토양에서 분리한 박테리아에 의한 Pinenes의 분해과정에 대한 연구

유선균, 조성용, 김도만, 한명희, 김태영, 최창남, 김병훈, 박병건, 김승재

Bioconversion pathway of pinenes by soil isolated bacetria

S.K.Yoo1, S.Y.Cho1, D.M.Kim1, C.N.Choi, B.H.Kim1,

B.G.Park2, H.M. Han, T.Y.Kim, S.J.Kim1

1 Engineering Research Institute, Chonnam National University

2 Dept. of Environmental and Health

Introduction

Pinenes (□and □ ) are bicyclic monoterpenes hydrocarbons. □-Pinene is the principal constituent of turpentine from most conifers and is a component of the wood and leaf oils of a wide variety of other plant [1].   □-Pinene is isomeric with a-pinene and accompanies the latter in most sources of the hydrocarbon [2]. The main components of turpentine derived from the pulp industry □- pinene (70%) and □-pinene (25%) [2]. Pinenes (□and □) are major components of commercial turpentine of great value as precursors for making flavor and fragrances. It is known that many Pseudomonas such as Pseudomonas PX1, Pseudomonas fluorescens NCIMB 11671, Pseudomonas pseudomallei, and Pseudomonas putida could carry out bioconversion of either □- or □- pinene although most of these could not  grow at high concentration of pinenes.  In previous study [3], we reported that a new strain that Pseudomonas sp. strain PIN isolated from terpene soaked soil could convert either □- or □-pinene to several bicyclic and p-menthene monoterpenes at relatively high concentration of □-and □-pinene (10%). In this study we report bioconversion of □- and □-pinene and related monoterpenes by resting cells of Pseudomonas sp. strain PIN and consider possible bioconversion pathway.

Materials and Methods

Terpenes, □-pinene, □-pinene, p-cymene, □-terpinolene, limonene, □-terpinene,  terpinene, verbenol, □-terpineol, endo-borneol, fenchyl alcohol, camphor, camphene, iso-borneol, mytenol, and carveol were obtained from Aldrich Chemical Company (Milwaukee, WI, USA).  Prior to the use of these compounds as sole carbon and energy source for bacterial growth, the oxidized impurities in terpenes were removed by passing through a silica gel column.  Pseudomonas sp. strain PIN was maintained on sterile S-2 mineral medium [4]. The medium was adjusted to pH 5.5 prior to sterilization.  A working culture was prepared by growing bacteria in 50 ml mineral medium containing 1% (v/v) of either □-pinene or □-pinene.  The culture was aerobically incubated in a model 3528 Environ-Shaker at 30oC and 200 rpm in the dark condition. One ml of working culture was aseptically transferred to a culture tube (25 x 150 mm) containing 10 ml mineral medium and a monoterpene as sole carbon and energy sources.  Tubes were shaken at 200 rpm and 30oC for 48 h.  Working cultures grown on either □-pinene or □-pinene was transferred into a 250 ml culture flask containing 45 ml of pH 5.5 mineral medium plus 5 ml of □- pinene.  Twenty-five ml lots of 24 h old culture were transferred into 6-litre flasks containing 225 ml of sterile pH 5.5 mineral medium plus 2 ml of pinenes.  Flasks were shaken at 200 rpm and 30oC for 48 h.  Culture broth (O.D.=1.0) was evenly dispensed into 6 x 250 ml sterilized centrifuge bottles.  Cells were harvested by centrifuging the culture broth for 20 min at 15,000 x g in a refrigerated (4□C) centrifuge.  After aseptically removing the supernatant from each bottle, cell pellets were washed twice with about 30 ml of sterile pH 5.5 and 50 mM of phosphate buffer solution, and then cell pellets were pooled and re-centrifuged.  The pooled cell pellet was washed with about 100 m1 of buffer solution.  Finally, the supernatant was decanted and the combined weight of the bottle plus cell pellet was measured.  The cell pellet was resuspended in buffer to give a concentration of ca. 1 g cells (wet basis) per 10 g buffer solution.  Transformation treatments were prepared using test tubes (25 x 150 mm) containing 5 ml of cell suspension and 50 m1 of pinenes.  All test tubes were extracted with 2 ml of diethyl ether containing tetradecane as an internal standard.  The ether layer was dried over anhydrous sodium sulphate and concentrated under a stream of nitrogen to about 1 ml.  Concentrated extracts were transferred into 2 ml amber vials equipped with teflon-lined caps and stored at- 20C until analysis. Five ml aliquots of inoculum from 24 h old -pinene grown cells were transferred into 250 ml side arm culture flasks containing 45 ml of mineral medium.  Each flask contained one of the following substrates at the indicated concentration: 1% (v/v) □-pinene, □-pinene, p-cymene, or limonene; 0.5% (v/v) □-terpineol, □-terpinolene or fenchyl alcohol; 0.5% (w/v) camphor or borneol.  Flasks were shaken at 200 rpm under optimal growth conditions. The above procedure was repeated for □-pinene grown cells.

The identification of monoterpenes metabolites were obtained by the previous reported method [3].  Two l of each extract was injected into a fused silica open tubular column (Supelcowax 10; 0.25 mm i.d. x 60 m length x 0.25 m df; Supelco, Inc., Bellefonte, USA) installed in a Hewlett-Packard (Palo Alto, CA, USA) 5790A gas chromatograph (GC).  Helium with a linear velocity of 25 cm/s was used as GC carrier gas.  The injector temperature was set at 200C. Column oven temperature programmed as follows: 40C for 10 min, and then a rise of 6C/min to 200C where it was held for 20 min.  The GC was coupled to an HP 5970B mass selective detector (MSD, Hewlett-Packard Co., USA) operated in the electron ionization (EI) mode with the ion source temperature at 200C, ionization energy at 70 eV, mass range at m/z 33-300, electron multiplier voltage at 1800 V, and scan rate at 1.6 scans/sec.  The MS interface was set at 200C. The amount of each product (analyte) was based on the ratio of its peak area to that of the internal standard (tetradecane).

