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Formaldehyde production with heat-treated cells of methanol yeast
[J. Ferment. Technol., Vol. 65, No. 4, 489-491. 1987]
Note
Formaldehyde Production with Heat-Treated Cells of Methanol Yeast YASUYOSHI SAKAI* a n d YOSHIKI TANI
Research Cent*rfor Cell and Tissue Culture and *Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University,Kyoto 606, Japan
Chemostat-grown cells of a methanol investigated for formaldehyde production. were improved by preincubation of the cells phate buffer (pH 7.5) containing 50 mlV[ formaldehyde after a 10-h reaction.
yeast, Candida boidinii $2 AOU-1, were The productivity and catalytic stability at 37°C for 24 h in 0.1 M potassium phosNaNa. These cells produced 1150 m M
In preceding papers, 1-3) we reported on producing an initial velocity of 240 m M / h . formaldehyde production by an alcohol The cell suspension was incubated at oxidase-enhanced m u t a n t strain AOU-1 of various temperatures and the formaldehyde Candida boidinii $2. In our previous study, 3) productivity was assayed. As shown in it was suggested that the m e m b r a n e permeability of the substrate limits the rate of the reaction because less improvement in the ©© initial velocity of formaldehyde production ° was seen compared to that for the final _ _ ~ lO0 ~o concentration or alcohol oxidase activity. m A T P production was established with Zymolyase®-treated or sorbitol-treated metha0 nol yeast cells. 4-e) In this paper, improve= ment in formaldehyde productivity of heattreated yeast cells will be described. 3'6 C. boidinii $2 AOU-1 cells were grown on I n c u b a t i o n t i m e (h) a methanol-limited cbemostat culture at the dilution rate of 0.075 h -1 as described Fig. 1. Change in formaldehyde productivity and viability of cells during treatment. previously.3) Cells were collected by centriThe cell suspension (20 mg as dry cell weight]ml fugation, washed with 0.1 M potassium of KPB) was incubated at 37°C for the period phosphate buffer, p H 7.5 (KPB), suspended indicated. Formaldehyde productivity was determined as described in the text. Formaldehyde in KPB, and subjected to heat-treatment. productivity with control cells is 100% and is The formaldehyde production reaction was expressed as relative activity. The viability of performed at 4°C and p H 6 . 0 in a 50-ml cells during the incubation was counted by Erlenmeyer flask equipped with a rubber spreading the diluted incubation mixture onto the glucose medium plate. balloon for continuous oxygen supply conFormaldehyde productivity: (3, the initial velotaining 3 rnl of the reaction mixture.3) city; e , the final amount of formaldehyde U n d e r these conditions, intact cells produced produced. Viability of cells: cells were incubated 950 m M formaldehyde after a 10-h reaction, at 28°C (A) or at 37°C (~).
490
Shg.al and TANI
[J. Ferment. Technol.,
Table 1. Effects of treatment at various temperatures on formaldehyde productivity. Incubation Temperature
Time (days)
Formaldehyde productivity Initial velocity Final concn. (mM/h) (mM)
4°C
1 2 3
220 220 215
950 950 930
16°C
1 2 3
190 185 178
850 830 779
28°C
1 2 3
180 176 160
665 65O 482
37°C
1 2 3
330 310 300
1090 1010 980
50°C
1 2 3
125 33 7
599 228 123
220
950
Intact cells
The cell suspension (20 mg as dry cell weight/ml of KPB) was incubated at the indicated temperature for 1, 2, or 3 days, and formaldehyde productivity was assayed as described in the text. T a b l e 1, e n h a n c e m e n t with respect to both the initial velocity and final formaldehyde concentration was observed with cells treated at 37°C. W h e n cells were incubated in the culture medium, the activities were lost. Therefore, p H and the concentration of potassium phosphate buffer in the treatment at 37°C were investigated. T r e a t m e n t with 0.1 M potassium phosphate buffer between p H 6 . 0 and 8.0 resulted in high activity. T h e o p t i m u m concentration o f potassium phosphate buffer for the treatment was I 0 0 500 raM. C h a n g i n g the ionic strength 7) by a d d i n g N a C I to the incubation mixture up to 2 M did not affect the productivity. T h e time course of cell activation for formaldehyde production d u r i n g the treatment is shown in Fig. I. T h e initial velocity reached a maxim u m o f 330 m M / h , w h i c h was i m p r o v e d by 50% after a 24-h reaction. 1090 m M formaldehyde was produced in the reaction
