WO1997016553A1 - MUTANT MONO-OXYGENASE CYTOCHROME P450cam - Google Patents
MUTANT MONO-OXYGENASE CYTOCHROME P450cam Download PDFInfo
- Publication number
- WO1997016553A1 WO1997016553A1 PCT/GB1996/002693 GB9602693W WO9716553A1 WO 1997016553 A1 WO1997016553 A1 WO 1997016553A1 GB 9602693 W GB9602693 W GB 9602693W WO 9716553 A1 WO9716553 A1 WO 9716553A1
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- WIPO (PCT)
- Prior art keywords
- mutant
- 450cam
- amino acid
- residue
- cysteine
- Prior art date
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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 mutant of the mono-oxygenase cytochrome P-450cam.
- Mono-oxygenases catalyse the selective oxidation of activated and unactivated carbon-hydrogen bonds using oxygen 1 , and are therefore of great interest for potential use in organic synthesis.
- progress in this area has been hampered by the difficulty in isolating sufficient quantities of the mono- oxygenase enzyme and/or the associated electron-transfer proteins.
- amino acid sequences of more than 150 different cytochrome P-450 mono-oxygenases to date structural date of only three are available 234 , and few have been successfully over-expressed in bacterial systems .
- cytochrome P-450 mono-oxygenase which is soluble and can be expressed in sufficient quantities, is the highly specific P-450cam from P. putida which catalyses the regio- and stereo- selective hydroxylation of camphor to 5-exo-hydroxycamphor 6 .
- the high resolution crystal structure of P-450cam has been determined 2 , and since the mechanism of action of this bacterial enzyme is believed to be very similar to that of its mammalian counterparts, it has been used as a framework on which structural models of mammalian enzymes are based.
- the nucleotide sequence and corresponding amino acid sequence of P-450cam have been described 5,7 .
- the location of an active site of the enzyme is known and structure-function relationships have been investigated 8,9 .
- Mutants of P-450cam have been described at the 101 and 185 and 247 and 295 positions 910 "- and at the 87 position 12 .
- a mutant in which tyrosine 96 (Y96) has been changed to phenylalanine 96 (the Y96F mutant) has been described 1113 ' 413 .
- the papers report effects of the mutations on the oxidation reactions of molecules which had previously been shown to be substrates for the wild-type enzyme. There is no teaching of how mutations might be used to provide biocatalysts for oxidation of different, novel substrates.
- the three dimensional structure of P-450cam shows the active site to provide close van der aals contacts with the hydrophobic groups of camphor as shown in Figure 1. Of particular significance are the contacts between camphor and the side chains of leucine 244, valine 247 and valine 295. Three aromatic residues (Y96, F87 and F98) are grouped together and line the substrate binding pocket, with a hydrogen bond between tyrosine 96 and the camphor carbonyl oxygen maintaining the substrate in the correct orientation to ensure the regio- and stereo- specificity of the reaction.
- the mutant F98A appeared to have the strongest binding interaction within the active site cavity accessible to the aromatic probe, with that of Y96A being slightly smaller, and that of F87A being substantially less. It was decided in the first instance to mutate tyrosine 96 to alanine as it is more central to the binding pocket, whereas phenylalanine 98 is in a groove to one side. Also, removal of tyrosine 96 should decrease the specificity of the enzyme towards camphor due to the loss of hydrogen bonding to the substrate.
- a mutant of the mono-oxygenase cytochrome p-450cam is provided in which the cysteine residue at position 334 is removed.
- the removal is by the substitution of another amino acid except cysteine for the cysteine residue.
- the removal is by the deletion of the entire cysteine 344 residue from the enzyme.
- the tyrosine residue at position 96 in the mutant is replaced by the residue of any amino acid except tyrosine.
- the amino acid is selected from any one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine and valine except that in the case of the cysteine residue at position 334, the amino acid is not cysteine and in the case of the tyrosine residue at position 96 the amino acid is not tyrosine.
