Microbial Conversion of p-lonone by Immobilized Aspergillus niger in the Presence of an Organic Solvent

Koji Sode and lsao Karube*' Research Center for Advanced Science and Technology, University of Tokyo, 4-6- 7 Komaba, Meguro-ku, Tokyo, 153 Japan

Reiko Araki Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227 Japan

Yoichi Mikarni Central Research Institute, Japan Tobacco lnc., 6-2 Umegaoka, Midori-ku, Yokohama 227, Japan

Accepted for publication June 28, 1988

Aspergillus niger JTS191 was capable of the conversion of p-ionone to a mixture of its derivatives that is utilized to an essential oil of tobacco. The authors attempted this microbial conversion in the presence of an organic solvent to improve its reaction rate. The addition of isooctane accelerated the microbial conversion of p- ionone. It took three days to complete the reaction whereas without isooctane it took more than six days. The addition of isooctane also improved the resistance of A. niger to the antifungal property of p-ionone. A. niger pellets were immobilized in hydrophobic polymer, PU-3, and applied for the microbial conversion of p- ionone. Further improvement of the resistance to the antifungal property of p-ionone was achieved by irnmo- bilization. PU-3 immobilized A. niger was repeatedly used for microbial conversion of p-ionone in the pres- ence of isooctane for more than 480 hours.

INTRODUCTION

Ionones and their derivatives are known to be important constituents of various essential oils. The authors have been studying the production of tobacco flavoring and fo- cused on the microbial conversion for this purpose,' since, microbial conversion normally creates this product with a naturally occurring structure. Mikami et al. have reported on the bioconversion of p-ionone by Aspergillus niger JTS 19 1 .2-5 They attempted the screening of microorgan- isms capable of ionone conversion, and found that A . niger JTS191 was capable of converting p-ionone to a mixture of its derivatives. These are ( R ) -4- hydroxy -p- ionone, (S) -2- hydroxy -p- ionone, 4- 0x0 -p- ionone, 2 - 0x0 - p - ionone, and the other five deriva- tives of p-ionone. This complex was found to be very ef-

* To whom all correspondence should be. addressed ' Isao Karube also affiliated with the Tokyo Institute of Technology

Biotechnology and Bioengineering, Vol. 33, Pp. 1191-1 195 (1989) 0 1989 John Wiley & Sons, Inc.

fective for tobacco flavoring at the pprn level. However, this microbial conversion took more than one week to complete the reaction. Furthermore, because of the anti- bacterial and antifungal properties of p-ionone much like other ionones, the substrate concentration should be less than 1.5 g L-'. Therefore, the improvement of these prop- erties is expected.

Bioconversion in the presence of an organic solvent is of great interest since, the application of organic solvent im- proved the solubility of the water immiscible substrate, and subsequently accelerated conversion velocity. In addi- tion, the biocatalysis normally exists in the water phase, therefore, the separation of the biocatalysis from the reac- tion mixture is relatively easy.

Regarding these advantages, many microbial conversions utilizing organic solvent have been achieved.6 Fukui et al. reported on the results of the several microbial conversions of steroids in the presence of an organic ~olvent .~- '~ It has been said that the polarity of the organic solvent was the major factor in the bioconversions using organic solvents. Takahashi and his group reported on the microbial con- version of alcohol in the presence of various organic sol- v e n t ~ . ' ~ , ' ~ They found that in the presence of isooctane, microbial conversion proceeded most efficiently.

Immobilization techniques were also attempted with rni- crobial conversion in the organic solvent. Fukui et al. de- veloped several polymers suitable for this They concluded that by optimizing the hydrophobicity of the polymer, both stability and reaction rate are improved.

The present study describes the improvement of the mi- crobial conversion of p-ionone using A . niger by adding several organic solvents. A . niger pellets were immobi- lized in a hydrophobic polymer and microbial conversion in the presence of an organic solvent was also attempted.

