Standard Deviation 3.9
6.2 3.3 8.6 5.4 3.7 5.1 6.1 4.1 10.9
Inhibition (% of control)
36.8 55.3 75.6 27.5 36.1 63.7 26.2 32.3 49.0
-R. glutinis, a yeast accumulates torularhodin abundantly, varied system of carotenoid biosynthesis and increased torularhodin productivity by oxygen stress.
These findings suggested that torularhodin plays a more important role in defensive against oxygen stress than other carotenoids in yeast. Hence, the anti oxidative properties of torularhodin were compared with ~-carotene. And author sought to estimate the biological function in yeast of torularhodin.
Peroxyl radical is most dominants radical in the process of lipid peroxide formation. And the chain reaction ends with formation of non-radicals owing to collision of peroxyl radicals with one another or scavenging by anti-oxidants.
Therefore, evaluation of the potency of scavenging of peroxyl radicals is very important in evaluation of lipid oxidation suppression.
The total carotenoids increased at the concentration of the AAPH, peroxyl radical generator, with which yeast growth is not inhibited. R. glutinis consumed torularhodin than ~-carotene with increase in loading by AAPH. But it cannot be declared that peroxyl radicals are main factor. Therefore, author examine scavenging of peroxyl radicals by carotenoids biosynthesized by R. glutinis. Although scavenging activity was not measured directly so far, Namikawa et al developed a novel procedure using ESR for detection of peroxyl radicals generated by the CHP-TPP-Fe(ill) reaction and pyrolysis of AIBN (13). This has enabled determination of
the peroxyl radicals scavenging activity of highly lipophilic antioxidants. In this method of direct measurement, torularhodin exhibited scavenging activity equivalent or superior to that of a-tocopherol, although the activity measured differed depending on the peroxyl radicals generator system used. The activity of torularhodin never surpassed that of a-tocopherol at any concentration in the system with pyrolysis of AIBN. This may be attributed to the heat stability of carotenoids. In the reaction of CHP and the TTP-Fe (III) system, activity of a-tocopherol decreases at high concentration. It was reported that a-tocopherol became proxidant at high concentration (4), and result obtained may support that. There are a few reports on scavenging of peroxyl radicals by ~-carotene, and partial oxygen pressure strongly affected this (5). Scavenging activity of ~-carotene was not detected under the present experimental conditions.
When torularhodin was added to rat brain homogenate, concentration-dependent suppression of MDA formation was observed, and the effect of torularhodin was stronger than those of a-tocopherol and ~-carotene. It is thought that scavenging of peroxyl radicals by ~-carotene is the main cause of suppression of the lipid peroxidation (5), and strong activity is related to stabilizing of hydroperoxide in a-tocopherol (6). Torularhodin showed strong activity compared with these compounds against lipid peroxide formation. The fact that torularhodin inhibited peroxide formation more effectively than a-tocopherol or ~-carotene is very interesting, considering the deep involvement of peroxyl radicals in the chain reaction of lipid peroxidation (7).
Although torularhodin exhibited potency in scavenging peroxyl radicals similar to that of a-tocopherol, it exhibited marked difference from it in suppression of lipid peroxide formation in rat brain homogenate. This finding suggested that torularhodin may contribute to the suppression of peroxide formation by an activity in addition to scavenging of peroxyl radicals. To explain to above results, more examination is needed.
It has been demonstrated that antioxidative activities are due to capturing or scavenging of active oxygen species by carotenoids, and their relation to structure has been discussed (8-10). There are two pathways of biosynthesis of carotenoids by yeast in Rhodotorula sp, which are known to produce carotenoids. One is from y-carotene to ~-carotene accompanying cyclization of ~-ionone ring, and the other is to torularhodin formation through desaturation and carboxylation (11). Since torularhodin is not cyclized in the process of biosynthesis from y-carotene, the carbon
chain that contributes to the stability of radicals appears to be long. It can therefore be presumed that the difference in activity between torularhodin and ~-carotene was related to the difference between them in length of carbon chain.
