Comparative study of the bioconversion process using R-(+)- and S-(–)-limonene as substrates for Fusarium oxysporum 152B
Introduction
The methods for obtaining flavour compounds include direct extraction from nature, chemical transformations and biotechnological transformations, such as microbial and enzymatic biotransformations, de novo synthesis and the use of genetic engineering tools (Berger, 2007). The scientific literature contains many examples of reviews that address the chemical reactions of terpenes to produce flavours (Swift, 2004) and the biotransformation of volatile terpenes for aroma production (Bicas et al., 2009, De Carvalho and Da Fonseca, 2006).
Despite the great number of industrial applications of aroma compounds produced via chemical synthesis, which is still responsible for a large portion of the market due to satisfactory yields, bioprocesses possess a number of inherent advantages compared with the classical chemical processing because they occur under mild conditions, present high regio- and enantio-selectivity, do not generate toxic waste and result in products that may be labelled “natural” (Bicas et al., 2009). Additionally, biotechnological processes usually involve less damaging processing conditions for the environment and yield desirable enantiomeric flavour compounds. Thus, bioflavours appeal to many sectors and have a high market value (Krings & Berger, 1998).
The biocatalytic conversion of a structurally related precursor molecule (bioconversion or biotransformation processes), is often a more adequate strategy that allows the significantly enhanced accumulation of a desired flavour product. As a prerequisite for this strategy, the precursor must be present in nature, and it must be easily feasible to isolate it in sufficient amounts from the natural source in an economically viable fashion (e.g., the monoterpenes limonene and α-pinene). Among the most targeted substrates for biotransformation/bioconversion approaches are the monoterpenes (Krings & Berger, 1998).
Several reports in the literature have addressed the biotransformation of limonene to many oxygenated derivatives; these have been reviewed by Maróstica and Pastore (2007a), and Duetz, Bouwmeester, Van Beilen, and Witholt (2003). Research and development on the degradation of limonene has continued to draw much attention, due to a wide variety of conversion products, such as perillic compounds, carveol and carvone at significant amounts, which could be more valuable in the fields of cosmetics, food ingredients, drug and chemical synthesis.
One interesting product obtained from the biotransformation of limonene is α-terpineol. This product is a stable alcohol that is widely distributed in nature and is typically used in household products, cosmetics, pesticide and flavour preparations. Additionally, it is one of the most commonly used perfume chemicals (Bauer, Garbe, & Surburg, 2001). The production of this monoterpene alcohol has been described using a wide range of microorganisms as catalysts (Adams et al., 2003, Bicas et al., 2008a, Bicas et al., 2008b, Molina et al., 2013b, Molina et al., 2013a, Tan et al., 1998). Another product obtained from the bioconversion of the monoterpenic substrate is limonene-1,2-diol, which has been described as possessing a cool minty aroma and is consumed mainly in flavours used in mint preparation, alcoholic and nonalcoholic beverages, chewing gum, gelatins/puddings and other food products (Burdock & Fenaroli, 2010).
A few reports have studied the bioconversion of both enantiomers of limonene, R-(+)- and S-(−)-limonene, including those of Adams et al., 2003, Bicas et al., 2008b, Demyttenaere et al., 2001, which aimed to compare the results, metabolic pathways and characterise their metabolism.
Thus, the main objective of this work was to characterise the biotransformation of S-(–)-limonene into limonene-1,2-diol, studying the improvements to this process using cell permeabilisation under anaerobic conditions and with the adoption of a biphasic system. In addition, these results were compared with the literature recently produced on the same microorganism, using R-(+)-limonene as the substrate for the production of α-terpineol. Furthermore, this study aimed to characterise and compare the bioconversion of R-(+)- and S-(–)-limonene using ultra-structural analysis.
Section snippets
Microorganism and chemicals
The microbial strain employed in this study was identified as Fusarium oxysporum 152B (Prazeres, Cruz, & Pastore, 2006). The fungal strain was maintained on yeast malt (YM) agar (in g L–1: agar = 20; glucose = 10; peptone = 5; yeast extract = 3; malt extract = 3, pH ∼ 6.7) and stored at 4 °C.
The chemical standards used as substrates in this study were R-(+)-limonene (98% purity, Sigma–Aldrich) and S-(−)-limonene (96% purity, Sigma–Aldrich). The reagents, n-hexadecane (99% purity, Sigma–Aldrich) and
Comparison of bioconversion process using different isomers of limonene
In previous studies, Bicas, Barros et al. (2008) described the optimisation of the ten main process variables involved in the biotransformation of R-(+)-limonene to R-(+)-α-terpineol by F. oxysporum 152B through a Plackett–Burman matrix with 16 assays, including the effects of the medium composition, substrate concentration, cultivation conditions and inoculum size and then the central composite design methodology. Thus, the authors improved significantly the production of R-(+)-α-terpineol,
Conclusion
The results collected so far with F. oxysporum 152B encourage further studies on this biocatalyst to better characterise the involvement of its enzyme system in the bioconversion of R-(+)-limonene into α-terpineol and S-(–)-limonene into limonene-1,2-diol, particularly considering the concentration of the products here achieved, 4.0 and 3.7 g L–1, respectively. In this sense, the present study represents an extensive comparative investigation into the bioconversion processes leading to these
Acknowledgements
The authors acknowledge Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP – Proc. no 2011/50687-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Proc. no 481487/2011-5 and Proc. no 473981/2012-2) for the financial support.
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