Ag | Novel cytochrome p450 monooxygenases and their use for the oxidation of organic compounds |
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2003
- 2003-05-09 DE DE10321082A patent/DE10321082A1/de not_active Withdrawn
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2004
- 2004-05-05 DE DE502004002309T patent/DE502004002309D1/de not_active Expired - Lifetime
- 2004-05-05 AT AT04739133T patent/ATE348150T1/de not_active IP Right Cessation
- 2004-05-05 ES ES04739133T patent/ES2278323T3/es not_active Expired - Lifetime
- 2004-05-05 US US10/555,719 patent/US7364884B2/en not_active Expired - Fee Related
- 2004-05-05 EP EP04739133A patent/EP1625215B1/de not_active Expired - Lifetime
- 2004-05-05 WO PCT/EP2004/004748 patent/WO2004099398A1/de active IP Right Grant
- 2004-05-05 JP JP2006505372A patent/JP5252805B2/ja not_active Expired - Fee Related
Non-Patent Citations (5)
Title |
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GILL IQBAL ECKERT HELLMUT (AV.-PROP.) ET AL, CHEMISTRY OF MATERIALS, , 13(10), 3404-3421, 261 REFS. ISSN: 0897-4756, 2001, XP002291608 * |
IWUOHA, EMMANUEL I. ET AL: "Reactivities of organic phase biosensors 3: electrochemical study of cytochrome P450cam immobilized in a methyltriethoxysilane sol-gel", ELECTROANALYSIS , 12(12), 980-986 CODEN: ELANEU; ISSN: 1040-0397, 2000, XP009034944 * |
MAURER S C ET AL.: "Immobilisation of P450 BM-3 and an NADP+ Cofactor Recycling System: Towards a Technical Application of Heme-Containing Monooxygenases in Fine Chemical Synthesis", ADVANCED SYNTHESIS AND CATALYSIS, vol. 345, no. 6-7, June 2003 (2003-06-01), pages 802 - 810, XP002291609 * |
MILES^A C S ET AL: "Protein engineering of cytochromes P-450", BIOCHIMICA ET BIOPHYSICA ACTA. PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, ELSEVIER, AMSTERDAM,, NL, vol. 1543, no. 2, 29 December 2000 (2000-12-29), pages 383 - 407, XP004279114, ISSN: 0167-4838 * |
URLACHER V B ET AL: "Microbial P450 enzymes in biotechnology", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 64, no. 3, April 2004 (2004-04-01), pages 317 - 325, XP002291610, ISSN: 0175-7598 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013076258A3 (en) * | 2011-11-23 | 2013-08-29 | Dsm Ip Assets B.V. | P450 bm3 mutants and their use for regio- and stereoselective hydroxylation of alpha-and beta-ionone |
WO2021168228A1 (en) * | 2020-02-21 | 2021-08-26 | Dow Silicones Corporation | Method of preparing silanols with selective cytochrome p450 variants and related compounds and compositions |
Also Published As
Publication number | Publication date |
---|---|
ES2278323T3 (es) | 2007-08-01 |
DE502004002309D1 (de) | 2007-01-25 |
US7364884B2 (en) | 2008-04-29 |
JP2006525799A (ja) | 2006-11-16 |
JP5252805B2 (ja) | 2013-07-31 |
DE10321082A1 (de) | 2004-11-25 |
EP1625215B1 (de) | 2006-12-13 |
EP1625215A1 (de) | 2006-02-15 |
US20060216802A1 (en) | 2006-09-28 |
ATE348150T1 (de) | 2007-01-15 |
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