R. glutinis increased production of torularhodin upon excessive aeration of culture medium. In addition, a high-torularhodin-producing mutant (TL12l) showed resistance to oxygen stress higher than another strain. Lactate dehydrogenase activity in medium of low resistance strain was high as compared with that of medium cultured with TL121. This indicated that the yeast membrane has received the damage by oxygen stress. Aeration and singlet oxygen generated by methylene blue addition are considered to be an initiator on the process of damage of yeast cells. It is reasonable to assume that a dominant factor is peroxyl radicals because cell membrane is rich in lipid. Although it is important to remove initiator to prevent growth inhibition of yeast, the deletion of peroxyl radicals becomes important in the stop of the chain reaction begun. As a result of examination which used ESR, torularhodin was found to be a strong scavenger of peroxyl radicals than ~-carotene at all concentrations tested.
It thus appears that torularhodin was biosynthesized to preserve yeast from oxygen stress. Further, the results that cannot be explained only by difference of the scavenging activity of peroxyl radicals was obtained from experiment of lipid peroxide formation. This result suggested that torularhodin have another effective activity or biological property such as affinity for the membrane.
In conclusion, torularhodin is an effective scavenger against peroxyl radicals, and appears to play an important role in protecting against injury by oxygen stress in R.
glutinis. Further investigation of this carotenoid is expected.
MATERIALS AND METHODS in Chapter 5
1) Culture condition
A glucose peptone broth (Nihonseiyaku Co. Ltd., Tokyo, GP medium) was used for yeast cultivation. A 500-ml flask containing 100 ml of GP medium was inoculated with yeast cells washed with phosphate-buffered saline (pH 5.6) and then incubated for 3 to 5 days at 30°C with reciprocal shaking at 120 rpm with a 5 cm span.
2,2'-Azobis(2-amidinopropane)-dihyrochloride (AAPH, Wako Co. Ltd., Osaka) was added as a generator of peroxyl radicals at various concentrations from O.OlmM to 1mM to the GP medium. Yeast growth was monitored by optical density at 610nm.
Carotenoids were isolated and analyzed quantitatively as described previously (9).
2) Electron spin resonance (ESR) spectrometry
Two reaction systems were used for generation of peroxyl radicals (15). The first system used cumene hydroperoxide (CHP) with 5,10,15,20-tetraphenyl-21 H,23 H-prophine iron(III) chloride (TPP-Fe(III)) (exp. 1). Fifty III of 0.1 mM TPP-Fe(III), 50111 of 0.2 mM 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap reagent, 50 III of test substances dissolved in dimethyl sulfoxide (DMSO) and O.4mM CHP were placed in a test tube and mixed using an automatic mixer. Exactly 7 minute after, the spin adducts formed by the reaction of peroxyl radicals were recorded by ESR spectrometry (JES FR-30, JOEL, Tokyo, Japan). The second system used pyrolysis of 2,2'-azobisisobutyronnitrile (AIBN) (exp. 2). Fifty III of 0.2 M AIBN, 50111 of 0.2 M N-tert-butyl-a-phenylnitrone (PBN) as a spin trap reagent, and 50 III of teat substances dissolved in DMSO were placed in a test tube and mixed using an automatic mixer.
After heating at 50°C for 10 min, recording of spin adducts was started using ESR spectrometry. Conditions for ESR spectrometry were as follows: magnetic field, 335.5 mT; power, 8.0 mW; response time, 0.10 sec; modulation, 0.2 mT; temperature, 298 K; sweep time, 1.0 min. Manganese oxide was used as an internal standard.
Scavenging activity was calculated as the ratio to blank sample that did not contain test compound.
3) Lipid peroxidation of rat brain homogenate
The brain was isolated from euthanized Sprague Dawley rats at 4 weeks age and stripped of its meninges. Five grams of brain was homogenized in 30ml phosphate
buffered saline (pH 7.4) and supernatant was obtained by centrifugation (750 X g, 10 min). Supernatant was diluted to one-fifth with the same buffer. Ten microliters of each test substance such as carotenoid and a-tocopherol in DMSO were added to 990 JlI of diluted supernatant in container. Containers were transferred to a 37°C water bath and incubated continuously for 1 h after addition of 0.02 mM FeCl2 (final concentration). Reaction was stopped in ice-cold water. Malonyldialdehyde (MDA) was quantified using the calibration curve from absorbance at 532 nm after thiobarbituric acid reaction (12).