mixture after a 10-h reaction. T h e viability o f the cells decreased during the incubation at 37°C and it was less than 2 % after 24 h (Fig. 1). I n contrast, viability after the 24-h incubation at 28°C was more than 90%. T h e stability of catalytic activity was c o m p a r e d between the heat-treated and intact cells. As shown in Fig. 2, the intact cells gradually lost their ability to produce formaldehyde during the incubation at 28°C. T h e a m o u n t o f formaldehyde produced fell to 70% after 1 day, and to less than half of the initial value after one week. I n contrast, heat-treated cells retained a high productivity after 5 days. Stabilization was also observed by U V - i r r a d i a t i o n which lead the cells to death. T h e catalytic activity o f the U V irradiated ceils was stabilized as for the heattreated cells, although an i m p r o v e m e n t in formaldehyde productivity was not observed in the U V irradiated cells. T h e stability of
~100C
U
50C 0 0 "I" -r
491
Formaldehyde Production by Methanol Yeast
Vol. 65, 1987]
0 I 1
I 3
I 5
I 8
(days) Fig. 2. Effects of the treatment on catalytic stability. , The suspension of heat-treated cells (©) or intact cells (0) in KPB (20 mg as dry cell weight/ml) was incubated at 28°C for the period indicated and formaldehyde productivity was assayed as described in the text. Incubation
time
the heat-treated cells or the U V - i r r a d i a t e d cells reflects alcohol oxidase activity. T h e activity is strictly controlled physiologically because of the toxicity of the reaction products formaldehyde and H202. 8,9) W h e n cells are alive, alcohol oxidase m a y be decreased by the conditions in the incubation mixture without methanol. F o r m a l d e h y d e production was inhibited by the addition of various Sift inhibitors, but not by p-chloromercuribenzoate (PCMB). *,8) However, the incubation of cells with 1 m M P C M B at 37°C for 2 4 h decreased the productivity. I n freezed-thawed or freezedried cells formaldehyde productivity was also slightly improved. N o significant differences in the activities of alcohol oxidase, catalase, and formaldehyde and formate dehydrogenases were detected between the intact and heat-treated ceils. These observations suggest that the i m p r o v e m e n t o f
formaldehyde productivity, especially for the initial velocity of heat-treated cells, was due to the increase in m e m b r a n e permeability o f the substrates. T h e addition of NaN3 during treatment at 37°C enhanced formaldehyde p r o d u c t i o n with the o p t i m u m concentration of NaN3 for formaldehyde production being 50 m M . T h e cells, which were heat-treated in K P B containing 50 m M NAN3, produced 1150 m M formaldehyde after a 10-h reaction. This was 21% higher than for the intact cells. W h e n NaN3 was added to the cell suspension, the cells turned dark red and then disappeared with the addition of methanol. T h e exact mechanism for the increase in formaldehyde productivity due to NaN3 is now u n d e r investigation.
Acknowledgments We would like to thank Professor H. Yamada of tile Department of Agricultural Chemistry, Kyoto University, for his encouragement throughout this work.
References 1) Tani, Y., Sakai, Y., Yamada, H.: Agric. Biol. Chem., 49, 2699 (1985). 2) Tani, Y., Sakai, Y., Yamada, H.: J. Ferment. Technol., 63, 443 (1985). 3) Sakai, Y., Tani, Y.: Agric. Biol. Chem., 50, 2615 (1986). 4) Tani, Y., Mitani, Y., Yamada, H.: Agric. Biol. Chem., 48, 431 (1984). 5) Tani, Y., Yonehara, T., Mitani, Y., Yamada, H.: J. Biotechnol., 1, 119 (1984). 6) Tani, Y., Mitani, Y., Yamada, H.: J. Ferment. Technol., 62, 99 (1984). 7) Chibata, I., Tosa, T., Sato, T.: Appl. Microbiol., 27, 878 (1974). 8) Kato, N., Omori, Y., Tani, Y., Ogata, K.: Eur. J. Biochem., 64, 341 (1976). 9) Veenhuis, M., van Dijken, J.P., Harder, W.: Adv. Microbial Physiol., 24, 1 (1983). (Received March 13, 1987)
Note
Formaldehyde Production with Heat-Treated Cells of Methanol Yeast YASUYOSHI SAKAI* a n d YOSHIKI TANI
Research Cent*rfor Cell and Tissue Culture and *Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University,Kyoto 606, Japan
Chemostat-grown cells of a methanol investigated for formaldehyde production. were improved by preincubation of the cells phate buffer (pH 7.5) containing 50 mlV[ formaldehyde after a 10-h reaction.
