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Essential Role of Mg

2+

in Flocculation of Saccharomyces diastaticus

Hiroshi

NISHIHARA*,

Chihiro

ONISHI

and Satoko

BABA

(Key words flocculation, Saccharomyces diastaticus, Mg

2+

-deficient)

Abstract

Flocculation of Saccharomyces diastaticus IFO 1958 was studied. Cells of IFO 1958 did not flocculate even in the stationary phase without Mg2+ although they began to flocculate strongly 18h after inoculation in the presence of Mg2+. Cycloheximide completely inhibited induction of floe-forming ability of Mg2+-deficient cells. Co-flocculation between flocculent cells and Mg2+-deficient cells was in- vestigated by treatment by proteolytic enzymes and chemical modification. Treatment of Mg2+ -deficient cells by proteolytic enzymes did not affect the co-flocculation with flocculent cells. Photo-oxidation or mercaptoethanol-reduction of Mg2+-deficient cells failed to diminish the co-flocculation with flocculent cells while treatment of Mg2+ -deficient cells by periodate caused a considerable loss of the co- flocculation. On the other hand, flocculent cells deflocculated by proteolysis or chemical modification of protein component failed to co-flocculate with Mg2+-deficient cells. These findigs suggest that Mg2+- deficient cells are non-flocculent because of lack of protein component essential for flocculation of cells of IFO 1958.

* Corresponding author.

phone: +81-(0) 87-832-1464

e-mail: nishihar@ed. kagawa-u. ac. jp fax: +81-(0) 87-832-1417

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INTRODUCTION

Flocculation of brewer's yeast cells is important and interesting from both biochemical and industrial standpoints. It is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended1 l. It is described that flocculation is caused by interaction between cell surface protein and mannan2' 3l • It was also reported that Mg2+ plays an essential part in flocculation of beer yeast IFO 2018, a flocculent strain of S. cerevisiae4-6).

Although S. diastaticus is now taxonomically regarded as S. cerevisiae, S. diastaticus is able to ferment starch to produce ethanol because it has ability to secret glucoamylase.

This paper describes an essential role of Mg2+ in flocculation of S. diastaticus.

MATERIALS AND METHODS

Yeast strain

S. diastaticus IFO 1958 was used throughout. The strain was obtained from Institute for Fermen- tation, Osaka.

Cultivation

The yeast cells, cultivated in the semi-synthetic medium described in the previous paper4l, were washed three times with sterilized deionized water and inoculated at a cell concentration of 1 µ g/ml into fresh medium of the same composition or fresh medium deficient in Mg2+ . Cultivation was carried out at 28 °C with shaking on a rotatory shaker. Yeast cells cultivated for appropriate time were harvested and washed three times with deionized water.

Estimation of flocculation

The degree of flocculation (D. F. value) of cells of a single strain and that of co-flocculation observed when flocculent cells and non-flocculent cells were mixed was estimated as described before3l.

Addition of cycloheximide into growing culture

After 1 µ g/ml of cycloheximide and Mg2+ was added into Mg2+ -deficient cell culture grown for 18h, cells grown for 21h after inoculation were harvested and D. F. values were determined.

Treatment of cells with proteolytic enzymes and chemical modification of cell surface

protein and carbohydrate components

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Proteolytic enzemes

10mg of cells was incubated with trypsin or chymotrypsin as described previously7l.

Mercaptoethanol-reduction

10mg of cells was treated with O. IM mercaptoethnol in the presence of SM urea, as described before8l.

Photo-oxidation

10mg of cells was photo-irradiated in the presence of methylene blue and SM urea at the room temperature, as described before8l.

Nal04

10mg of cells was treated with 20mM NalO4 at 0°C for 30min in the dark, as described previouly3l.

After appropriate treatments described above, cells were washed three times with deionized water and then used in the flocculation and co-flocculation experiments.

RESULTS AND DISCUSSION

Time-course of flocculation of cells of S. diastaticus IFO 1958

Figure 1 shows a time course of flocculation of cells of S. diastaticus IFO 1958 in the presence and absence of Mg2+. Cells cultivated in the presence of Mg2+ began to flocculate 18h after inoculation while cells grown in the absence of Mg2+ did not flocculate until 24h after inoculation.

Cells cultivated for 21h in the presence of Mg2+ and in the absence of Mg2+ were designated as flocculent cells and Mg2+ -deficient cells, respectively.

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4

~ Ol 3

.s

..c

!