- amino acid residue at one or more of the positions 87, 98, 101, 185, 193, 244, 247, 295, 297, 395 and 396 is replaced by another amino acid residue.
- the C334A mutation has the obvious benefit of removing unwanted protein dimerisation, thus ensuring the presence of a single species in solution at all times.
- wild-type P-450cam shows aggregation upon standing. The reasons why proteins aggregate are not clear, but the P-450cam aggregates are insoluble and catalytically inactive.
- the wild-type and C58A, C85A, C136A and C148A mutants all showed dimerisation as well as aggregation upon storage at 4"C, and even in 50% glycerol solutions at -20°C. Aggregation will also occur during turnover, especially at the higher P-450cam concentrations required in any economically viable industrial application in, for example, synthesis of organic molecules.
- the C334A mutant did not show any evidence of aggregation even at mM concentrations at room temperature over a period of three days. Thus, the C334A mutation has beneficial effects in protein handling, storage, and increased catalyst lifetime.
- amino acid at one or more of these positions may be replaced by: a small hydrophobic amino acid so as to enlarge the active site,- or a large hydrophobic amino acid so as to reduce the size of the active site,- or by an amino acid having an aromatic ring to interact with a corresponding aromatic ring of a substrate.
- the enzyme system typically includes putidaredoxin and putidaredoxin reductase together with NADH as co-factors in addition to the mutant enzyme.
- the example of cyclohexylbenzene oxidation is described in the experimental section below.
- Various classes of organic compounds are envisaged and described below.
- the wild-type P-450cam is active towards the oxidation of a number of molecules included in the following sections. However, in all cases the mutant P-450cam proteins show much higher turnover activities.
- the organic compound is an aromatic compound, either a hydrocarbon or a compound used under conditions in which it does not inactivate or denature the enzyme. Since the mutation has been effected with a view to creating an aromatic-binding pocket in the active site of the enzyme, the mutant enzyme is capable of catalysing the oxidation of a wide variety of aromatic compounds. Oxidation of example aromatic and polyaromatic compounds is demonstrated in the experimental section below and is believed very surprising given that the wild-type enzyme has been reported to catalyse the oxidation of only members of the camphor family and shows low activity towards a few other molecules such as styrene 19 , ethylbenzene 910 , a tetralone derivative 20 , and nicotine 21 .
- the organic compound may be a hydrocarbon, e.g. aliphatic or alicyclic, carrying a functional group (see Scheme l) .
- An aromatic protecting group is attached to the functional group prior to the oxidation reaction and removed from the functional group after the oxidation reaction.
- a suitable aromatic group is a benzyl group.
- the protecting group serves two purposes: firstly it makes the substrate more hydrophobic and hence increases binding to the hydrophobic enzyme pocket; secondly it may help to hold the substrate in place at the active site. Thus, with the correct aromatic protection group, both regio- and stereo-selective hydroxylation of the substrate may be achieved.
- Examples of monofunctionalised hydrocarbons are cyclohexyl, cyclopentyl and alkyl derivatives (Scheme 1) .
- the oxidation products of these compounds are valuable starting materials for organic synthesis, particularly when produced in a homochiral form.
- a range of aromatic protecting groups are envisaged, e.g. benzyl or naphthyl ethers and benzoyl ethers and amides (Scheme 1) .
- Of interest are also benzoxazole groups as carboxyl protecting groups and N- benzyl oxazolidine groups as aldehyde protecting groups. Both can be easily cleaved after the enzymatic oxidation and have previously been described in the literature for the microbial oxidations of aldehydes and acids 22 .
- the organic compound is a C4 to C12 aliphatic or alicyclic hydrocarbon. Oxidation of cyclohexane and linear and branched hydrocarbons is demonstrated in the experimental section below. We have found that wild-type P-450cam is also capable of oxidising these molecules, but the activities are low and in all cases the mutants show substantially higher activities.
- the organic compound is a halogenated aliphatic or alicyclic hydrocarbon. Oxidation of lindane (hexachlorocyclohexane) is also describe below.