CCC 0006-3592/89/091191-05$04.00

MATERIALS AND METHODS

Chemicals

p-ionone was purchased from Takasago Koryo Inc. p- glucuronidase was obtained from Sigma Corp. (from Helix pomatia, type H-1). Urethane prepolymer (PU-3) was kindly provided by Dr. Atsuo Tanaka (Faculty of Engineering, Kyoto Univ., Kyoto, Japan). All other reagents were of analytical grade.

Microorganisms and Cultivation

Aspergillus niger JTS 191 was utilized in this study. Spores of A . niger were inoculated in the medium with the following composition, and cultivated using a rotary shaker at 28"C, 150 rpm; sucrose 30 g, NaNO, 2 g, K2HP0, 1 g, yeast extract 1 g, MgSO, 7H,O 0.5 g and KCl 0.5 g (in 1 L distilled water, pH 7.0). After 48 hours of incubation, pellets of 2-3 mm in diameter were formed. These pellets were utilized for the bioconversion.

Immobilization Procedure

A . niger pellets were immobilized as follows. 10 g of A . niger pellets were suspended in 40 mL of 0.05M, pH 5.0 succinate buffer containing 20 mg of p-glucuronidase. This suspension was incubated at 28°C for 15 hours, to de- composite the pellet formation. Treated pellets were then sonicated (20 kHz) for 5 min at O"C, and centrifuged (20,000 g, 30 min). 2.0 g of PU-3 was mixed with 4 mL water, and kept at 60°C. Then, 2 g of centrifuged treated pellets were resuspended in this polymer solution. This mixture was kept at 0°C for 1 hour to form the gel. The gel was cut with blade to form cubes with 2-3 mm edges.

p-lonone Conversion

5.5 g of A . niger pellets were suspended in 100 mL of the reaction medium with the following composition: su- crose 15 g, HNO, 1 g, K2HP04 1 g, yeast extract 1 g, MgSO, 7H20 0.2 g and KCl 0.2 g (in 1 L of distilled water, pH 4.0). The reaction was initiated by adding sev- eral amounts of p-ionone. The solution was incubated at 28"C, 150 rpm. Organic solvent was added with p-ionone addition.

The microbial conversion by immobilized A . niger was attempted by using gels immobilizing 2 g of pellets. Other conditions were the same as above. After a period of time was allowed for bath conversion, gels immobilizing A . niger were harvested by mesh, washed with 0.9% saline, and uti- lized for the next batch in a fresh medium with p-ionone.

Assay Procedure

Two mL of sample was extracted by adding 1 mL of ethylacetate. The extract was concentrated and analyzed by gas-chromatography (Shimazu Corp. GC 3BF), The

analytical parameters were as follows; detector: flame ionization detector, column: PEG 20M (S%)/Chromosorb W 3 mm x 2 m, carrier gas: Ar 30 mL min-', 210°C.

Since this microbial conversion resulted in the complex of the derivatives of p-ionone, the authors used the ratio (%) of formed ( R ) -4- hydroxy -p- ionone (4-OH- p-ionone) to the initial concentration of p-ionone, as the indicator of the extent of the conversion. Throughout this article, the extent of conversion was expressed this ratio (%).

RESULTS

The Effect of Organic Solvent Addition

Several organic solvents and surfactants were added to the medium, and the microbial conversion of p-ionone was attempted. The results are summarized in Table I. The ad- dition of Triton X-100 and SDS caused the inactivation, and no conversion was achieved. Methanol was often used to improve the reaction rate of the bioconversion of steroids. However, the p-ionone bioconversion was inhib- ited by methanol addition. Among the organic solvents tested, isooctane resulted in the improvement of the extent of conversion after 45 hours of incubation. The conversion extent in the presence of isooctane, was 3.3 times higher than the control. The other water immiscible organic sol- vent caused no significant improvement.

The structural correlation of organic solvents and their effect on microbial conversion was then examined. Obvi- ously, the organic solvent with a branched structure re- tained the most positive effect on the p-ionone conversion. In this experiment, addition of isooctane resulted in the highest conversion extent.