yeast, Candida boidinii $2 AOU-1, were The productivity and catalytic stability at 37°C for 24 h in 0.1 M potassium phosNaNa. These cells produced 1150 m M
In preceding papers, 1-3) we reported on producing an initial velocity of 240 m M / h . formaldehyde production by an alcohol The cell suspension was incubated at oxidase-enhanced m u t a n t strain AOU-1 of various temperatures and the formaldehyde Candida boidinii $2. In our previous study, 3) productivity was assayed. As shown in it was suggested that the m e m b r a n e permeability of the substrate limits the rate of the reaction because less improvement in the ©© initial velocity of formaldehyde production ° was seen compared to that for the final _ _ ~ lO0 ~o concentration or alcohol oxidase activity. m A T P production was established with Zymolyase®-treated or sorbitol-treated metha0 nol yeast cells. 4-e) In this paper, improve= ment in formaldehyde productivity of heattreated yeast cells will be described. 3'6 C. boidinii $2 AOU-1 cells were grown on I n c u b a t i o n t i m e (h) a methanol-limited cbemostat culture at the dilution rate of 0.075 h -1 as described Fig. 1. Change in formaldehyde productivity and viability of cells during treatment. previously.3) Cells were collected by centriThe cell suspension (20 mg as dry cell weight]ml fugation, washed with 0.1 M potassium of KPB) was incubated at 37°C for the period phosphate buffer, p H 7.5 (KPB), suspended indicated. Formaldehyde productivity was determined as described in the text. Formaldehyde in KPB, and subjected to heat-treatment. productivity with control cells is 100% and is The formaldehyde production reaction was expressed as relative activity. The viability of performed at 4°C and p H 6 . 0 in a 50-ml cells during the incubation was counted by Erlenmeyer flask equipped with a rubber spreading the diluted incubation mixture onto the glucose medium plate. balloon for continuous oxygen supply conFormaldehyde productivity: (3, the initial velotaining 3 rnl of the reaction mixture.3) city; e , the final amount of formaldehyde U n d e r these conditions, intact cells produced produced. Viability of cells: cells were incubated 950 m M formaldehyde after a 10-h reaction, at 28°C (A) or at 37°C (~).
490
Shg.al and TANI
[J. Ferment. Technol.,
Table 1. Effects of treatment at various temperatures on formaldehyde productivity. Incubation Temperature
Time (days)
Formaldehyde productivity Initial velocity Final concn. (mM/h) (mM)
4°C
1 2 3
220 220 215
950 950 930
16°C
1 2 3
190 185 178
850 830 779
28°C
1 2 3
180 176 160
665 65O 482
37°C
1 2 3
330 310 300
1090 1010 980
50°C
1 2 3
125 33 7
599 228 123
220
950
Intact cells
The cell suspension (20 mg as dry cell weight/ml of KPB) was incubated at the indicated temperature for 1, 2, or 3 days, and formaldehyde productivity was assayed as described in the text. T a b l e 1, e n h a n c e m e n t with respect to both the initial velocity and final formaldehyde concentration was observed with cells treated at 37°C. W h e n cells were incubated in the culture medium, the activities were lost. Therefore, p H and the concentration of potassium phosphate buffer in the treatment at 37°C were investigated. T r e a t m e n t with 0.1 M potassium phosphate buffer between p H 6 . 0 and 8.0 resulted in high activity. T h e o p t i m u m concentration o f potassium phosphate buffer for the treatment was I 0 0 500 raM. C h a n g i n g the ionic strength 7) by a d d i n g N a C I to the incubation mixture up to 2 M did not affect the productivity. T h e time course of cell activation for formaldehyde production d u r i n g the treatment is shown in Fig. I. T h e initial velocity reached a maxim u m o f 330 m M / h , w h i c h was i m p r o v e d by 50% after a 24-h reaction. 1090 m M formaldehyde was produced in the reaction