- Mutants were constructed in which active site substitutions were combined with the surface mutation of cysteine at position 334 to alanine and contained alanine, leucine, valine, or phenylalanine instead of tyrosine at position 96 (Y96) . Lastly several active site mutations and the surface mutation were combined to constitute mutant enzymes with multiple mutations.
- the genes encoding cytochrome P-450cam, and its natural electron-transfer partners putidaredoxin and putidaredoxin reductase, were amplified from the total cellular DNA of P. Putida using the polymerise chain reaction (PCR) .
- PCR polymerise chain reaction
- coli host combinations employed were pRH1091 23 in strain JM109 for P-450cam, pUC 118 in strain JM109 for putidaredoxin, and pGL Wll in strain DH5 for putidaredoxin reductase.
- Oligonucleotide-directed site-specific mutagenesis was carried out using an M13 mp 19 subclone by the method of Zoller and Smith 24 , and mutant selection was by the method of Kunkel 25 .
- Binding of potential substrates was investigated by spectroscopic methods.
- the wild-type enzyme in the absence of substrate is in the 6-co-ordinated, low-spin form with a weakly bound water occupying the sixth co-ordination site, and shows a characteristic Soret maximum at 418 n .
- Binding of camphor and the substrate analogues adamantanone, adamantane and norbornane fully converted the haem to the 5-co-ordinated, high-spin form which has a characteristic Soret band at 392 nm.
- This haem spin-state shift is accompanied by an increase in the haem reduction potential which enables the physiological electron- transfer partner putidaredoxin to reduce P-450cam and initiate the catalytic hydroxylation cycle 26 .
- the haem spin state shift is thus a qualitative indication of the likelihood of molecules shown in Tables 1 and 2 being oxidised by the wild- ype and mutant P-450cam enzymes.
- a buffered solution (50 mM Tris.HCI, pH 7.4), typically 3ml in volume, containing lOuM putidaredoxin, 2 uM putidaredoxin reductase, 1 uM cytochrome P-450cam mono-oxygenase (wild-type or mutant) , 200 mM KCI, 50 ug/ml bovine liver catalase (Sigma) , and 1 mM target organic compound such as cyclohexylbenzene (added as a 0.1 M stock in ethanol) was preincubated at 30"C for 5 minutes. The enzymatic reaction was initiated by adding NADH to a total 11 concentration of 2 mM.
- the chloroform extracts are evaporated to dryness under a stream of nitrogen.
- the residues were extracted with hexane and the oxidation products separated by high performance liquid chromatography, eluting with a hexane/isopropanol gradient.
- the purified products were then identified by mass spectroscopy and particularly nuclear magnetic resonance spectroscopy.
- the amount of substrate added to the incubation mixtures varies from 0.2 mM to 4 mM final concentration.
- the NADH concentration can be monitored at 340 nm and, in all cases, more substrates and NADH are added during the incubation.
- the results for C334A and C334A-Y96A are set out in Table 1 and 2, in which structurally related molecules are grouped together.
- Table 1 details the NADH consumption for oxidation of small linear, branched and cyclic hydrocarbons by the mutant Y96A- C334A.
- Tables 2(a) to 2(h) details the product distributions for mutant and substrate combinations where these have been elucidated to date.
- the cysteine residue at position 344 can be deleted by any well known and freely available standard restriction techniques and will therefore not be described in detail herein.