2

&

0 5 10 15

Time(h)

20 25

100

80

60

40

20

0 30

Figure 1. Time-course of Growth and Flocculation of Cells of

S. diastaticus IFO 1958 Grown with and without

Mg2+

Effect of cycloheximide on induction of floe-forming ability of

Mg2+

-de~cient cells

100 ~ - - - ~

80

l 60

~ ~ 40

u.:

ci 20

17 18 19

Mg2' and CHI None

20 21 22

Time(h)

Figure 2. Effect of Cycloheximide on Induction of Floe-forming Ability of Mg2+ -deficient Cells.

Figure 2 shows effect of cycloheximide on induction of floe-forming ability of growing non-flocculent Mg2+ -deficient cells. When Mg2+ was added to Mg2+ -deficient cell culture grown for 18h, cells flocculated strongly after 3 h. Cycloheximide strongly inhibited the induction of floe-forming ability of Mg2+ -deficient cells by Mg2+, suggesting that de novo protein synthesis at ribosomes is necessary for the induction of floe-forming ability of Mg2+ -deficient cells by Mg2+.

Effect of treatment with proteolytic enzymes and chemical modification of cell surface protein and carbohydrate components on co-flocculation

As shown in Figure 3, strong co-flocculation was observed when non-flocculent Mg2+ -deficient cells and flocculent cells were mixed.

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Figure 3 shows also the effect of treatment with proteolytic enzymes of cell surface protein of non-flocculent Mgz+ -deficient cells on co-flocculation between Mgz+ -deficient cells and flocculent cells.

Treatment of Mgz+ -deficient cells with trypsin or chymotrypsin failed to affect the co-flocculation significantly.

Mg2+-deficient cells

None

Trypsin-treated

Chymotrypsin-treated

0 20 40 60 80 100

D.F. value(%)

Figure 3. Effect of Treatment with Proteolytic Enzymes of Non-flocculent Mg2+ -deficient Cells on Co-flocculation with Flocculent Cells.

Mg2+-deficient cells

Non-treated

photo-oxidized

0 20 40 60 80 100

D.F. value(%)

Figure 4. Effect of Photo-oxidation of Non-flocculent Mg2+ -deficient Cells on Co-flocculation with Flocculent Cells.

Figure 4 illustrates effect of photo-irradiation of Mgz+ -deficient cells in the presence of methylene blue on the co-flocculation between Mgz+ -deficient cells and flocculent cells. It is known that photo-irradiation in the presence of methylene blue preferentially brings about modification of imidazole groups of histidyl residues in proteins9l. It has also been described that floe-forming ability of flocculent cells of S. cerevisiae IFO 2018 is lost by photo-irradiation in the presence of a photo-sensitizer because of the destruction of the steric structure of a surface protein component essential for flocculation8l. Photo-irradiation did not bring about a loss of the co-flocculation but somewhat enhanced the co-flocculation.

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Figure 5 shows effect of reduction with mercaptoethanol of cell surface protein of non-flocculent Mg2+ -deficient cells on co-flocculation between Mg2+ -deficient cells and flocculent cells. Mercapto- ethanol-reduction of the Mg2+ -deficient cells, in particular, in the presence of SM urea stimulated co-flocculation with flocculent cells strongly.

Mg2+-deficient cells

Non-treated

Mercaptoethanol-reduced

0 20 40 60 80 100

D.F. value(%)

Figure 5. Effect of Reduction with Mercaptoethanol of Non-flocculent Mg2+ -deficient Cells on Co-flocculation with Flocculent Cells.

Mg2+-deficient cells

Non-treated

periodate-oxidized

0 20 40 60 80 100

D.F value(%)

Figure 6. Effect of Oxidation with Periodate of Non-flocculent Mg2+ -deficient Cells on Co-flocculation with Flocculent Cells.

Treatment with periodate is known to result in the C-C bond cleavage of vicinal dihydroxy compounds including carbohydrates. As shown in Figure 6, periodate-oxidation of Mg2+ -deficient cells diminished considerably co-flocculation with flocculent cells.

These results suggest strongly that not surface protein components but surface carbohydrate components of Mg2+ -deficient cells are essential for co-flocculation with flocculent cells.

Next, effect of proteolytic treatment and chemical modification of flocculent cell surface components on co-flocculation between Mg2+ -deficient cells and flocculent cells was investigated.

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As shown in Figure 7, flocculent cells lost the floe-forming ability by treatment with protelytic enzymes. Cells deflocculated by the treatment with proteolytic enzymes failed to co-flocculate with non-flocculent Mg2+ -deficient cells.

