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Properties of sake yeast mutants resistant to isoamyl monochloroacetate
JOURNAL OPFERMENTATIONANDBIOENCIINEERING Vol. 80, No. 3, 291-293.1995
Properties of Sake Yeast Mutants Resistant to Isoamyl Monochloroacetate MUTSUMI WATANABE,* HIDE0 NAGAI,
AND
KYOICHI KONDO
Research Laboratory, Hakutsuru Sake Brewing Co. Ltd., 4-5-5 Sumiyoshiminami-machi, Higashinada-ku, Kobe, Hyogo 658, Japan
Received10April 19951Accepted 14June 1995 Isoamyl monochloroacetate (i-AmOClAc) inhibited the growth of sake yeast. AOFlO, which is resistant to isoamyl monofluoroacetate and has low esterase activity toward isoamyl acetate (i-AmOAc), also showed resistance to i-AmOClAc. Monochloroacetic acid formed by an esterase through the hydrolysis of I-AmOCk was more effective at glycolysis inhibition than i-AmOClAc. Forty five mutants resistant to i-AmOClAc were isolated from a parental haploid strain (I-IL-69) derived from sake yeast. Three had less than 20% of the original activity of the e&erase. AU mutants fermented sake mash as well as the parent strain, I&69, and accumulated 1.3 to l.Ffold the levels of i-AmOAc and 1.8 to 2.4-fold the levels of isobutyl acetate accumulated by EL-69. Surprisingly one mutant (CL-90), which accumulated 1.7-fold the amount of i-AmOAc and l.ffold the amount of ethyl acetate, exhibited increased alcohol acetyltransferase (EC 2.3.1.84) activity (l.dfold). Esterase patterns were also examined by gel electrophoresis and activity staining. Changes in the intensity of the strongest band (EsT2) were closely related to the activity of the esterase toward i-AmOAc. [Key words: isoamyl monochloroacetate, sake yeast, esterase, isoamyl acetate, activity staining]
On the other hand, non-fluorine monohaloacetic acids (XAcOH) inhibit yeast growth by a substantially different mechanism to that of FAcOH. XAcOH, which are sulfhydryl reagents, inhibit SH-enzymes such as aldolase and alcohol dehydrogenase, thereby stopping glycolysis. XAcOH directly attack SH-groups in enzymes, thus there is no need to metabolize it into other compounds, nor is there competition by AcOH. The use of monohaloacetic esters seems to be preferable for the isolation of mutants which are lacking esterase. In this paper, mutants with low esterase activity were isolated by selection based on resistance to isoamyl monochloroacetate (i-AmOClAc), and their improved iAmOAc productivity was confirmed in a small scale sake brewing test. Esterase patterns were also examined by polyacrylamide gel electrophoresis (7, 8). XAcOH reactivity depends greatly on the halogen species (9, 10). i-AmOClAc (bp. 92-94’C/34mm Hg) and isoamyl monobromoacetate (i-AmOBrAc, bp. 86-87”C/ 11 mm Hg) were chosen and synthesized. Since the actions of these esters are determined by the halogen atom present at 2-position of the acetyl group, the cleavage of their ester bonds is not necessary in order to express their toxicity. First, it was determined whether or not AOFlO exhibited resistance to these esters. Cultivation was performed at 30°C for 3 d on a reciprocal shaker at 120 rpm in a test tube (15 ml) containing 1 ml of SD medium (0.67% Difco Yeast Nitrogen Base, 2% glucose) and sealed with Para Film to prevent evaporation of the esters. Table 1 shows that both esters inhibited the growth of yeast. i-AmOBrAc exhibited a strong inhibitory effect, even at 1% of the concentration of i-AmOClAc. This finding confirms that bromine is a much better leaving group than chlorine (9, lo), and suggests that the action of these esters is due to alkylation of the SH-group. Free XAcOH did not inhibit yeast growth when the same concentration as these esters was employed. The permeability of the esters into the cell therefore must have been increased by esterification.
