Biotransformation of 1,8-cineole, the main product of Eucalyptus oils
Financial support: CSIC, PDT-DINACYT, PEDECIBA and OPCW.
Keywords: Biocatalysis, Monoterpenes, Eucalyptus, Green Chemistry.
The forest industry
development of the forest industry in
species account for 74% of the forested area, with Eucalyptus
globulus being the main cultivar showing an increase of
leaves are rich in essential oils (FAO, 1995). These
oils have a recognized allelopathic action (Romagni et
al. 2000a; Romagni et al. 2000b)
for this reason its extraction before returning the leaves to the
soil would be an ecologically advisable practice. Eucalyptus essential
oil, that is commercialized at a price of U$S
main component of Eucalyptus essential oil in most species
is 1,8-cineole, representing about 70% of the total oil by gas chromatography
Production of these derivatives implies the stereo specific introduction of molecular oxygen in not activated carbon atoms, which continues to be a challenge in organic synthesis (Liu and Rosazza, 1990; Roberts et al. 2002). The use of microorganism that carries out this type of reactions constitutes an interesting alternative. Microbial hydroxylations have advantages over classic organic synthesis procedures since they are carried out in soft conditions, they use biodegradable reagents and they are generally stereo selective resulting in the production of an optically pure synthon (Faber, 1995).
In addition, the products obtained by this type of methodology can be labelled and commercialized as GREEN products.
In this work the results on the biotransformation of 1,8-cineole using a native Rhodococcus sp. strain isolated from soil obtained from beneath Eucalyptus sp. trees are presented.
native Rhodococcus sp. strain was isolated by our laboratory
from soil obtained from beneath Eucalytus sp. trees. This strain
is maintained in our microbial collection at
Biotransformations of 1,8-cineole were carried out by fermentation under different conditions.
methodology for biotransformation: Rhodococcus sp. strain was
plated in LB and incubated for 5 days at
The cells in the culture media were sonicated for 5 min and extracted with an equal volume of dichloromethane. The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure.
The obtained products were analyzed by TLC, GC, GC-MS, GC-GC.
chromatography was carried out in a HEWLLET PACKARD 5890 serie II
equipped with a FID detector and a CARBOWAX capillary column (
optical purity was determined in a GC-GC Shimadzu GC 17A. The first
GC is equipped with a SE52 column and the second one with a modified
β-ciclodextrin chiral capillary column. Temperature program:
1,8-cineole was provided by the Center of Agroindustrial Technology of Cochabamba, Bolivia.
Limonene Oxide: Aldrich, 97%.
During the initial screening three biotransformation products were detected and identified as 2-endo-hydroxy-1,8-cineole, 2-exo-hydroxy-1,8-cineole and 2-oxo-1,8-cineole (Figure 1).
Chiral GC analysis indicated that all these compounds are optically pure.
In order to optimize biotransformation parameters to obtain the best percent conversion we analyzed two variables. First, the influence of the inoculum’s size was analyzed and it was found that the best conversion is achieved for the larger inoculum’s size, being 2-endo-hydroxy-1,8-cineole the major product obtained (Figure 2).
We also analyzed the effect of the reaction time on the biotransformation yield and product profile. These studies were conducted using the larger inoculum’s size from the previous assay, and following the production of biotransformation products at different reaction times. The results indicate that the better yield is obtained at 24 hrs, with 2-endo-hydroxy-1,8-cineole accounting for 56% of the total product (Figure 3).
Table 1 summarizes the best results obtained with the Rhodococcus sp. strain isolated by our group as well as previous reports with other strains of bacteria, including a Rhodococcus sp. strain. The Rhodococcus sp. strain isolated by our group presents a major percent conversion that the strain described before; although it produces a third metabolite. An important feature of the biocatalyst described in this paper is the stereo selectivity, since only one enantiomer of each product is obtained.
A Rhodococcus sp. strain capable of metabolizing 1,8-cineole was isolated from soil beneath Eucalyptus sp. Three compounds were obtained from the biotransformation of 1,8-cineole with this strain and they were identified as 2-endo-hydroxy-1,8-cineole, 2-exo-hydroxy-1,8-cineole and 2-oxo-1,8-cineole. Chiral GC analysis indicated that these three compounds were optically pure. The biotransformation conditions were optimized to reach 98% bioconversion with this strain, what represents a better percent conversion than those previously reported for the biotransformation of 1,8-cineole with other bacterial strains. Despite this advantage, our strain shows low stereo selectivity since both the 2-endo- and the 2-exo-hydroxy-1,8-cineole are produced and conditions should be optimized to achieve only one stereoisomer.
The authors wish to thank Dr. Carmen Rossini and Dr. Andrés González for technical support. They also want to thank Bach. Daniel Lorenzo and Dr. Eduardo Dellacasa for chiral GC analysis.
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