The torularhodin used in these studies was isolated and purified from R. glutinis no. 21. d,l-a-Tocopherol and ~-carotene were purchased from Wako Pure Chemical Industries as comparative controls. Addition of DMSO without test substances was performed as a negative control.
REFERENCES
1) Palozza, P., Moualla, S., and Krinsky, N.I.: Effects of ~-carotene and -tocopherol on radical-initiated peroxidation of microsomes. Free Radic. BioI. Med., 13, 127-136(1992).
2) Tsuchihashi, H., Kigoshi, M., Iwatsuki, M., and Niki, E.: Action of ~-carotene as an antioxidant against lipid peroxidation. Arch. Biochem. Biophys., 323, 137-147(1995).
3) Kennedy, T.A., and Liebler, D.C.: Peroxyl radical scavenging by ~-carotene in lipids bilayers. J. BioI. Chern., 267, 4658-4663(1992).
4) Takahashi, M., Yoshikawa, Y., and Niki E.: Oxidation of lipids. XVII. Crossover effect of tocopherols in the spontaneous oxidation of methyl linoleate. Bull. Chern.
Soc. Jpn., 62, 1885-1890(1989).
5) Terao, J.: Antioxidant activity of ~-carotene-related carotenoids in solution. Lipids, 24, 659-661(1989).
6) Nakano, M., Sugioka, K., Nakamura, T., and Oki, T.: Interaction between an organic hydroperoxide and unsaturated phospholipid and a-tocopherol and vitamin E acetate. Biochim. Biophys. Acta., 619, 274-286 (1980).
7) Yamamoto, Y., Niki, E., Eguchi, J., Kamiya, Y., and Shimasaki, H.: Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythtocyte ghost membranes by oxygen. Biochim. Biophys. Acta., 819, 29-36(1985).
8) Jorgensen, K., and Skibsted, L.H.: Carotenoid scavenging of radicals. Z. Lebensm.
Vters. Forsch., 196,423-429(1993).
9) Kennedy, T.A., and Liebler, D.C.: Peroxyl radical oxidation of ~-carotene. Chern.
Res. ToxicoI., 4, 290-295(1991).
10)Woodall, A.A., Lee, S.W., Weesie, R.J., Jackson, M.J., and Britton, G.: Oxidation of carotenoids by free radicals. Biochim. Biophys. Acta., 1336, 33-42(1997).
I1)Simpson, K.L., Nakayama, T.O.M., and Chichester, e.O.: Biosynthesis of yeast carotenoids. J.Bacteriol., 88, 1688-1694(1964).
12)Ko, F.N., Liao, C.H., and Wu, C.L.: Marchantiquinone, isolated from Reboulia hemisphaerica, as inhibitor of lipid peroxidation and as free radical scavenger.
Chemico. BioI. Interact., 98, 131-143(1995).
EFFECT OF WEAK WHITE LIGHT IRRADIATION ON YEAST GROWTH AND TORULARHODIN BIOSYNTHESIS
6.1 Introduction
In this chapter, as a part of studies examining biological significance of torularhodin, the author sought to evaluate the effect of sunlight using R. glutinis.
Therefore, a change in the amount of carotenoids produced in R. glutinis no.2l was investigated using an irradiation of white light. The ability to quench singlet oxygen was also investigated.
6.2 Growth inhibition and activation of torularhodin productivity by weak white light
Effects of light irradiation on the growth of yeast are shown in Table 6-1. It is clear that the growth of S. cerevisiae is not inhibited by light irradiation based upon the results that the same growth of S. cerevisiae as that under the dark was obtained even when white light was irradiated at 3,500 Ix. Although the irradiation of strong white light causes bactericidal action in general, this irradiation at 3,500 Ix was not so strong as affecting the growth after penetrating a glass. On the other hand, the growth of R.
glutinis was slightly inhibited by light irradiation under the same conditions with a delay of the growth rate at logarithmic growth phase. It is clarified that even a weak light irradiation not affecting the growth of other yeast may affect on the growth of R.