Isoamyl acetate (i-AmOAc) has a fruit-like flavor and plays an important role in determining the quality of premium Japanese sake (rice wine), called Ginjo. This ester is formed from isoamyl alcohol and acetyl CoA by alcohol acetyltransferase (AATase, EC 2.3.1.84) (l), and is hydrolyzed at the same time by esterase in sake mash (2, 3). The accumulation of i-AmOAc is thought to depend on the ratio of activities of AATase and esterase toward i-AmOAc (4). In a previous paper (5), AOFlO was isolated by selection based on its resistance to isoamyl monofluoroacetate (i-AmOFAc) from a haploid strain, HL-69 (MATa), derived from sake yeast Kyokai no. 10. AOFlO exhibited reduced esterase activity toward i-AmOAc. AOFlO fermented sake mash as efficiently as HL-69, and the resultant sake contained 1.5 to 2.0 times higher amounts of i-AmOAc and isobutyl acetate (i-BuOAc) than that obtained with HL-69. These findings are similar to those of a previous study in which mutants selected by diazo staining of colonies were observed to have low esterase activity (4). Since the i-AmOFAc method is a positive selection technique, a high isolation efficiency for mutants with low esterase activity can be expected. However, monofluoroacetic acid (FAcOH) is extremely toxic (oral lethal dose, 2-5 mg/kg) (6), and i-AmOFAc is more volatile than FAcOH. Therefore, special precautions and handling are required when using i-AmOFAc. Cleavage of the ester bond is necessary for i-AmOFAc to express its toxicity. The resultant FAcOH must be metabolized into monofluorocitric acid in order to inhibit aconitase in the Krebs cycle. Many i-AmOFAc resistant mutants which have the same activity as their parent strain accumulated acetic acid in the cell. This accumulation is believed to compete with the metabolism of FAcOH. These mutants acquired their resistance to i-AmOFAc through an unknown mechanism which was independent of the deletion of the esterase. *
Corresponding author.
291
292
J. FERMENT. BIOENG.,
WATANABE ET AL.
TABLE 1.
Strain
Inhibition of AOFlO and HL-69 growth by i-AmOCIAc and i-AmOBrAc i-AmOClAc
@pm)
i-AmOBrAc (ppm)
Component None
60
50
40
30
0.75
0.50
0.25
AOFlOa
-
tt
tt
i+
-
-
it
++
HL-69
-
-
+
it
-
tt
ft
it
Growth index, it; good growth, +; slightly lower growth, -; no growth. * AOFIO is an i-AmOFAc resistant mutant which has 12% of the original esterase activity toward i-AmOAc.
AOFlO was more resistant to i-AmOClAc than HL69. This was most likely caused by monochloroacetic acid formed by the esterase through the hydrolysis of i-AmOClAc, because it is more effective at inhibiting glycolysis than i-AmOClAc in the cell. It was possible to isolate mutants with low esterase activity by selection based on resistance to i-AmOClAc. The reverse was found for i-AmOBrAc, in other words, HL-69 was more resistant than AOFlO. In this case, the concentration of i-AmOBrAc was very low. Therefore, the total amount of monobromoacetic acid in the cell may have been reduced by the action of the esterase present in the periplasm, and which hydrolyzed the majority of iAmOBrAc outside of the plasma membrane (11). HL-69 was mutagenized with 3% ethyl methane sulfonate at 30°C for 6Omin, in order to obtain mutants resistant to i-AmOClAc. About 106 cells were spread on a CL plate (0.67% Difco Yeast Nitrogen Base, 2% glucose, 2% agar, pH 7) containing 70-90 ppm i-AmOClAc. Immediately after spreading, the plates were sealed with Para Film and incubated at 30°C for 6d. The colonies which developed were isolated as mutants resistant to iAmOClAc. The screening of mutants with lowered esterase activity toward i-AmOAc was performed as previously described (5). Three independent experiments were carried out. In each experiment, one mutant which had less than 20% of the original esterase activity was obtained among 15 mutants resistant to i-AmOClAc (CL70, CL-80 and CL-90 in Table 2). This result confirmed that mutants with low esterase activity could be isolated by selection based on their resistance to i-AmOClAc. These mutants, however, had slightly higher esterase activity than AOFlO. The small scale sake brewing test was performed according to a method previously described (5). Sake mash containing 400g of rice was fermented at 12.5’C for 19d. All of the mutants fermented the sake mash as efficiently as HL-69. The components of the brewed sake are shown in Table 3. The values for sake meter, alcohol concentration, acidity and amino acidity in the sake TABLE 2. Strains CL-70 CL-80 CL-90 AOFlO HL-69 (narent)
TABLE 3.
