Enzymatic Synthesis of Vanillin

Heuvel, Robert H. H. van den; Fraaije, Marco W.; Laane, C.; Berkel, Willem J.H. van

doi:10.1021/jf010093j

Cited by 89 publications

(65 citation statements)

References 24 publications

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“…This spectrum, indicative of the flavin N-5-creosol adduct (21), was stable for a few minutes, after which it changed to a spectrum with an emission maximum at 425 nm (Fig. 3).…”

Section: Directed Evolution Of Vanillyl-alcohol Oxidasementioning

confidence: 96%

“…does not limit catalysis (21). Table V summarizes the data of the reductive half-reaction of wild-type VAO and its mutants with creosol at pH 7.5 and 10.…”

Section: Directed Evolution Of Vanillyl-alcohol Oxidasementioning

confidence: 99%

“…Thus, at basic pH values, the reduction rate does not limit catalysis for wild-type VAO, F454Y, and T505S. FAD Substrate Adduct in VAO Mutants-Enzyme-monitored turnover experiments and fluorescence spectroscopy have indicated that the FAD cofactor of wild-type VAO forms covalent flavin N-5 adducts with both p-cresol and creosol and that these adducts become more stable at higher pH (21,27). Because the evolved VAO mutants, especially at basic pH values, converted creosol to vanillin much better than the wild-type enzyme, it was of interest to investigate adduct formation with the mutant enzymes with these techniques.…”

Section: Directed Evolution Of Vanillyl-alcohol Oxidasementioning

confidence: 99%

“…Therefore, because of the increasing interest in natural products, alternative processes are being developed to produce natural vanillin (15, 18 -20). One possible approach involves the use of enzymes such as VAO (21). This flavoprotein is able to produce vanillin from creosol (see Fig.…”

mentioning

confidence: 99%

“…The two enzymes catalyze similar reactions, but p-cresol (4-methylphenol), the physiological substrate of PCMH (30), is an extremely poor substrate for VAO (27). Kinetic studies have shown that the low activity of VAO with p-cresol and also with creosol is due to the formation of an air-stable flavin N-5 substrate adduct (21,27,31). From the available crystallographic data, it is not obvious which structural features are involved in determining the different reactivity of both enzymes to 4-methylphenols.…”

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confidence: 99%

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Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (k cat /K m ) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.The increased use of enzymes and other proteins in the pharmaceutical, chemical, and agricultural industry has generated considerable interest in the design of proteins with new or improved properties. Two different but complementary technologies have been applied to this goal: (i) rational design, which relies on the availability of the three-dimensional structure and knowledge about the relationship between sequence, structure, and mechanism, and (ii) directed evolution methods, which use random mutagenesis of the gene encoding the protein or recombination of gene fragments to create diversity and then experimental screening of the libraries generated for the desired properties. Rational design has been used to elucidate and change enzyme mechanism, substrate and product specificity, enantioselectivity, cofactor specificity, and protein stability (1-3). Directed evolution has been applied to increase catalytic activity; to invert or improve enantioselectivity; and to alter substrate and product specificity, protein stability, pH optimum, and tolerance against organic solvents (1, 4 -7). An obvious advantage of directed evolution methods over sitedirected mutagenesis is that enzymes can be tailored for the production of (intermediate) products without detailed knowledge of protein structure and structure-function relationships.In this study, we engineered the flavoenzyme vanillyl-alcohol oxidase (VAO) 1 by random mutagenesis such that the evolved mutants are capable of producing natural vanillin (4-hydroxy-3-methoxybenzaldehyde) from the precursor creosol (2-methoxy...

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“…This spectrum, indicative of the flavin N-5-creosol adduct (21), was stable for a few minutes, after which it changed to a spectrum with an emission maximum at 425 nm (Fig. 3).…”

Section: Directed Evolution Of Vanillyl-alcohol Oxidasementioning

confidence: 96%

“…does not limit catalysis (21). Table V summarizes the data of the reductive half-reaction of wild-type VAO and its mutants with creosol at pH 7.5 and 10.…”

Section: Directed Evolution Of Vanillyl-alcohol Oxidasementioning

confidence: 99%

Section: Directed Evolution Of Vanillyl-alcohol Oxidasementioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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Sustainable Development and Chemistry

Cavani

Centi

2011

Kirk-Othmer Encyclopedia of Chemical Technology

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The review covers the new trends in the design of industrial chemical processes that can reduce the impact on environment and at the same time be sustainable from the economic point of view. Significant attention is also given to the opportunities given from new technologies, catalysts and materials, and process control to develop sustainable industrial processes. Aspects that are covered include the use of alternative or renewable raw materials (and biomasses), the reducing of the risks and number of steps in chemical processes, the reduction of energy consumption (and the use of alternative energy sources in chemical processes), the design of intrinsically safe processes, the down‐sizing of the chemical production and the opportunities offered from micro‐reactor and integrated reaction/separation technologies, the possibilities deriving from process intensification, and the reduction of waste by introducing new catalytic routes and/or solvents. Emphasis is given to the role of catalysis to develop new sustainable industrial processes, with also an overview of the new opportunities given from advanced catalytic materials and technologies.

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Flavoenzyme-Catalyzed Oxygenations and Oxidations of Phenolic Compounds

Moonen

Fraaije

Rietjens

et al. 2002

Advanced Synthesis & Catalysis

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Flavin‐dependent monooxygenases and oxidases play an important role in the mineralization of phenolic compounds. Because of their exquisite regioselectivity and stereoselectivity, these enzymes are of interest for the biocatalytic production of fine chemicals and food ingredients. In our group, we have characterized several flavoenzymes that act on phenolic compounds, including 4‐hydroxybenzoate 3‐hydroxylase, 3‐hydroxyphenylacetate 6‐hydroxylase, 4‐hydroxybenzoate 1‐hydroxylase (decarboxylating), hydroquinone hydroxylase, 2‐hydroxybiphenyl 3‐monooxygenase, phenol hydroxylase, 4‐hydroxyacetophenone monooxygenase and vanillyl‐alcohol oxidase. The catalytic properties of these enzymes are reviewed here, together with insights obtained from site‐directed and random mutagenesis.

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Enzymatic Synthesis of Vanillin

Cited by 89 publications

References 24 publications

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Sustainable Development and Chemistry

Flavoenzyme-Catalyzed Oxygenations and Oxidations of Phenolic Compounds

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