glutinis. There was no large difference between LDH activities in the medium under light irradiation and the dark, suggesting that light irradiation under these conditions may not bactericidal to R. glutinis. The amount of carotenoids biosynthesized by R.
glutinis was increased by light irradiation. Especially, the amount of torularhodin was increased to 180 % by light irradiation compared with that under the dark. The amounts of a route of carboxylation by introduction of oxygen into both torulene and torularhodin were 37.1 mg/lOOg cells dry weight (cdw) under the dark and 46.4 mg/lOOg cdw under light irradiation, demonstrating approximately 9.3 mg/lOOg cdw (25 %) increase in latter. On the other hand, ~-carotene was also increased by light
irradiation, but its increasing rate was only 0.5 mg/lOOg cdw (14 %). That is, approximately 95 % of the produced carotenoids increased by light irradiation (9.8mg) were related to the increased amount of carboxylation. A mutant, TL121 strain which increases the production of torularhodin has been made from R. glutinis no. 21. Then, it was previously confirmed that the inhibition of growth occurred in this strain by such weak light irradiation as exerting the growth inhibition in wild type strain no. 21, and the production of torularhodin was additively investigated. The relationship between the length of light irradiation from the start of cultivation and the amount of carotenoids produced was investigated. The result showed that torularhodin was remarkably increased in proportion to the length of light exposure (Fig.6-l), suggesting more clearly that light irradiation may cause the production of torularhodin.
Furthermore, growth inhibition and the amount of torularhodin at stationary phase were investigated when light irradiation was divided into each l2-hour period of the growth (Table 6-2). It restricted to the time which was irradiating light, and there are few increases in the number of yeast. It was checked that growth is inhibited by irradiation of weak white light. Although the amount of torularhodin increased depending on the number of yeast in irradiation time, there were few increases in yeast just before a logarithmic growth phase is completed. That is, it is likely that the production of torularhodin may not only be dependent on the number of yeast cells exposed with light irradiation but may be also affected by their physiological conditions.
Table 6-1 Effect of white light irradiation on the growth of red yeast and non-pigmented yeast and change in amount of carotenoids.
Doubling LDH activity Carotenodis
(U/L) (mgllOOg cell dry weight)
Strain Condition time
and % of dark
(h) condition /3-Carotene Torulene Torularhodin
Dark 2.4 22040 - -
-S. cerevisiae
White light 2.5 25480 (116%) - -
-R. glutinis Dark 3.6 73960 3.6 29.2 7.9
White light 4.8 61920 (84%) 4.1 32.2 14.2
CultIvatIOn on dark condItIons was carned out by shadmg completely. When It took out from ..
incubator for measurement, it worked under yellow light. White light was irradiated from a flask to IOcm distance using the fluorescent light. It kept applying irradiation while yeasts were incubating.
In the non-pigmented yeast, quantitative analysis of carotenoids was not performed.
Table 6-2 Growth inhibition while light irradiation and amount of torularhodin after reaching stationary phase
Irradiated Proliferation every 12 h (X108)
Torularhodin period (h) 0-12 12-24 24-36 36-48 (mg/lOOg cells dry weight)
0-12 0.02 0.35 1.31 1.85 27.1
12-24 0.02 0.20 1.72 1.99 46.7
24-36 0.02 0.33 0.68 2.24 62.3
36-48 0.02 0.31 2.04 1.09 28.8
Yeast growth reached the statIOnary phase 60 h after It began to have mcubated. Torularhodm was quantitatively analyzed immediately after reaching stationary phase.
4.0 x
"CI
8 l!I.
] ~ 3.0- x - 107 ~
CD
Q. 0a; CD
:g ~ :0
E! ~ ... ~-~
i
~ 2.0- x[e
[3 "8
_.
~'5 8 bII x X ~q !!
~ ~ 1.0- [
~ 106 J.
<
0.0
12 15 18 21 24 Period of irradiation (h)
Fig.6-1 Relationship between irradiation period and carotenoid content in mutant TLl21.
White light irradiation was carried out from the start of incubation. Cell number (x) measured at the end of the irradiation period is shown . • : Torularhodin; 0: Torulene;
0: ~-Carotene.