Esterase and AATase activities
Esterase (ppm/h/mg protein)
AATase @pm/h/mg protein)
0.20 0.19 0.30 0.17 1.44
4.0 3.8 5.5 3.7 3.5
a CL-70, CL-80 and CL-90 were resistant to i-AmOClAc, while AOFlO was resistant to i-AmOFAc. All mutants were derived from the parent strain HL-69.
Components of sake brewed with various strains Straina CL-70 CL-80 CL-90 AOFlO HL-69
Sake meter +4.0 Alcohol (%) 19.1 Acidity (ml) 2.1 Amino acidity (ml) 2.0 n-Propyl alcohol (ppm) 93.8 i-Amy1 alcohol (ppm) 234 i-Butyl alcohol (ppm) 71.8 i-Amy1 acetate (ppm) 11.3 i-Butyl acetate (ppm) 1.2 Ethyl acetate (ppm) 105 Ethylcaproate(ppm) 1.3
+4.0 19.2 2.2 1.8 95.4 239 68.3 10.5 1.l 101 1.3
+1.8 19.1 2.6 1.9 132 237 71.9 14.2 0.9 123 1.1
+4.0 19.2 2.2 1.9 92.7 235 73.0 11.7 1.3 107 1.1
f4.3 19.2 2.2 1.9 97.3 244 71.3 8.3 0.5 94.6 1.3
KlO f4.0 19.2 2.5 1.7 159 228 68.4 7.1 0.4 99.3 0.9
a KlO is Kyokai no. 10. Other strains are the same as in Table 2.
brewed with mutants, except CL-90, were similar to those of the parent strain HL-69. In the sake brewed with CL-90, the sake meter was slightly lower and the acidity was slightly higher. Mutants resistant to iAmOClAc produced levels of i-AmOAc 1.3 to 1.7-fold greater and levels of i-BuOAc 1.8 to 2.4-fold greater than HL-69, while the contents of isoamyl alcohol and isobutyl alcohol were not different. These results are identical to those for AOFlO, and suggest that the improved i-AmOAc productivity of the mutants was due to their lowered esterase activity (4, 5). The increase in iBuOAc content has been suggested to be a characteristic of these mutants because the esterase hydrolyzed iBuOAc faster than i-AmOAc (12). CL-90 accumulated the highest amount of i-AmOAc, in spite of having the highest esterase activity among these mutants. Only CL-90 produced an increased amount (1.3-fold) of ethyl acetate (Table 3). The activity of AATase, which catalyzes the formation of acetic esters, was determined according to the method of Fujii et al. (13). CL-90 had AATase activity which was 1.6 times greater than the other strains (Table 2). Recently, molecular cloning of the ATFZ gene, which encodes AATase, has been reported (13). Transformants carrying multicopies of ATFI expressed elevated levels of AATase activity and produced significant amounts of ethyl acetate and i-AmOAc. It is believed that the high i-AmOAc productivity of CL-90 was partly due to the increase in AATase activity. AATase is an SH-enzyme containing a relatively high number of cysteine residues (1, 13). i-AmOClAc has a structure very similar to the AATase product i-AmOAc. Based on these observations, the relationship between i-AmOClAc and AATase activity was suggested. Many investigators have reported that yeast possess several isozymes of esterase (2, 3, 7, 8, 11, 12, 14-16). Wohrmann and Lange (7) demonstrated using gel electrophoresis that there were four loci (ESTl-4) for esterase isozymes in wine yeast. Esterase patterns were examined by polyacrylamide gel electrophoresis in order to identify which isozyme was deleted in our mutants. The electrophoresis was performed at 150 V for 2 h. The volume of each sample applied was adjusted to 400 pg of protein. Immediately after the electrophoresis, the gel was stained with 250ppm 1-naphthyl acetate and 400 ppm Fast blue salt B (Merck) in 0.1 M potassium phosphate buffer (pH 7.0) at 30°C for 20min. AOFll and AOF16, which have already been isolated (5), were also examined simultaneously.
