FEMS Microbiology Letters 27 (1985) 103-105 103 Published by Elsevier

FEM 02045

Superoxide dismutase in methylotrophic yeasts (Methanol; superoxide radical; hydrogen peroxide)

Anna Kuyumdzhieva-Savova, Valentin A. Savov *, Ljubka K. Genova and Sofia P. Peikova

Bulgarian Academy of Sciences, Institute of Microbiology, Acad, G. Bonchev BL 26, 1113, Sofia, and * University of Sofia, Faculty of Biology, Bul. D. Tzancov 8, Sofia, Bulgaria.

Received 26 October 1984 Revision received 17 January 1985

Accepted 18 January 1985

1. SUMMARY 3. MATERIALS AND METHODS

Superoxide dismutase (SOD) activity of 4 strains of methylotrophic yeasts of the genera Candida, Torulopsis, Hansenula and Pichia was demon- strated. Strains tested were grown on glucose or methanol. Yeasts grown on methanol possessed considerably higher levels of SOD- and catalase activity than cells cultivated on glucose.

2. INTRODUCTION

In methanol-utilizing yeasts, methanol is oxidized to carbon dioxide by alcohol oxidase, catalase, formaldehyde dehydrogenase and for- mate dehydrogenase [6,12,14]. Eggeling and Sahm [3] suggested that the synthesis of these enzymes was controlled by derepression and partial catabolite repression. In studies on the biochem- istry of primary oxidative metabolism of methanol by yeasts, we observed high levels of superoxide dismutase (SOD), a protective enzyme for aerobic cells against oxygen toxicity [4].

The present study was undertaken to investigate the role of SOD in the primary oxidation of methanol by yeasts.

3.1. Organisms and media Torulopsis domerqii CC-8, Hansenula poly-

morpha 76-26, Candida boidinii 77-1 and Piehia pastoris 72-13 were from our laboratory collection. Medium MK4 [7] was used for the cultivation of yeast strains. Carbon sources were glucose (15 g/l) or methanol (10 g/l), both at pH 4.5. The cells were precultured at 28°C for 48 h on slant medium. 2 Tubes of slant culture were inoculated into an erlenmeyer flask containing 100 ml glucose or methanol medium, and cultivated at 28°C on a reciprocal shaker. The culture was harvested in the early stationary phase by centrifugation at 4°C and 5000 x g for 15 rain. Cell-free extracts from these samples were than assayed for protein con- tent and SOD- and catalase activities.

To determine the changes in activity during growth, T. domerqii CC-8 was grown on glucose for 24 h and then transferred to methanol medium in a 75 1 laboratory fermentor (New Brunswick, New Jersey, U.S.A.) Cultivation was at 28°C and pH 4.5. Agitation was maintained at 800 rev./min, and pOE was 80%. Samples were withdrawn every 3 h and cultures were harvested by centrifugation at 4°C and 5000 × g for 15 rain. Cell-free extracts prepared from these samples were assayed for

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protein content and SOD- and catalase activities. Cell growth was estimated by absorbance at 610 am.

3.2. Enzyme preparation and assay The harvested cells were washed twice with a

0.05 M phosphate buffer, pH 7.8, and suspended in a small amount of the same buffer. Cells were disrupted with glass beads in a vibration homo- genisator VH GI. Homogenates were centrifugated at 4°C and 13000 x g for 15 min to remove cells and cell debris.

SOD activity was assayed by the method de- scribed in [2]. One unit of SOD is defined as the quantity of the enzyme which caused 50% inhibi- tion of the NBT-reduction, and was expressed as U . m g protein -~. Catalase activity was de- termined spectrophotometrically by measuring the change in absorbance at 240 nm [1]. Enzyme activ- ity was expressed as A E m i n - l . m g protein -~. Protein was determined by the method of Lowry et al. [8].

4. RESULTS

4.1. Activation of SOD by methanol In all 4 yeast strains, cells grown on methanol

had higher activities of SOD and catalase than cells grown on glucose (Table 1). The SOD from these strains was sensitive to inhibition by 1 mM potassium cyanide, and insensitive to 5 and 10 mM NaN 3. These results suggested the presence of Cu-Zn containing SOD in methanol-grown cells.

