Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.10 No.4, Issue of October 15, 2007 © 2007 by Pontificia Universidad Católica de Valparaíso -- Chile Received November 3, 2006 / Accepted April 19, 2007 DOI: 10.2225/vol10-issue4-fulltext-9 RESEARCH ARTICLE Electrogeneration of hydrogen peroxide applied to the peroxide-mediated oxidation of (R)-limonene in organic media Camilo Enrique La Rotta Hernández* Electrochemistry and Electroanalysis Laboratory Department of Inorganic Chemistry Institute of Chemistry Federal University of Rio de Janeiro CT, Bloco A Sala 634-A, Ilha do Fundão CEP 21949-900, Rio de Janeiro, RJ, Brazil Tel: 55 021 25627149 E-mail: [email protected] Diogo Simon Werberich Electrochemistry and Electroanalysis Laboratory Department of Inorganic Chemistry Institute of Chemistry Federal University of Rio de Janeiro CT, Bloco A Sala 634-A, Ilha do Fundão CEP 21949-900, Rio de Janeiro, RJ, Brazil Tel: 55 021 25627149 E-mail: [email protected] Marcio Contrucci Saraiva de Mattos Department of Organic Chemistry Institute of Chemistry Federal University of Rio de Janeiro CT, Bloco A Sala 613, Ilha do Fundão CEP 21949-900, Rio de Janeiro, RJ, Brazil Tel: 55 021 25627129 E-mail: [email protected] Eliane D'Elia Electrochemistry and Electroanalysis Laboratory Department of Inorganic Chemistry Institute of Chemistry Federal University of Rio de Janeiro CT, Bloco A Sala 634-A, Ilha do Fundão CEP 21949-900, Rio de Janeiro, RJ, Brazil Tel: 55 021 25627813 E-mail: [email protected] Financial support: We acknowledge the financial support of a FAPERJ research grant for Dr. C.E. La Rotta post-doctoral studies. Keywords: Armoracia rusticana, bioelectrochemistry, carveol, carvone, horse-radish peroxidase, hydrogen peroxide electrogeneration, (R)-limonene. Abbreviations: 2,4-DCP: 2,4-dichlorophenol 4-AAP: 4-aminoantipyrine AE: auxiliary electrode CO: chemical oxidation CPO: chloroperoxidase CW: copper web DA: direct addition EG: electrogeneration EG AC: anodic direct oxidation bioelectrochemical processes EG CC: bioelectrooxidation at the cathodic chamber FID: flame ionization detector GC: gas chromatography HRP: horse radish peroxidase RVCF: reticulated vitreous carbon foam SCE: saturated calomel electrode Vap: apparent volume WE: working electrode *Corresponding author This paper is available on line at http://www.ejbiotechnology.info/content/vol10/issue4/full/9/ La Rotta, C.E. et al. Horse radish peroxidase (HRP) from Armoracia carvone, carveol and perillyl alcohol, used in fine chemistry rusticana catalyses the oxidation of (R)-limonene into (Lerner, 2003). the oxidized derivatives carveol and carvone. This study compares the direct addition (DA) of hydrogen peroxide Although carvone can be obtained by extraction and with its continuous electrogeneration (EG) during the purification from the essential oils of caraway, dill and enzymatic oxidation of (R)-limonene. Reaction mixtures spearmint seeds, it can also be produced by chemical and containing HRP, (R)-limonene as substrate, and biotechnological synthesis. A few papers concerning the hydrogen peroxide, added directly or electrogenerated, chemical synthesis of carvone were published after 1990. in 100 mM sodium-potassium phosphate buffer pH 7.0, Applications of carvone as fragrance and flavour, potato at 25ºC were studied. Two electrochemical systems for sprouting inhibitor, antimicrobial agent, building block and the hydrogen peroxide electrogeneration were biochemical environmental indicator, along with its evaluated, both containing as auxiliary electrode (AE) a relevance in the medical field, justify the interest in this platinum wire and saturated calomel electrode (SCE) as monoterpene. reference. Reticulated vitreous carbon foam (RVCF) and an electrolytic copper web (CW) were evaluated as Chemical oxidation (CO) of limonene in sodium citrate- working electrodes (WE). Results were compared in aqueous buffers, with and without low density terms of hydrogen peroxide electrogeneration, (R)- polyethylene, was reported by Kutty et al. 1994. Limonene limonene residual concentration or conversion and oxidation end-products included (4S)-(+)-carvone, carveol, product selectivity. Best results in terms of maximum limonene oxide, perilaldehyde, linalool and the hydrolysis product, α-terpineol (Figure 1). Wacker oxidation of H2O2 concentration (1.2 mM) were obtained using the limonene, using PdCl /CuCl /O , carried out by Silva et al. CW electrode at -620 mVSCE, and continuous aeration. 