NOTES
origin
293
The authors are grateful to Professor Takahito Suzuki of Nara Women’s University for his suggestions concerning monoiodoacetic acid. REFERENCES
cEST1 cEST2
hont FIG. 1. Polyacrylamide gel electrophoresis of esterase. The esterase activity toward I-naphthyl acetate was stained with Fast blue salt B. Lane 1, K10; lane 2, HL-69; lane 3, CL-90; lane 4, CL-80; lane 5, CL-70; lane 6, AOFlO; lane 7, AOF16; lane 8, HL-68; lane 9, AOFll. KlO is sake yeast Kyokai no. 10. HL-68 and HL-69 are haploid strains derived from KlO. CL-70, CL-80 and CL-90 are mutants resistant to i-AmOClAc, while AOFlO, AOFll and AOF are mutants resistant to i-AmOFAc. All mutants had less than 20% of the original esterase activity toward i-AmOAc,exceptAOFll which
wasderivedfrom HL-68 and had 60%. Figure 1 presents the results of activity staining for the esterase toward 1-naphthyl acetate. The strongest band and the band having the smallest mobility appeared to be EST2 and ESTI, respectively (7). AOFlO, AOF16, CL-70 and CL-80 completely lost the EST2 band, while CL-90, which had slightly higher esterase activity toward i-AmOAc than the other four mutants exhibited a faint EST2 band. AOFll, which had 60% of this activity, showed a little weak EST2 band. Therefore, it can be seen that the band intensity of EST2 changed in close relation to the activity of the esterase toward i-AmOAc. This esterase has been considered to correspond to EST2 (8). Mutants isolated by diazo staining of colonies, which had low esterase activity toward i-AmOAc, also lacked the EST2 band (Fukuda, K. et al., Abstr. Annu. Meet., Japan Sot. Biosci. Biotech. Agrochem., p.348, 1993), leading to the conclusion that their mutants and our mutants must have been missing the same EST2 isozyme. Other minor bands which appeared in the gel exhibited no differences between the mutant and parent strains, suggesting that they were not involved in iAmOAc hydrolysis.
1. Yoshioka, K. and Hashinfoto, N.: Ester formation by alcohol acetyltransferase from brewers’ yeast. Agric. Biol. Chem., 45, 2183-2190 (1981). 2. Schermers, F. IX., Dues, J. H., and Macleod, M.: Studies on yeast esterase. J. Inst. Brew., 82, 170-174 (1976). 3. Parkkioen, E. and SuomaIainea, H.: Esterases of baker’s yeast. III. The ester/acid ratio in model solutions. J. Inst. Brew., 82, 170-174 (1982). 4. Yanagiucki, T., KIyokawa, Y., and WakaI, Y.: Isolation of sake-yeast strains accumulating large amounts of isoamyl acetate. Hakkokogaku, 67, 159-165 (1989). 5. Watanabe, M., Tanaka, N., MIshIma, H., and Takemnra, S.: Isolation of sake yeast mutants resistant to isoamyl monofluoroacetate to improve isoamyl acetate productivity. J. Ferment. Bioeng., 76, 229-231 (1993). 6. WiadhoIz, M., BudavarI, S., Stroumtsos, L. Y., and FertIg, M. N. (ea.): The Merck index (9th ed.), p. 539. Merck & Co., Rahway, USA (1976). 7. Wahrmann, K. and Lange, P.: The polymorphism of esterases in yeast. J. Inst. Brew., 86, 174 (1980). 8. Yanagiucki, T., Kiyokawa, Y., and WakaI, Y.: Isoamyl acetate accumulation in sake mash and isoamyl acetate hydrolysis activity of sake-yeast strains. Hakkokogaku, 67, 419-425 (1989). 9. Nippon Kagaku-kai: Shin jikken kagaku koza, vol. 20, Seibutsu kagaku I, p. 191-197. Nippon Kagaku-kai, Tokyo (1978). (in Japanese) 10. Barron, E.S.G.: Thiol groups of biological importance, p. 201-266. In Nord, F. F. (ed.), Adv. enzymol., vol. 11. Interscience Publishers, New York, USA (1951). 11. ParkkInen, E., Oura, E., and SuomaIaInen, H.: The esterases of baker’s yeast. I. Activity and localization in the cell. J. Inst. Brew., 84, 5-8 (1978). 12. Wakai, Y., Yanagiuchi, T., and Kiyokawa, Y.: Properties of an isoamyl acetate hydrolytic enzyme from a sake yeast strain. Hakkokogaku, 68, 101-105 (1990). 13. Fnjii, T., Nagasawa, N., Iwamatsu, A., Bogaki, T., Tamai, Y., and Hamachi, M.: Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene. Appl. Environ. Microbial., 60, 2786-2792 (1994). 14. ParkkInen, E.: Multiple forms of carboxyesterases in baker’s yeast. Cellul. Molec. Biology, 26, 147-154 (1980). IS. KurIyama, K., Askida, S., Saito, Y., Rata, Y., Suginnmi, K., and Imayasu, S.: Ethyl caproate synthesis and h