- Phenanthrene Products (%) for mutants: Products WT Y96A Y96F Y96L Y96V F87A-F96G
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP9517168A JP2000508163A (en) | 1995-11-01 | 1996-11-01 | Monooxygenase cytochrome P450cam mutant |
EP96935162A EP0906431A1 (en) | 1995-11-01 | 1996-11-01 | MUTANT MONO-OXYGENASE CYTOCHROME P450cam |
PL96326445A PL326445A1 (en) | 1995-11-01 | 1996-11-01 | Mutant of cytochrome p450cam mono-oxygenase |
NZ320497A NZ320497A (en) | 1995-11-01 | 1996-11-01 | Mutant mono-oxygenase cytochrome p450cam |
AU73236/96A AU716583B2 (en) | 1995-11-01 | 1996-11-01 | Mutant mono-oxygenase cytochome P450cam |
SK555-98A SK55598A3 (en) | 1995-11-01 | 1996-11-01 | Mutant mono-oxygenase cytochrome p450cam |
US09/068,132 US6117661A (en) | 1995-11-01 | 1996-11-01 | Mutant mono-oxygenase cytochrome P450cam |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9522407.7 | 1995-11-01 | ||
GB9522407A GB2294692B (en) | 1994-11-03 | 1995-11-01 | Enzyme mutant and method |
AUPCT/GB95/02588 | 1995-11-02 | ||
PCT/GB1995/002588 WO1996014419A1 (en) | 1994-11-03 | 1995-11-02 | MUTANT MONO-OXYGENASE CYTOCHROME P-450¿cam? |
Publications (1)
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WO1997016553A1 true WO1997016553A1 (en) | 1997-05-09 |
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PCT/GB1996/002693 WO1997016553A1 (en) | 1995-11-01 | 1996-11-01 | MUTANT MONO-OXYGENASE CYTOCHROME P450cam |
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JP (1) | JP2000508163A (en) |
CN (1) | CN1212015A (en) |
CA (1) | CA2236381A1 (en) |
GB (1) | GB2306485B (en) |
SK (1) | SK55598A3 (en) |
WO (1) | WO1997016553A1 (en) |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000078973A1 (en) * | 1999-06-18 | 2000-12-28 | Isis Innovation Limited | Process for oxidising aromatic compounds |
US6902918B1 (en) * | 1998-05-21 | 2005-06-07 | California Institute Of Technology | Oxygenase enzymes and screening method |
US7211420B1 (en) | 1998-11-19 | 2007-05-01 | Isis Innovation Limited | Process for oxidizing terpenes |
US7435570B2 (en) | 2003-08-11 | 2008-10-14 | California Institute Of Technology | Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use |
US7465567B2 (en) | 2001-04-16 | 2008-12-16 | California Institute Of Technology | Peroxide-driven cytochrome P450 oxygenase variants |
US7524664B2 (en) | 2003-06-17 | 2009-04-28 | California Institute Of Technology | Regio- and enantioselective alkane hydroxylation with modified cytochrome P450 |
US7691616B2 (en) | 2001-07-20 | 2010-04-06 | California Institute Of Technology | Cytochrome P450 oxygenases |
US8026085B2 (en) | 2006-08-04 | 2011-09-27 | California Institute Of Technology | Methods and systems for selective fluorination of organic molecules |
US8252559B2 (en) | 2006-08-04 | 2012-08-28 | The California Institute Of Technology | Methods and systems for selective fluorination of organic molecules |
US8715988B2 (en) | 2005-03-28 | 2014-05-06 | California Institute Of Technology | Alkane oxidation by modified hydroxylases |
US9133443B2 (en) | 2007-10-08 | 2015-09-15 | Isis Innovation Limited | Mutant enzymes |
US9322007B2 (en) | 2011-07-22 | 2016-04-26 | The California Institute Of Technology | Stable fungal Cel6 enzyme variants |
US9737425B2 (en) | 2005-07-07 | 2017-08-22 | Nellix, Inc. | System and methods for endovascular aneurysm treatment |
US11214817B2 (en) | 2005-03-28 | 2022-01-04 | California Institute Of Technology | Alkane oxidation by modified hydroxylases |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MY126592A (en) | 1999-07-27 | 2006-10-31 | Basf Ag | Novel cytochrome p450 monooxygenases and their use for the oxidation of organic compounds |
