Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation

Farinas, Edgardo T.; Schwaneberg, Ulrich; Glieder, Anton; Arnold, Frances H.

doi:10.1002/1615-4169(200108)343:6/7<601::aid-adsc601>3.0.co;2-9

Cited by 162 publications

(72 citation statements)

References 2 publications

(3 reference statements)

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“…One of the variants contained a single point mutation of a Glu residue in the surface of the protein by a His residue. The effect of this mutation was surprising, given the fact that this residue is located relatively far from the haem group [139].…”

Section: Ec 113 and 114: Oxygenases And Monooxygenasesmentioning

confidence: 99%

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Vidal

Kelly

Mordaka

et al. 2018

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

235

147

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A B S T R A C T NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P) + . NAD(P)Hdependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.

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Section: Ec 113 and 114: Oxygenases And Monooxygenasesmentioning

confidence: 99%

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Vidal

Kelly

Mordaka

et al. 2018

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

235

147

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“…These and other properties, such as solvent tolerance, substrate versatility, and temperature spectrum, can now be improved remarkably in a targeted manner by enzyme engineering. [162] With modern genetic methods it is possible to produce several thousand mutants per day. To make it possible to screen such a large number of enzyme variants as quickly as possible, sophisticated screening systems must be developed.…”

Section: Enzyme-catalyzed Resolutionmentioning

confidence: 99%

Industrial Methods for the Production of Optically Active Intermediates

Breuer¹,

Ditrich²,

Habicher³

et al. 2004

Angew Chem Int Ed

1,318

625

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Enantiomerically pure amino acids, amino alcohols, amines, alcohols, and epoxides play an increasingly important role as intermediates in the pharmaceutical industry and agrochemistry, where both a high degree of purity and large quantities of the compounds are required. The chemical industry has primarily relied upon established chemical methods for the synthesis of these intermediates, but is now turning more and more to enzymatic and biotechnological fermentation processes. For the industrial implementation of many transformations alternative methods are available. The advantages of the individual methods will be discussed herein and exemplified by syntheses of relevant compounds.

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“…[13,14] Recently cloning and characterisation of two further fusion enzymes from Bacillus subtilis, which exhibit high homology to CYP102A1, was reported. [15][16][17] While a large number of publications describing novel evolved CYP102A1 mutants with altered substrate specificity exist, [18][19][20][21][22][23][24][25][26] there are only very few reports dealing with the use of these biocatalysts in preparative organic synthesis. [7,27] Beside strong doubts concerning the operational stability of the enzyme class, the high cost of the nicotinamide cofactor NADPH which has to be added in stochiometric or even higher amount (if uncoupling is taken into account) seems to make their in vitro application impractical.…”

Section: Introductionmentioning

confidence: 99%

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

et al. 2005

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Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP + -dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD + -dependent FDH was used.Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.

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Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation

Cited by 162 publications

References 2 publications

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Industrial Methods for the Production of Optically Active Intermediates

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

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