Flocculent cells Trypsin-treated cells (1) Chymotrypsin-treated cells (2) Mg2+-deficient cells Co-flocculation between (1) and (3) Co-flocculation between (2) and (3)

0 20 40 60 80 100

D.F. value(%)

Figure 7. Effect of Treatment with Proteolytic Enzymes of Flocculent Cells on Co-flocculation with Mg2+ -deficient Cells.

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Flocculent cells

photo-oxidized flocculent cells (1)

Mg2+-deficient cells (3)

Co-flocculation between (1) + (3)

0 20 40 60 80

D.F. value(%)

Figure 8. Effect of of Photo-oxidation of Flocculent Cells on Co-flocculation with Mg2+ -deficient Cells.

Flocculent cells

Mercaptoethanol-reduced Mg2+-deficient cells(1)

Mg2+-deficient cells (3)

Co-flocculation between (1) + (3)

0 20 40 60 80

D. F. value (%)

Figure 9. Effect of Reduction with Mercaptoethanol of Flocculent Cells on Co-flocculation with Mg2+ -deficient Cells.

100

100

As shown m Figure 8 and Figure 9, flocculent cells lost the floe-forming ability by photo-oxidation and mercaptoethanol reduction. Both cells deflocculated by photo-oxidation and mercaptoethanol-reduction did not co-flocculate with non-flocculent Mg2+ -deficient cells. These results suggest that surface protein components of flocculent cells are essential for self-flocculation of flocculent cells and co-flocculation with Mg2+ -deficient cells.

Figure 10 shows effect of periodate oxidation of flocculent cells on self-flocculation of flocculent cells and co-flocculation with Mg2+ -deficient cells. Flocculent cells lost the floe-forming ability by periodate oxidation, suggesting that carbohydrate components (mannan) on the flocculent cell surface also play an important part in the floe-forming ability of flocculent cells. Flocculent cells deflocculated

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Flocculent cells

Periodate-oxidized flocculent cells (1)

Mg2+-deficient cells (3)

Co-flocculation between (1) + (3)

0 20 40 60 80

D.F. value(%)

Figure 1 O. Effect of Oxidation with Periodate of Flocculent Cells on Co-flocculation with Mgz+ -deficient Cells.

Flocculent cells Periodate-oxidized flocculent cells (1) Mg2+-deficient cells (2) Mercaptoethanol • treated early phase cells (3 Co-flocculation between ( 1) + (2)

Co-flocculation between (1) + (3)

0 20 40 60 80

D.F. value(%)

100

100

Figure 11 . Effect of Mercaptoethanol-reduction of Mg2+ -deficient Cells on Co-flocculation with Flocculent Cells Oxidized with Periodate.

by periodate oxidation failed to co-flocculate with Mg2+ -deficient cells. Flocculent cells defocculated by periodate oxidation, however, co-flocculated with Mg2+ -deficient cells after treatment of Mg2+ - deficient cells with mercaptoethanol, as shown in Figure 11.

Therefore, it is evident that both protein and carbohydrate components on the cell surface play the essential roles in the flocculation of flocculent cells of S. diastaticus IFO 1958. On the other hand, Mg2+ -deficient cells are non-flocculent since they are not able to produce surface protein component essential for flocculation while they possess essential carbohydrate component.

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References

1) Stewart, G. G., Brewer's Digest, 1975, 50, 42.

2) Miki, B. L., Poon, N. H., James, A. P. & Seligy, V. L., Journal of Bacteriology, 1982, 150, 878.

3) Nishihara, H. & Toraya, T., Agricultural and Biological Chemistry, 1987, 51, 2721.

4) Nishihara, H., Toraya, T. & Fukui, S., Journal of Fermentation Technology, 1976, 54, 351.

5) Nishihara, H., Toraya, T. & Fukui, S., Journal of Fermentation Technology, 1976, 54, 356.

6) Nishihara, H. , Takao, M. , Senba, E. & Matsumoto, N. , Memoirs of the Faculty of Education, Kagawa University (Part II), 1988, 38, 9.

7) Nishihara, H., Miyake, K. & Kageyama, Y., Journal of the Institute of Brewing, 2002, 108, 187.

8) Nishihara, H., Toraya, T .. & Fukui, S., Archives of Microbiology, 1977, 115, 19.

9) Forman, H. J., Evans, H. J., Hill, R. L. & Fridovich, I., Biochemistry, 1973, 12, 823.

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