The abilities of ~-carotene and torularhodin to quench singlet oxygen were evaluated. In this case, 2,5-diphenyl-3,4-benzofran (DPBF) decomposition by singlet oxygen generated from EPA was monitored (Table 6-3). When the ~-carotene and
torularhodin were added by the same mole concentration, the decomposition of the DPBF in torularhodin was slower than the ~-carotene. This indicated that torularhodin showed a more effective ability to quench singlet oxygen than ~-carotene.
It has been demonstrated that carotenoids specific to marine organisms exhibit more potent abilities to quench singlet oxygen with longer polyene chain (1). Torularhodin has a non-cycled ~-ionone ring and a longer polyene chain than that of ~-carotene. It is likely that the more potent ability may be due to the longer polyene chain. In previous study, it has been demonstrated that torularhodin content in yeast was increased by aeration to excesses and also exhibits a more tolerance to oxygen stress by addition of methylene bleu to cultivation medium. The result indicates that this ability may also be effective on the protection against oxidative damage.
Table 6-3 Inhibitory effect of torularhodin on decomposition of DPBF by singlet oxygen.
Decomposition of DPBF (mM/h)
Control 0.023
~-Carotene
Torularhodin
DPBF: 2,5-diphenyl-3,4-benzofran
0.013 0.002
Many studies have been performed on the protective mechanism against light damage in yeast. In S. cerevisiae, detection of the systems for the protection and identification of repair gene have been already reported (2-5). It has been known that the protective carotenoids exist against light damage in yeasts which biosynthesize carotenoids (6). However, the effect of an individual carotenoid has not been studied yet, and the biological significance of typical carotenoids has not been clarified yet, either. In R. glutinis, ~-carotene and torularhodin are biosynthesized as final products.
It has been already reported that carotenoids are important in the protection against oxidative stress in Rhodotorula sp. (7). Torularhodin has a structure with non-cycled
~-ionone ring and a carboxyl group as mentioned above, and a specific carotenoid observed in yeasts and bacteria. In R. glutinis no. 21, torularhodin has been demonstrated to exhibit a characteristic behavior against oxidative stress and has been estimated to be important for the protection among the carotenoids produced in yeasts.
In this study, an inhibition of the growth was observed when weak light was irradiated, and simultaneously a conspicuous production of torularhodin was also observed.
Consequently, torularhodin is demonstrated to be important for the protection against light damage. It is clear that torularhodin may also play an important role for the protection against oxidative damage from the results reported up to now. The protection against light damage is an essential function to distribute in the nature world.
R. glutinis distributes not only in the soil but also in various places (8). It is likely that this yeast can distribute widely in the nature world by possessing the function to biosynthesize torularhodin.
Table 6-4 Inhibition of uric acid formation in the xanthin and xanthinoxidase mixture
Torularhodin (mM) Alloprinol
1
I
10I
100 ICso% inhibition 3
I
11I
7 3.97 J.lMOn allopunol, 50% mhIbItIon concentratIOn (IC.. so) was calculated from regressIOn line. ICso was not obtained from the result of torularhodin in this study range.
Production of uric acid by xanthineoxidase remained unchanged up to the torularhodin concentration of 100 11M, and the inhibition rate was 11 % at best (Table 6-4). This result suggests that torularhodin do not effect directly on superoxide anion.
MATERIALS AND METHODS in Chapter 6
1) Light irradiation to liquid culture broth
A glucose peptone broth (Nihonseiyaku Co, Ltd., Tokyo) was used for cultivation.
Cultivation was performed according to the method indicated by the previous report (Chapter 4). A fluorescent lamp set on 10 cm from flask was used for white light irradiation. The intensity of the illumination, which was measured by illumination meter (IM-2D, Toshiba Co. Ltd., Tokyo), was 3,500 Ix. Cell number counted using the Thoma plate and the optical density at 610 nm monitored yeast growth. After ultra-sonication, yeast carotenoids were isolated and analyzed quantitatively as described previously (Chapter 3). Lactate dehydrogenase (LDH) activity in the cultivation medium used as index of injury (9) was determined according to the procedure described previously (10).