EP3319987B1 (en) * | 2015-07-07 | 2021-05-05 | Codexis, Inc. | Novel p450-bm3 variants with improved activity |
Citations (3)
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WO1995016041A1 (en) * | 1993-12-08 | 1995-06-15 | Ciba-Geigy Ag | Cytochrome p-450 monooxygenases |
WO1995034679A2 (en) * | 1994-06-16 | 1995-12-21 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Defects in drug metabolism |
WO1996014419A1 (en) * | 1994-11-03 | 1996-05-17 | British Gas Plc | MUTANT MONO-OXYGENASE CYTOCHROME P-450¿cam? |
-
1996
- 1996-11-01 JP JP9517168A patent/JP2000508163A/en active Pending
- 1996-11-01 CA CA002236381A patent/CA2236381A1/en not_active Abandoned
- 1996-11-01 SK SK555-98A patent/SK55598A3/en unknown
- 1996-11-01 WO PCT/GB1996/002693 patent/WO1997016553A1/en not_active Application Discontinuation
- 1996-11-01 CN CN96199231A patent/CN1212015A/en active Pending
- 1996-11-01 GB GB9622819A patent/GB2306485B/en not_active Expired - Fee Related
Patent Citations (3)
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WO1995016041A1 (en) * | 1993-12-08 | 1995-06-15 | Ciba-Geigy Ag | Cytochrome p-450 monooxygenases |
WO1995034679A2 (en) * | 1994-06-16 | 1995-12-21 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Defects in drug metabolism |
WO1996014419A1 (en) * | 1994-11-03 | 1996-05-17 | British Gas Plc | MUTANT MONO-OXYGENASE CYTOCHROME P-450¿cam? |
Non-Patent Citations (6)
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C. DI PRIMO ET AL.: "Mutagenesis of a single hydrogen bond in cytochrome p450 alters cation binding and heme solvation.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 265, no. 10, 5 April 1990 (1990-04-05), pages 5361 - 5363, XP002025959 * |
C.A.D. SMITH ET AL: "Debrisoquine hydoxylase gene polymorphism and susceptibility to Parkinson's disease.", THE LANCET, vol. 339, no. 8806, 6 June 1992 (1992-06-06), pages 1375 - 1377, XP000565682 * |
DAWSON E ET AL: "An association study of debrisoquine hydroxylase (CYP2D6) polymorphisms in schizophrenia.", PSYCHIATRIC GENETICS 4 (4). 1994. 215-218. ISSN: 0955-8829, XP000565684 * |
S. F. TUCK ET AL.: "Active sites of the cytochrome p450cam {CYP101} F87W and F87A mutants.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 1, 5 January 1993 (1993-01-05), pages 269 - 275, XP002025961 * |
W. M. ATKINS ET AL.: "Tyrosine-96 as a natural spectroscopic probe of the cytrochrome P-450cam active site", BIOCHEMISTRY, vol. 29, no. 5, 6 February 1990 (1990-02-06), pages 1271 - 1275, XP002025960 * |
W.M. ATKINS ET AL.: "The role of active site hydrogen bonding in cytochrome P-450 cam as revealed by site-directed mutagenesis.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 263, no. 35, 15 December 1988 (1988-12-15), pages 18842 - 18849, XP002025958 * |
Cited By (30)
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US6902918B1 (en) * | 1998-05-21 | 2005-06-07 | California Institute Of Technology | Oxygenase enzymes and screening method |
US7211420B1 (en) | 1998-11-19 | 2007-05-01 | Isis Innovation Limited | Process for oxidizing terpenes |
US6794168B1 (en) | 1999-06-18 | 2004-09-21 | Isis Innovation Limited | Process for oxidising aromatic compounds |
WO2000078973A1 (en) * | 1999-06-18 | 2000-12-28 | Isis Innovation Limited | Process for oxidising aromatic compounds |
US7704715B2 (en) | 2001-04-16 | 2010-04-27 | California Institute Of Technology | Peroxide-driven cytochrome P450 oxygenase variants |