Properties of Sake Yeast Mutants Resistant to Isoamyl Monochloroacetate MUTSUMI WATANABE,* HIDE0 NAGAI,
AND
KYOICHI KONDO
Research Laboratory, Hakutsuru Sake Brewing Co. Ltd., 4-5-5 Sumiyoshiminami-machi, Higashinada-ku, Kobe, Hyogo 658, Japan
Received10April 19951Accepted 14June 1995 Isoamyl monochloroacetate (i-AmOClAc) inhibited the growth of sake yeast. AOFlO, which is resistant to isoamyl monofluoroacetate and has low esterase activity toward isoamyl acetate (i-AmOAc), also showed resistance to i-AmOClAc. Monochloroacetic acid formed by an esterase through the hydrolysis of I-AmOCk was more effective at glycolysis inhibition than i-AmOClAc. Forty five mutants resistant to i-AmOClAc were isolated from a parental haploid strain (I-IL-69) derived from sake yeast. Three had less than 20% of the original activity of the e&erase. AU mutants fermented sake mash as well as the parent strain, I&69, and accumulated 1.3 to l.Ffold the levels of i-AmOAc and 1.8 to 2.4-fold the levels of isobutyl acetate accumulated by EL-69. Surprisingly one mutant (CL-90), which accumulated 1.7-fold the amount of i-AmOAc and l.ffold the amount of ethyl acetate, exhibited increased alcohol acetyltransferase (EC 2.3.1.84) activity (l.dfold). Esterase patterns were also examined by gel electrophoresis and activity staining. Changes in the intensity of the strongest band (EsT2) were closely related to the activity of the esterase toward i-AmOAc. [Key words: isoamyl monochloroacetate, sake yeast, esterase, isoamyl acetate, activity staining]
On the other hand, non-fluorine monohaloacetic acids (XAcOH) inhibit yeast growth by a substantially different mechanism to that of FAcOH. XAcOH, which are sulfhydryl reagents, inhibit SH-enzymes such as aldolase and alcohol dehydrogenase, thereby stopping glycolysis. XAcOH directly attack SH-groups in enzymes, thus there is no need to metabolize it into other compounds, nor is there competition by AcOH. The use of monohaloacetic esters seems to be preferable for the isolation of mutants which are lacking esterase. In this paper, mutants with low esterase activity were isolated by selection based on resistance to isoamyl monochloroacetate (i-AmOClAc), and their improved iAmOAc productivity was confirmed in a small scale sake brewing test. Esterase patterns were also examined by polyacrylamide gel electrophoresis (7, 8). XAcOH reactivity depends greatly on the halogen species (9, 10). i-AmOClAc (bp. 92-94’C/34mm Hg) and isoamyl monobromoacetate (i-AmOBrAc, bp. 86-87”C/ 11 mm Hg) were chosen and synthesized. Since the actions of these esters are determined by the halogen atom present at 2-position of the acetyl group, the cleavage of their ester bonds is not necessary in order to express their toxicity. First, it was determined whether or not AOFlO exhibited resistance to these esters. Cultivation was performed at 30°C for 3 d on a reciprocal shaker at 120 rpm in a test tube (15 ml) containing 1 ml of SD medium (0.67% Difco Yeast Nitrogen Base, 2% glucose) and sealed with Para Film to prevent evaporation of the esters. Table 1 shows that both esters inhibited the growth of yeast. i-AmOBrAc exhibited a strong inhibitory effect, even at 1% of the concentration of i-AmOClAc. This finding confirms that bromine is a much better leaving group than chlorine (9, lo), and suggests that the action of these esters is due to alkylation of the SH-group. Free XAcOH did not inhibit yeast growth when the same concentration as these esters was employed. The permeability of the esters into the cell therefore must have been increased by esterification.