Table I. Effect of additional organic solvents and surfactants on the mi- crobial conversion of p-ionone. The extent of conversion indicated are the ratio (%) of (R)-4-hydrooxy-P-ionone (4-OH-P-ionone) to the initial concentration of p-ionone after 45 hours of incubation. DMSO; dimethyl- sulfoxide, SDS; sodium dodecyl sulfate

Solvent Extent of Relative conc. (%, v/v) conversion (%) activity (%) Structure

none methanol (1) DMSO (2) ethylmethylketone (2) Triton X-100 (1) SDS (1) n-hexane (2) n-heptane (2) n-octane (2)

isooctane (2) 2,2-dimethyl (2)

pentane

2.5-dimethyl (2) hexane

16 0

18 4 0 0 8

14 19

53 32

42

100 0

112 25 0 0

50 /W 88

119

331 200 XA

263

1192 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 33, APRIL 1989

Figure I shows the time course of the p-ionone micro- bial conversion in the presence of 2% (v/v) isooctane. Normally, in the batch using freely suspended pellets, it took more than six days to complete the conversion. Ap- parently, the addition of isooctane accelerated the reaction rate, and the conversion was finished within three days.

Figure 2 shows the effect of isooctane concentration on the extent of conversion. By increasing the concentration of isooctane up to 2%, the conversion extent after 45 hours of incubation was increased. Higher isooctane concentra- tion caused a decrease in the extent of conversion.

Figure 3 shows the effect of the p-ionone concentration on the conversion extent. Without isooctane in the reaction medium, p-ionone conversion was very sensitive to the added p-ionone concentration. A p-ionone concentration higher than 2.0 mg mL-' inhibited the microbial conver- sion. In the presence of isooctane, the microbial conversion of p-ionone was achieved even at a p-ionone concentration higher than 2.5 mg mL-'. As was previously mentioned, the addition of isooctane accelerated the conversion rate.

1.0 . 1 -

4

'- 0.75 E cn E -

0.5G .- u m L t. I=

0.25 cz 0 0

r I 1 . - I I

0 50 100 150 Time ( h )

Figure 1. Time course of p-ionone microbial conversion by A. niger in the medium with 1.0 mg rnI-Ip-ionone. The lines show the concentra- tion of each component. (-); in the presence of 2% isooctane, ( - - - ) ; without isooctane.

4 0

20

I I

t

I I I 1 I 1 0 1 . 0 2,o 3 . 0 4.0

I s o o c t a n e C o n c e n t r a t i o n ( % V / V )

Figure 2. Effect of isooctane concentration on the microbial conversion of p-ionone by A . niger pellets. The extent of conversion was measured after 45 hours of incubation in the medium with 1 .O mg mL-' p-ionone.

I

6-o 60 v

i. c a, u 2 40 c 0

v) L

.-

$ 20 L

0 0

0 1.0 2.0 p - I o n o n e c o n c e n t r a t i o n (ing rn1-l)

Figure 3. Effect of p-ionone concentration on the microbial conversion by A . niger pellets. The extent of conversion was measured after 45 hours of incubation in the medium. (--); in the presence of 2% (v/v) isooc- tane, (-A-); without isooctane.

The reported data after 45 hours of incubation showed that the conversion extent at each p-ionone concentration was higher than reaction without isooctane. These results sug- gested the addition of isooctane caused the activation of catalytic activity of A . niger, allowed the use of a higher substrate concentration.

p-lonone Conversion Utilizing Immobilized A. niger in the Presence of lsooctane

The authors examined the reusability of A. niger pellets for P-ionone microbial conversion, however, the activity gradually decreased with the number of operations. The application of immobilization techniques would improve the stability. Fukui et al. have reported on the microbial con- version of steriods in the presence of an organic They have developed several types of hydrophobic poly- mers suitable for the immobilization of microorganisms. It was, therefore, attempted to utilize one of these hydropho- bic polymer, PU-3, for the immobilization of A . niger. Then, the p-ionone conversion in the presence of isooctane by PU-3 immobilized A . niger was investigated.