2) Scavenging activity of singlet oxygen
3-(1,4-epidioxyl-4-methyl-1,4-dehydro-1-naphtyl) propionic acid (EPA), as a singlet oxygen generator, and 2,5-diphenyl-3,4-benzofran (DPBF) were dissolved in ethanol at the concentrations of 0.46 mM and 0.043 mM respectively. Concurrently, 0.01 mM of carotenodis were added and then it was incubated at 35°C for 1 h.
Optical density at 411 nm measured disassembly of a DPBF under reaction (11).
3) Effect on superoxide anion radical
Effect of carotenoids on superoxide anion radicals was investigated according to the previous procedure using xanthineoxidase (12). Mixture of xanthine, dissolved in phosphate buffer (pH 7.5) at concentration of 160 JlM, and carotenoid, dissolved in 1 % dimethyl sulfoxide, were incubated for 30 min at 25°C. Absorbance at 290 nm was used to measure the uric acid in the reaction. Allopuriol, known xanthineoxidase inhibitor, was used for positive control. On allopuriol, 50% inhibition concentration (ICso) was calculated from regression line.
REFERENCES
1) Shimidzu, N., Goto, M., and Miki, W.:Carotenoids as singlet oxygen quenchers in marine organisms, Fisheries Sci.,62,134-137(1996).
2) Mieczkowski, P., Dajewski, W., Podlaska, A., Skoneczna, A., Ciesla, Z., and Sledziewska-Gojska, E,.:Expression of UMP1 is inducible by DNA damage and required for resistance of S. cerevisiae cells to UV light. Curf. Genet.,38,53-59(2000).
3) Halas, A., Policinska, Z., Baranowska, H., and Jachymczyk, W.J.: The essential DNA polymerases delta and epsilon are involved in repair of UV -damaged DNA in the yeast Saccharomyces cerevisiae. Acta. Biochim. PoI.,46,289-298(1999).
4) Padula, M., and Boiteux, S.:Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae. Braz. J. Med. BioI. Res., 32, 1063-1071(1999).
5) Tijsterman, M., Verhoeven, E.E., Jong, J.G., and Brouwer, J.: Enzymatic detection of ultraviolet-induced pyrimidine (6-4) pyrimidone photoproducts at nucleotide resolution in Saccharomyces cerevisiae. Anal. Biochem.,260,1l0-1l3(1998).
6) Weeks, O.B., Saleh, F.K., Wirahadikusumah M., and Berry RA.: Photoregulated carotenoid biosynthesis in non-photosynthetic microorganisms. Pure Appi.
Chem.,35,63-80(1973).
7) Moore, M.M., Breedveld, M.W., and Autor, A.P.:The role of carotenoids in preventing oxidative damage in the pigmented yeast, Rhodotorula mucilaginosa.
8) Fell, J.W., Tallman, A.S., and Ahearn, D.G.: The Yeast,(Kreger-van Rij,N.J.W), 3rd ed., 893-894,Elsevier Science Publishers B.W.,Amsterdam(1984).
9) Pizarro RA.: UV-A oxidative damage modified by environmental conditions in Escherichia coli. Int. J. Radiat. BioI.,68,293-299(1995).
10) Babson, A.L., and Pillips, G.E.: A rapid colorimetric assay for serum lactic dehydrogenase. Clin. Chim. Acta,12,210-215(1965).
l1)Robert H. Young, Kathy Wehrly, and Robert L.M.: Solvent effects in dye-sensitized photo oxidation reactions, J .A.Chem.Soci. ,93,5774-5779(1971).
12)Hatano, T., Yasuhara, T., Yoshihara, R, Agata, 1., Noro, T., Okuda, T.: Effects of interaction of tannins with co-existing substances. VII. Inhibitory effects of tannins and related polyphenols on xanthine oxidese. Chem.Pharm.Bull., 38. 1224-1229 (1990).
DISCUSSION
R. glutinis no. 21 was isolated as a yeast viable with DL-Iactic acid as carbon source. This yeast efficiently assimilated the D-Iactic acid supplied and could not assimilate L-Iactic acid as a single energy source. However L-Iactic acid decreased in medium containing DL-Iactic acid, indicating that this yeast assimilated L-Iactic acid after the conversion to D-Iactic acid. To my certain knowledge, this is a first case of D-Iactic acid utilization as a single energy source, because D-Iactic acid is not available for organisms in general.