Table 1 Specific activities of SOD and catalase of yeast strains during growth on methanol or glucose

Organism Methanol Glucose

SOD a Catalase b SOD Catalase

T. domerqii CC-8 75.01 44.07 2.12 1.06 H. polymorpha 76-26 64.51 25.00 3.33 1.56 P. pastoris 72-13 66.62 27.00 5.24 4.08 C. boidinii 77-1 53.24 33.34 2.85 1.55

a SOD activity: units- mg protein- 1. b Catalase activity: AE24o.min-l.mg protein -1.

O. 600

E C 0.500

0 o.~.oo

0.300 c" O

..O 0.200

0.100

, J ~ t , , i i , b

3 6 9 12 15 18 21 2~, 27 30 t i m e

(hours)

:li :1 0 50 O~ 50

E

3o-E 30

20 20 O O o i-i

Fig. 1. Growth of Torulopsis domerqii on methanol and activi- ties of superoxide dismutase and catalase: growth, specific activity of SOD, - e - ; specific activity of catalase, - C ) - .

4.2. SOD- and catalase activities during growth Cells of T. domerqii CC-8 pre-grown on glucose

showed a marked increase in the activity of SOD and catalase during growth in methanol (Fig. 1). Enzyme activities increased faster than the pro- liferation of cells. In the first 9 h, during the lag phase in cell growth, there was a 25-fold increase in SOD activity and only a 2-fold increase in turbidity.

5. DISCUSSION

McCord and Fridovich [10] suggest that the physiological function of SOD is to catalyse the scavenging of 0 2 . The presence and ubiquity of SOD suggested that 0 2 was an important by- product of oxidative metabolism.

Methanol oxidation proceeds via formaldehyde and formate to CO2. During this oxidation yeast cells synthesize considerable amounts of methanol oxidase [11]. This enzyme, which catalyses the first step in the oxidation of methanol, is a flavoprotein [13]. As a result of its reaction with methanol, reduced ravin and aldehyde are formed. Reduced flavin dehydrogenase reacts with molecular oxygen, and the products of this reaction are hydrogen peroxide, or flavin and peroxide radicals ("O2H).

The latter may dissociate to form the superoxide radical (O~-) [9].

It was assumed that methanol oxidase is a H202-producing enzyme, localized with catalase in specialized organdies, peroxisomes [13]. The pres- ence of a high S O D activity in methanol-grown cells suggest that more than one reaction proceeds with reduced flavin. The alternative reaction prob- ably also utilizes flavin, to produce substrate for SOD. Moreover, the H202 which was obtained may also be the source of this substrate [4,5]. The high levels of SOD and catalase activities in methanol-grown cells observed here, may therefore be a consequence of the growth of these methyl- otrophic yeasts on methanol.

R E F E R E N C E S

[1] Aehi, H. (1970) in Methoden der Enzymatischen Analyse (Bergemeyer, H.V., Ed.) pp. 634-641, Acad. Verl., Berlin.

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[2] Beaushamp, C. and Fridovich, I. (1970) J. Biol. Chem. 245, 4641-4646.

[3] Eggeling, L. and Sahm, H. (1978) Eur. J. Appl. Microbiol. Biotechnol. 5, 197-202.

[4] Fridovich, I. (1976) in Free Radicals in Biology, (W. Pryor, Ed.) pp. 239-277, Academic Press, New York.

[51 Fridovich, I. (1978) Photochem. Photobiol. 28, 733-741. [6l Kato, N. and Tani, Y. (1973) Agr. Biol. Chem. 38, 675-677. [7] Kuyumdzhieva, A. and Denchev, D. (1979) Acta Micro-

biol. Bul. 5, 66-72. [8l Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall,

R.L. (1951) J. Biol. Chem. 193, 265-275. [9l Yamasaki, M., and Yamano, T. (1973) Biochem. Biophys.

Res. Commun. 51, 612-619. [10] McCord, J.M. and Fridovich, I. (1979) J. Biol. Chem. 244,

604%6055. [11] Sabra, H. (1967) Adv. Biochem. Eng. 6, 77-103. [12] Tam, Y., Miya, T., Nishikawa, and Ogata, K. (1972) Agr.

Biol. Chem. 36, 68-74. [13] Van Dijken, J.P. (1982) Ann. New York Acad. Sci.

208-216. [14] Van Dijken, J.P., Veenhuis, M., Kreger-van Rij, N.J.M.

and Harder, W. (1975) Arch. Microbiol. 102, 41-44.