mixture after a 10-h reaction. T h e viability o f the cells decreased during the incubation at 37°C and it was less than 2 % after 24 h (Fig. 1). I n contrast, viability after the 24-h incubation at 28°C was more than 90%. T h e stability of catalytic activity was c o m p a r e d between the heat-treated and intact cells. As shown in Fig. 2, the intact cells gradually lost their ability to produce formaldehyde during the incubation at 28°C. T h e a m o u n t o f formaldehyde produced fell to 70% after 1 day, and to less than half of the initial value after one week. I n contrast, heat-treated cells retained a high productivity after 5 days. Stabilization was also observed by U V - i r r a d i a t i o n which lead the cells to death. T h e catalytic activity o f the U V irradiated ceils was stabilized as for the heattreated cells, although an i m p r o v e m e n t in formaldehyde productivity was not observed in the U V irradiated cells. T h e stability of
~100C
U
50C 0 0 "I" -r
491
Formaldehyde Production by Methanol Yeast
Vol. 65, 1987]
0 I 1
I 3
I 5
I 8
(days) Fig. 2. Effects of the treatment on catalytic stability. , The suspension of heat-treated cells (©) or intact cells (0) in KPB (20 mg as dry cell weight/ml) was incubated at 28°C for the period indicated and formaldehyde productivity was assayed as described in the text. Incubation
time
the heat-treated cells or the U V - i r r a d i a t e d cells reflects alcohol oxidase activity. T h e activity is strictly controlled physiologically because of the toxicity of the reaction products formaldehyde and H202. 8,9) W h e n cells are alive, alcohol oxidase m a y be decreased by the conditions in the incubation mixture without methanol. F o r m a l d e h y d e production was inhibited by the addition of various Sift inhibitors, but not by p-chloromercuribenzoate (PCMB). *,8) However, the incubation of cells with 1 m M P C M B at 37°C for 2 4 h decreased the productivity. I n freezed-thawed or freezedried cells formaldehyde productivity was also slightly improved. N o significant differences in the activities of alcohol oxidase, catalase, and formaldehyde and formate dehydrogenases were detected between the intact and heat-treated ceils. These observations suggest that the i m p r o v e m e n t o f
formaldehyde productivity, especially for the initial velocity of heat-treated cells, was due to the increase in m e m b r a n e permeability o f the substrates. T h e addition of NaN3 during treatment at 37°C enhanced formaldehyde p r o d u c t i o n with the o p t i m u m concentration of NaN3 for formaldehyde production being 50 m M . T h e cells, which were heat-treated in K P B containing 50 m M NAN3, produced 1150 m M formaldehyde after a 10-h reaction. This was 21% higher than for the intact cells. W h e n NaN3 was added to the cell suspension, the cells turned dark red and then disappeared with the addition of methanol. T h e exact mechanism for the increase in formaldehyde productivity due to NaN3 is now u n d e r investigation.
Acknowledgments We would like to thank Professor H. Yamada of tile Department of Agricultural Chemistry, Kyoto University, for his encouragement throughout this work.
References 1) Tani, Y., Sakai, Y., Yamada, H.: Agric. Biol. Chem., 49, 2699 (1985). 2) Tani, Y., Sakai, Y., Yamada, H.: J. Ferment. Technol., 63, 443 (1985). 3) Sakai, Y., Tani, Y.: Agric. Biol. Chem., 50, 2615 (1986). 4) Tani, Y., Mitani, Y., Yamada, H.: Agric. Biol. Chem., 48, 431 (1984). 5) Tani, Y., Yonehara, T., Mitani, Y., Yamada, H.: J. Biotechnol., 1, 119 (1984). 6) Tani, Y., Mitani, Y., Yamada, H.: J. Ferment. Technol., 62, 99 (1984). 7) Chibata, I., Tosa, T., Sato, T.: Appl. Microbiol., 27, 878 (1974). 8) Kato, N., Omori, Y., Tani, Y., Ogata, K.: Eur. J. Biochem., 64, 341 (1976). 9) Veenhuis, M., van Dijken, J.P., Harder, W.: Adv. Microbial Physiol., 24, 1 (1983). (Received March 13, 1987)