R. glutinis no. 21 obtained after culture with lactic acid is slightly reddish compared with one obtained after culture with glucose as a carbon source. The difference in color tone was attributable to quantitative difference between two finally obtained carotenoids, ~-carotene and torularhodin. Thereafter, it became apparent that the increases in torulene and torularhodin are associated with dissolved oxygen concentration that was adjusted by aeration. Effect of active oxygen species on the productivity of torularhodin by R. glutinis no.21 was examined. Methylene blue, methyviologen and AAPH [2,2-azo-bis (2-amidinopropane)-dehyrochloride] were used as generators of singlet oxygen, superoxide anion radical and peroxy radical, respectively. Addition of these generators to culture medium gave almost no influence on the biosynthesis of ~-carotene, whereas marked enhancement on those of torulene and torularhodin was observed. These findings led to the assumption that R.
glutinis no.21 increases its production of torularhodin for protection against membrane impairment by activated oxygen species, suggesting that torularhodin may potentially act as an antioxidant.
The biological function of carotenoid was actively researched after it became clear that there is strong antioxidative activity (1-5). Yeast carotenoids also turned out to play an important role against oxidative stress (6). However, an individual evaluation was not examined though yeast synthesizes plural kind of carotenoids as final products. Torularhodin is a carotenoid that is classified to the carboxyl group, and is isolated mainly from the fungi and a part of bacteria. R. glutinis biologically synthesizes j3-carotene and torularhodin as final products (7). This suggests that torularhodin itself has actions different from those of ~-carotene. In this respect, it is tempted to have interest in basic study of torularhodin.
A mutant (TLl21) which produces large amounts of torularhodin was constructed
and its tolerance against oxidative stress was investigated. This mutant was capable of producing large amounts of torularhodin in response to irradiation with blue light.
The mutant, incubated under irradiation with white light that resulted in an increased production of torularhodin, exhibited resistance to growth inhibition induced by the addition of methylene blue as the generator of singlet oxygen. Leakage of lactate dehydrogenase to the growth medium from the mutant was not increased as compared with that from a parent strain and a high-~-carotene-producing mutant. It is suggested that torularhodin has a potent prevent to oxygen stress in yeast cells.
Peroxyl radicals scavenging by torularhodin known property in carotenoids was evaluated using electron spin resonance. Scavenging activity by torularhodin was greater than that of J3-carotene in different generation system of peroxyl radicals, reaction of CHP and pyrolysis of AIBN.
Addition of MB, which generates singlet oxygen, gives rise to increased production of torularhodin. Since carotenoids would eliminate singlet oxygen by own oxidative degradation (16), the increased production observed after addition of MB may possibly be due to the removal of singlet oxygen. However, it may also be attributable to removal of peroxyl radicals, since photooxidation is also able to trigger the chain reaction for lipid peroxidation. That is, it is impossible to specify the cause of increasing of torularhodin. It is difficult to know how long the generators added at the beginning of the culture would retain their effect. Moreover, various reactions would simultaneously occur at the beginning of the chain reaction, and it is difficult to attribute to an effect of a specific radical or reactive oxygen species. However, estimation of this effect may become possible by increasing the effect of a specific oxidative load by means of continuous addition of a generating agent to the medium.
Examination of the effect of continuous MB supply on carotenoid biosynthesis revealed the increased production of torularhodin. This increase is apparently greater than that produced by single addition at the onset of culture, indicating that oxidative load gives rise to increased production of torularhodin. The effect of singlet oxygen was increased under the conditions of continuous addition. An experiment using EPA clarified that torularhodin inhibited DPBF decomposition by singlet oxygen quenching more strongly than did J3-carotene. This result is consistent with the report that carotenoids having a longer polyene chain may exhibit a more potent ability to quench singlet oxygen (14,18).
Since torularhodin showed increased production even after MV was added to culture medium, torularhodin was expected to have the elimination capacity against