Figure 4 presents the effect of isooctane concentration on the microbial conversion by PU-3 immobilized A . niger. It is noteworthy that, with the increase of isooctane concentration the extent of conversion after 45 hours of in- cubation also increased. As mentioned before regarding the results from Figure 2 , in the microbial conversion with freely suspended pellets, the increase in the isooctane con- centration inhibits the reaction rate. This phenomenon might be dependent upon the character of PU-3.

Figure 5 shows the effect of p-ionone concentration on the bioconversion by PU-3 immobilized A . niger in the presence of 2% of isooctane. At a higher substrate con- centration above 2.0 mg mL-' caused the decrease in the extent of conversion. Compared with the results from Fig- ure 3, however, the decrease in catalytic activity was not drastical. Therefore, by using PU-3, further improvements in the substrate concentration were achieved.

SODE ET AL.: MICROBIAL CONVERSION OF p-IONONE 1193

c -0 20

s 10 v) L

W > c

0 2.0 4.0 6 . 0 8,O

Isooctane concentration ( % V / V ) Figure 4. Effect of isooctane concentration on the microbial conversion of p-ionone by PU-3 immobilized A. niger pellets. The extent of conver- sion was measured after 60 hours of incubation in the medium with 1 .O mg mL-' p-ionone.

I -30 w - c, c a, +J a,

c 0

vl L

x 20

.-

p 10 l= 0 V

1 I I 1

0 1.0 2.0 3.0 4.0 p-Ionone concentration (mg m 1 - l )

Figure 5. Effect of p-ionone concentration on the microbial conversion by PU-3 immobilized A . niger. The extent of conversion was measured after 60 hours of incubation in the medium with 2% (v/v) isooctane.

The authors then examined the reusability of PU-3 im- mobilized A . niger, with several concentrations of isooc- tane and 0-ionone . Mycelia leakage from the immobilized gel was focused on because, leakage caused several prob- lems in the long term operation. The results are summa- rized in Table 11. At a p-ionone concentration of 1.0 mg mL-' (the extent of conversion is shown in Figure 4), mycelia leakage occurred so that these gels could not be utilized repeatedly. The batch with a p-ionone concentra- tions of 2.0 and 3.0 mg mL-' caused no significant myce- lia growth, so that these gels were repeatedly used. At a p-ionone concentration of 4.0 mg mL-', with 4% isooc- tane medium, no microbial conversion was achieved. A medium with 8% of isooctane, bioconversion was possible even at 4.0 mg mI-' of p-ionone.

The authors then examined the long term stability of PU-3 immobilized A. niger. The results are shown in Fig- ure 6. The conversion extents presented were after 60 hours

Table 11. Effect of isooctane and &ionone concentration on the conver- sion extent and reusability of PU-3 immobilized A . niger mycelia. The extent of conversion are expressed as the ratio (%) of 4-OH-@-ionone to the initial concentration of p-ionone after 45 hours of incubation. [-I; not tested

Concentration Conversion isooctane p-ionone extent (%) ("r, v/v) (mg d - 7 1st 2nd

2.0 4.0 8.0 4.0 8.0 4.0 8.0

2.0 2.0 2.0 3 .O 3.0 4.0 4.0

19 29 20 25 21 0

14

~

-

38 33 39 30

0 -

- 60 ho - Y c a, w

a c

x 40

0,

9 20 u, i

c 0 U

0

.... 0 0 .

0

I I 1 1 1 1 1 1

1 2 3 4 5 6 7 8

Batch number (run) Figure 6. Reusability of PU-3 immobilized A. niger pellets for the mi- crobial conversion of P-ionone. The extent of conversion was determined after 60 hours of incubation in the medium with 2.0 mg mL-] p-ionone and 2% (v/v) isooctane.

of incubation. Apparently, with the increase of batch num- ber up to 4, the extent of conversion rate also increased. Then this conversion extent was maintained for a further 240 hours of operations. This result suggests that PU-3 im- mobilized A . niger retained a high activity and its stability was maintained for the long term operation of more than 480 hours.