US7465567B2 (en) | 2001-04-16 | 2008-12-16 | California Institute Of Technology | Peroxide-driven cytochrome P450 oxygenase variants |
US8367386B2 (en) | 2001-07-20 | 2013-02-05 | California Institute Of Technology | Cytochrome P450 oxygenases |
US7691616B2 (en) | 2001-07-20 | 2010-04-06 | California Institute Of Technology | Cytochrome P450 oxygenases |
US8076114B2 (en) | 2001-07-20 | 2011-12-13 | California Institute Of Technology | Cytochrome P450 oxygenases |
US9322001B2 (en) | 2001-07-20 | 2016-04-26 | California Institute Of Technology | Cytochrome P450 oxygenases |
US8722371B2 (en) | 2001-07-20 | 2014-05-13 | California Institute Of Technology | Cytochrome P450 oxygenases |
US8741616B2 (en) | 2003-06-17 | 2014-06-03 | California Institute Of Technology | Regio- and enantioselective alkane hydroxylation with modified cytochrome P450 |
US7524664B2 (en) | 2003-06-17 | 2009-04-28 | California Institute Of Technology | Regio- and enantioselective alkane hydroxylation with modified cytochrome P450 |
US7863030B2 (en) | 2003-06-17 | 2011-01-04 | The California Institute Of Technology | Regio- and enantioselective alkane hydroxylation with modified cytochrome P450 |
US8343744B2 (en) | 2003-06-17 | 2013-01-01 | The California Institute Of Technology | Regio- and enantioselective alkane hydroxylation with modified cytochrome P450 |
US9145549B2 (en) | 2003-06-17 | 2015-09-29 | The California Institute Of Technology | Regio- and enantioselective alkane hydroxylation with modified cytochrome P450 |
US7435570B2 (en) | 2003-08-11 | 2008-10-14 | California Institute Of Technology | Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use |
US9404096B2 (en) | 2005-03-28 | 2016-08-02 | California Institute Of Technology | Alkane oxidation by modified hydroxylases |
US8715988B2 (en) | 2005-03-28 | 2014-05-06 | California Institute Of Technology | Alkane oxidation by modified hydroxylases |
US9963720B2 (en) | 2005-03-28 | 2018-05-08 | California Institute Of Technology | Alkane oxidation by modified hydroxylases |
US10648006B2 (en) | 2005-03-28 | 2020-05-12 | California Institute Of Technology | Alkane oxidation by modified hydroxylases |
US11214817B2 (en) | 2005-03-28 | 2022-01-04 | California Institute Of Technology | Alkane oxidation by modified hydroxylases |
US9737425B2 (en) | 2005-07-07 | 2017-08-22 | Nellix, Inc. | System and methods for endovascular aneurysm treatment |
US8252559B2 (en) | 2006-08-04 | 2012-08-28 | The California Institute Of Technology | Methods and systems for selective fluorination of organic molecules |
US8026085B2 (en) | 2006-08-04 | 2011-09-27 | California Institute Of Technology | Methods and systems for selective fluorination of organic molecules |
US9133443B2 (en) | 2007-10-08 | 2015-09-15 | Isis Innovation Limited | Mutant enzymes |
US9834759B2 (en) | 2007-10-08 | 2017-12-05 | Oxford University Innovation Limited | Mutant enzymes |
US10501727B2 (en) | 2007-10-08 | 2019-12-10 | Isis Innovation Limited | Mutant enzymes |
US11155790B2 (en) | 2007-10-08 | 2021-10-26 | Oxford University Innovation Limited | Mutant enzymes |
US9322007B2 (en) | 2011-07-22 | 2016-04-26 | The California Institute Of Technology | Stable fungal Cel6 enzyme variants |
Also Published As
Publication number | Publication date |
---|---|
GB2306485A8 (en) | 1997-05-19 |
GB2306485B (en) | 1998-12-09 |
CN1212015A (en) | 1999-03-24 |
GB9622819D0 (en) | 1997-01-08 |
JP2000508163A (en) | 2000-07-04 |
GB2306485A (en) | 1997-05-07 |
CA2236381A1 (en) | 1997-05-09 |
SK55598A3 (en) | 1999-04-13 |
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