2 2 2 Use of the EG system under optimized conditions, which (2002), also resulted in the formation of cis-carvyl acetate, included the use of acetone (30% v/v) as a cosolvent in a trans-carvyl acetate, trans-carveol, carvone and α-terpinyl 3 hrs enzymatic reaction, lead to a 45% conversion of acetate. Oxidation of limonene in t-butanol using (R)-limonene into carveol and carvone (2:1). In PdCl2/CuCl2/t-butyl hydroperoxide was also done, and comparison to the results obtained with DA, the use of higher yields of oxidation products obtained in the presence EG also improved the half-life of the enzyme. of added chloride ions. On the other hand, during the CO of limonene with dioxygen in acetic acid solutions containing The success of a new aroma or flavoring additive depends catalytic amounts of Pd(OAc)2, benzoquinone and on a partnership between consumers and the industry to get M(OAc)2 (M = Cu, Co or Mn) reported by Gonçalves and an adequate product to the requirements of the market. The Gusevskaya (2004) three isomeric allylic acetates, as such volume of additives grows daily in the Western European, trans-2-acetoxy-p-mentha-1(7),8-diene, perilla acetate and American and Japanese markets and in the Latin American, trans-carveyl acetate were obtained. Limonene oxidation Asian and Eastern European emerging markets. Among over V2O5/TiO2 catalysts yielded polymers, limonene oxide these, Brazil, China, India and Mexico are the most and limonene glycol as the main reaction products, as well attractive. The flavor industry spends approximately 250 as carveol and carvone obtained in small amounts (Oliveira million dollar per year in research and development. The et al. 2006). functional food area, which moved 10 billion dollar up-to 2005, offers opportunities in this industry. Krings and Berger (1998) demonstrated the advantages of biotechnological flavors and fragrances, among which are: Essential oils from fruits are an abundant source of volatile (i) the ability to label microbiologically produced terpenes, substances which are widely distributed in nature. compounds “natural product”, making them attractive to The terpenes derived from essential oils are little increasingly health- and nutrition-conscious consumers; (ii) functionalized and present chains of C10 (fraction of low the potential of using industrially designed biochemical boiling point) and C15 (fraction of high boiling). The pathways for up-regulating metabolism; (iii) the possibility orange juice industry, that moves around 2 billion dollar of producing chiral compounds responsible for odour annually, has its market dominated by Brazil and the United perception; (iv) independence from agricultural and local States. The essential oil from orange is one of the main by- conditions and (v) the usually straightforward product products coming from orange juice processing, and is recovery. Furthermore, biocatalysis allows the production employed by the food, pharmaceutical, cosmetics and of enantiomericaly pure compounds, the modification of a cleaning products industries. molecule at chemically inert carbons and the selective modification of a specific functional group in (R)-limonene is the main constituent of orange and other multifunctional molecules. The disadvantages are related to citrus fruits skin oils (92 to 96%), and is produced in common low water solubility, chemical instability, amounts of up to 50,000 tons per year. Due to its low price, cytotoxicity and high volatility of terpenes. Some works which may vary from US$ 0.66 to 1.45 per kg, this have already been published describing successful compound is an attractive starting point for the production production of carvone with whole cells or purified enzymes of economically more interesting derivatives such as (Carvalho and Fonseca, 2006). 522 Electrogeneration of hydrogen peroxide applied to the peroxide-mediated oxidation of (R)-limonene in organic media Biotransformation of limonene to carvone were previously Peroxidase activity determination carried out using immobilized plant cells (Vanek et al. 1999) marine microalgae (Wise et al. 2002), fungi as For the determination of peroxidase activity, 2,4- Pleurotus ostratus (Onken and Berger, 1999), bacteria like dichlorophenol (2,4-DCP) was used as substrate which is Rhodococcus globerulus and Rhodococcus opacus (Duetz oxidized by the enzyme in the presence of hydrogen et al. 2001). peroxide and 4-aminoantipyrine (4-AAP) to produce colored products. Absorbance increase is proportional to Although, the enzymatic oxidation of (R)-limonene has the red derivative concentration, and it was followed at 510 been successfully achieved using whole microbial cells and nm (ε = 7100 M-1 cm-1). One unit of peroxidase activity was pure enzymes as mentioned above, the use of peroxidases defined as the amount (µmoles) of red oxidized derivative has not been widely explored. On the other hand, the use of formed per min at pH 7.0 and 25ºC (Metelitza et al. 1991). peroxidases in biocatalysis was also limited by the use of hydrogen peroxide