DISCUSSION This study described the improvement of p-ionone mi-

crobial conversion by adding isooctane. Obviously, the ad- dition of isooctane accelerated the reaction rate, and also increased the substrate concentration. The following two roles of isooctane addition in the p-ionone microbial con- version have been assumed: 1) the partition coefficient of isooctane toward P-ionone might change the concentration of p-ionone in the water phase, 2) interaction of isooctane toward the A . niger cell wall might change the permeabil- ity andfor reaction rate of p-ionone.

p-ionone partition coefficiency to heptane was similar to that of isooctane. However, the addition of heptane caused

1194 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 33, APRIL 1989

no significant change in the reaction rate (Table I). Fur- thermore, only the organic solvent with a branched struc- ture improved the microbial conversion. Therefore, it was assumed that the interaction of isooctane to the cell wall might effect the permeability and/or enzymatic reaction.

The immobilization of A . niger was then attempted using a hydrophobic polymer, PU-3, to improve the reusability of biocatalysis. PU-3 was developed by Fukui et al.7-’2 The microorganisms immobilized in this gel improved the catalytic activity in the bioconversion with the organic sol- vent. It was expected that the hydrophobicity of the gels would improve the diffusion of p-ionone in the gels. The conversion extent after 60 hours of incubation indicated, however, a decrease in the conversion extent. Figure 4 shows that the effect of immobilization was reflected on the improvement in the resistance to the isooctane concen- tration. The allowance to apply high isooctane concentra- tion for the conversion subsequently resulted in an increase in the maximum concentration of p-ionone utilized for the conversion. In other words, the advantages associated with the hydrophobicity of gels might contribute in only a high concentration of organic solvent.

The combination of isooctane addition and immobili- zation by PU-3 improved the conversion efficiency, with regards to the reaction rate and reusability. Further opti- mization in the processes will lead to the continuous mi- crobial conversion of p-ionone in the presence of isooctane.

References 1 . K . Sode, K. Kajiwara, E. Tamiya, I. Karube, Y. Mikami, N. Hori,

2. Y. Mikami, E. Watanabe, Y. Fukunaga, and T. Kisaki, Agric. Biol.

3. Y. Mikami, Y. Fukunaga, M. Arita, and T. Kisaki, Appl. Environ.

4. Y . Mikami, Y. Fukunaga, T. Hieda, Y. Obi, and T. Kisaki, Agric.

5 . Y. Mikami, Y. Fukunaga, M. Arita, Y. Obi, and T. Kisaki, Agric.

6. P. J. Hailing, Biotech. Adv., 5, 47 (1987). 7. S. Fukui, A. Tanaka, T. Iida, and E. Hasegawa, FEBS Lett., 66, 179

8. S. Fukushima, T. Nagai, K. Fujita, A. Tanaka, and S. Fukui, Bio-

9. T. Omata, T. Iida, A. Tanaka, and S. Fukui, Eur. J . Appl. Microbio.

and T. Yanagimoto, Biocatalysis, 1, 77 (1987).

Chem., 42, 1075 (1978).

Microbiol., 41, 610 (1981).

B i d . Chem., 45, 331 (1981).

B i d . Chem., 45, 791 (1981).

(1976).

technol. Bioeng., 20, 1465 (1978).

Biotechnol., 8, 143 (1979). 10. S. Fukui and A. Tanaka, Actu Biorechnol., 1, 339 (1981). 11. S. Fukui and A. Tanaka, Adv. Biochem. Eng.lBiorechnol., 29, 1

12. S. Fukui and A. Tanaka, Endeavour, 9, 10 (1985). 13. Y. Takazawa, S. Sato, and J. Takahashi, Agric. Biol. Chem., 48,

14. M. Ueda, S. Mukataka, S. Sato, and J. Takahashi, Agric. B i d . Chem.,

(1984).

2489 (1984).

50, 1533 (1986).

SODE ET AL.: MICROBIAL CONVERSION OF P-IONONE 1195