Coupling of Biocatalytic Asymmetric Epoxidation with NADH Regeneration in Organic–Aqueous Emulsions

Hofstetter, Karin; Lutz, Jochen; Lang, Irene; Witholt, Bernard; Schmid, Andreas

doi:10.1002/anie.200353338

Cited by 92 publications

(69 citation statements)

References 44 publications

(11 reference statements)

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“…[54] While volumetric productivities reported here are quite low (maximum 0.15 g l -1 h -1 ), higher values found in literature (up to 1 g l -1 h -1 ) [8] are usually calculated for reaction times of 10 h or less compared to up to 100 h in our P450-mediated approach.Switching the cofactor specificity of CYP102A1 to NADH allows to reduce the cofactor costs to about 20%. Further investigations concerning the stability and general applicability of the NAD + -dependent reaction system are required.…”

Section: Discussioncontrasting

confidence: 51%

“…The total turnover numbers for the biocatalyst exceed the value of up to 2867 reported for the asymmetric epoxidation of styrene by styrene monooxygenase (StyAB) in a two-phase dodecane / aqueous buffer system. [8] In this case for the cofactor NAD + a maximum turnover number of 87 was detrmined. Lutz et al investigated the preparative application of 2-hydroxybiphenyl 3-monooxygenase with enzymatic cofactor regeneration.…”

Section: Discussionmentioning

confidence: 95%

“…The cofactor recycling system is based on a mutant of FDH from Pseudomonas sp which has been proven as highly stable in organic solvents. [8,41] The stability of this dehydrogenase in a 1:1 mixture of FDH reaction buffer and cyclohexane under vigorous stirring was tested. FDH activity assays revealed a halflife of at least one day for this cofactor regenerating enzyme in the two-phase system.…”

Section: Biphasic Reaction System Cyclohexane / Aqueous Buffermentioning

confidence: 99%

“…Considering these problems, the use of isolated oxygenases in enzyme reactors could be advantageous. [6][7][8][9] Regarding the application of isolated P450 monooxygenases in fine chemical synthesis, enzymes from bacterial sources turned out as more suitable than those originating from plants, fungi or vertebrates: besides a much higher activity compared to eucaryotic enzymes, bacterial P450s exhibit in many cases higher stability. [10] P450 enzymes consisting of a heme domain fused to an FAD-and FMN-containing P450 reductase domain are -in contrast to the majority of P450s -"self-sufficient", i.e., they do not have to be supplied with additional redox partners (reductases) apart from NADPH.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussioncontrasting

confidence: 51%

Section: Discussionmentioning

confidence: 95%

Section: Biphasic Reaction System Cyclohexane / Aqueous Buffermentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

et al. 2005

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“…Yet, only a few such processes have been scaled to production level, since biotransformation rates typically are rather unstable and activities during long-term biotransformations are often lower than maximal activities obtained in short-term assays with whole cells (6,10,48,54), cell extracts (13), and enriched enzyme preparations (26). The physiology of whole-cell redox biocatalysts during long-term biotransformations is largely unknown, although it is influenced by biocatalysis and may at least in part be responsible for limited biocatalyst activities and stabilities.…”

Section: Discussionmentioning

confidence: 99%

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Bühler

Park

Blank

et al. 2008

Appl Environ Microbiol

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Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 mol per min per g cells (dry weight), the absence of limitations by O 2 and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.Oxygenases catalyze regio-and enantioselective oxyfunctionalization reactions and have a considerable potential in the area of asymmetric organic synthesis (2, 35). These enzymes typically depend on coenzymes such as NAD(P)H and are unstable outside cells, and they often consist of multiple components. Thus, for synthetic applications, their use in whole cells is favored over the use of isolated enzymes (7,18).The productivity of oxygenase-based whole-cell biocatalysts is influenced by various factors, such as maximal enzyme activity, enzyme level, substrate mass transfer, substrate and/or product inhibition/toxicity, and cofactor regeneration (7,13,54,68). Enzyme synthesis and cofactor regeneration are related to host metabolism, which in turn may be affected by the toxicity of organic substrates and products. Such toxicity can efficiently be attenuated by regulated substrate feeding (1, 11) and in situ product removal (7,38,61,66,74). Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via...

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Formation of Ethers by Alkane, Arene, Alkene, and Alkyne Substitutions and Additions

Larock

Kesharwani

2018

Comprehensive Organic Transformations

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Coupling of Biocatalytic Asymmetric Epoxidation with NADH Regeneration in Organic–Aqueous Emulsions

Abstract: Cell‐free enzymatic oxidations: Styrene monooxygenase (StyA) was used as a reagent for the gram‐scale preparation of enantiopure epoxides. The catalyst is highly stable in a biphasic system and results in conversions of more than 88 %. R1=H, Cl; R2=R3=H, CH3.

Cited by 92 publications

References 44 publications

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Formation of Ethers by Alkane, Arene, Alkene, and Alkyne Substitutions and Additions

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