Results and Discussion

The growth and adaptation study for individual metabolites was undertaken to gain information on the relative utilization of metabolites using growing cells either adapted to □-pinene or □-pinene as sole carbon and energy source.  The identified bioconversion products are listed in Table 1. Data are not presented in this article, pinenes, □-terpinolene, and p-cymene supported good growth of □-pinene adapted cells.  Limonene supported consistent growth after a relatively long lag period (12 h).  In this regard, the pathway of degradation of □-pinene may be mainly carried out via p-menthene derivatives after opening of the bicyclic ring structure.  □-Terpinolene, p-cymene, and □-pinene also supported good growth of □-pinene adapted cells.  Limonene supported consistent growth after a long lag phase.  No growth was found in cultures added with □-terpineol and borneol.  These growth patterns were essentially the same as those observed for □-pinene adapted cells.  From the above data it is possible to rationalize the pathways for degradation of □- and □-pinene with main energy yielding sequence of reactions, involving p-menthene derivatives.  Bicyclic metabolites, endo-borneol and camphor were derived from □-pinene and endo-borneol and fenchyl alcohol from □-pinene.  From the results of growth studies, it is clear that these compounds do not play a role in the energy yielding cycle or otherwise do not serve as carbon sources.  Data are listed in Table 2 showing the growth of Pseudomonas sp. strain PIN on different structure of monoterpenes.  The reaction sites of terpenoid substrates, which are highly susceptible to chemical reactions are also easily attacked by corresponding enzyme systems.  In this regard, the first chemical or enzyme attack may occur at the double bond of either pinene.  Chemical attack of electrophilic reagents on pinenes proceeds preferentially from the less hindered side of the molecule to produce initially a cation, and then this cation migrates to a position on the cyclohexane ring [5].  This cation presumably migrates to form a borneol cation through the Wagner-Meerwein prototrophic rearrangement.  Alternatively, this cation also can undergo the above reaction after reaction with the applied nucleophile, i.e. the fenchyl derivative. In the present study, it is reasonable to propose that camphor may have resulted from the oxidation of borneol, while fenchyl alcohol may have formed from cleavage of the cyclobutane ring of the pinyl cation.  However, the occurrence of these compounds must be studied further to determine whether these reactions are formed chemically or biologically.  While no degradation of camphor, borneol, and fenchyl alcohol by Pseudomonas cepacia was observed in spite of adaptation studies, several researchers have reported degradation of some of these compounds. To explain the formation of monocyclic p-menthene derivatives from □- and □-pinene, it is necessary to first postulate the cleavage of the cyclobuthane ring of □- or □-pinene.  In view of this point, Shukla and Bhattacharyya [6] suggested the existence of a monocyclic cation, which is the probable precursor of all the p-menthene and p-menthenoid compounds.  This postulation may be valid on the basis of chemical rationale.  In the present study, monocyclic p-menthene derivatives were p-cymene, limonene, and □-terpinolene as well as oxygenated monocyclic p-menthenoid derivatives □-terpineol, □-terpinene-4-ol, and p-cymene-8-ol.  As limonene supported good growth of this strain, it is possible to postulate further degradation to acidic materials, which are liable to attack by □-oxidation.  Actually, the result of bioconversion of limonene as a sole carbon source gave rise to a large amount of perillic acid.  Pseudomonas gladioli is known to convert limonene to perillic acid [7].  In the present study conversion metabolites of -terpinolene was not clearly observed although previous researchers have reported further degradation of □-terpinolene.  It was postulated that the hydration of □-terpinolene resulted in □-terpinene -4-ol by addition of a hydroxyl group to double bond at the 4-carbon position.  In this study, a large accumulation of p-cymene allowed for a feasible reaction scheme different than previous related studies.  Further study of Psedumonas strain sp. PIN using p-cymene as a sole substrate and energy source showed a great deal of cumic acid accumulation.  Pseudomonas putida and other soil isolated bacteria shows similar metabolism pattern.  The hydroxylation of p-cymene might result in p-cymene-8-ol.  Hydroxylation of "unactivated" centers in hydrocarbons can be potentially the most useful of all bioconversions.  For example, 1,4-cineole gives 8-hydroxy cineole as the major product on incubation with Streptomyces griseus.  It was presumed that the hydroxylation of monocyclic cation or addition of a hydroxyl group to limonene resulted in □-terpineol accumulation.  Other strains, Penicillium digitatum NRRL 1202 (Tan series) and Pseudomonas gladioli are known to produce -terpineol from limonene.  From the above results, postulated pathway of □- and □-pinene conversion to p-menthene derivatives based on our own results and other observation is shown in below figure.

Figure Pathway proposed for the bioconversion of pinenes to bicyclic terpenes and to monocyclic terpenes by Pseudomonas sp.

1.  □-Pinene

2.  □-Pinene

3.  Fenchyl alcohol

4.  Borneol

5.  Camphor

6.  □-Terpinolene

7.  □-Terpinen-4-ol

8.  P-Cymene

9.  P-Cymene-4-ol

10. Cumic acid

11. □-Terpineol

12. Perillic acid

13. Limonene

Acknowledgement

This research was supported by a Korea Research Foundation Grant (KRF-YOO-290).

References

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