Isoamyl acetate (i-AmOAc) has a fruit-like flavor and plays an important role in determining the quality of premium Japanese sake (rice wine), called Ginjo. This ester is formed from isoamyl alcohol and acetyl CoA by alcohol acetyltransferase (AATase, EC 2.3.1.84) (l), and is hydrolyzed at the same time by esterase in sake mash (2, 3). The accumulation of i-AmOAc is thought to depend on the ratio of activities of AATase and esterase toward i-AmOAc (4). In a previous paper (5), AOFlO was isolated by selection based on its resistance to isoamyl monofluoroacetate (i-AmOFAc) from a haploid strain, HL-69 (MATa), derived from sake yeast Kyokai no. 10. AOFlO exhibited reduced esterase activity toward i-AmOAc. AOFlO fermented sake mash as efficiently as HL-69, and the resultant sake contained 1.5 to 2.0 times higher amounts of i-AmOAc and isobutyl acetate (i-BuOAc) than that obtained with HL-69. These findings are similar to those of a previous study in which mutants selected by diazo staining of colonies were observed to have low esterase activity (4). Since the i-AmOFAc method is a positive selection technique, a high isolation efficiency for mutants with low esterase activity can be expected. However, monofluoroacetic acid (FAcOH) is extremely toxic (oral lethal dose, 2-5 mg/kg) (6), and i-AmOFAc is more volatile than FAcOH. Therefore, special precautions and handling are required when using i-AmOFAc. Cleavage of the ester bond is necessary for i-AmOFAc to express its toxicity. The resultant FAcOH must be metabolized into monofluorocitric acid in order to inhibit aconitase in the Krebs cycle. Many i-AmOFAc resistant mutants which have the same activity as their parent strain accumulated acetic acid in the cell. This accumulation is believed to compete with the metabolism of FAcOH. These mutants acquired their resistance to i-AmOFAc through an unknown mechanism which was independent of the deletion of the esterase. *
Corresponding author.
291
292
J. FERMENT. BIOENG.,
WATANABE ET AL.
TABLE 1.
Strain
Inhibition of AOFlO and HL-69 growth by i-AmOCIAc and i-AmOBrAc i-AmOClAc
@pm)
i-AmOBrAc (ppm)
Component None
60
50
40
30
0.75
0.50
0.25
AOFlOa
-
tt
tt
i+
-
-
it
++
HL-69
-
-
+
it
-
tt
ft
it
Growth index, it; good growth, +; slightly lower growth, -; no growth. * AOFIO is an i-AmOFAc resistant mutant which has 12% of the original esterase activity toward i-AmOAc.
AOFlO was more resistant to i-AmOClAc than HL69. This was most likely caused by monochloroacetic acid formed by the esterase through the hydrolysis of i-AmOClAc, because it is more effective at inhibiting glycolysis than i-AmOClAc in the cell. It was possible to isolate mutants with low esterase activity by selection based on resistance to i-AmOClAc. The reverse was found for i-AmOBrAc, in other words, HL-69 was more resistant than AOFlO. In this case, the concentration of i-AmOBrAc was very low. Therefore, the total amount of monobromoacetic acid in the cell may have been reduced by the action of the esterase present in the periplasm, and which hydrolyzed the majority of iAmOBrAc outside of the plasma membrane (11). HL-69 was mutagenized with 3% ethyl methane sulfonate at 30°C for 6Omin, in order to obtain mutants resistant to i-AmOClAc. About 106 cells were spread on a CL plate (0.67% Difco Yeast Nitrogen Base, 2% glucose, 2% agar, pH 7) containing 70-90 ppm i-AmOClAc. Immediately after spreading, the plates were sealed with Para Film and incubated at 30°C for 6d. The colonies which developed were isolated as mutants resistant to iAmOClAc. The screening of mutants with lowered esterase activity toward i-AmOAc was performed as previously described (5). Three independent experiments were carried out. In each experiment, one mutant which had less than 20% of the original esterase activity was obtained among 15 mutants resistant to i-AmOClAc (CL70, CL-80 and CL-90 in Table 2). This result confirmed that mutants with low esterase activity could be isolated by selection based on their resistance to i-AmOClAc. These mutants, however, had slightly higher esterase activity than AOFlO. The small scale sake brewing test was performed according to a method previously described (5). Sake mash containing 400g of rice was fermented at 12.5’C for 19d. All of the mutants fermented the sake mash as efficiently as HL-69. The components of the brewed sake are shown in Table 3. The values for sake meter, alcohol concentration, acidity and amino acidity in the sake TABLE 2. Strains CL-70 CL-80 CL-90 AOFlO HL-69 (narent)
TABLE 3.
Esterase and AATase activities
Esterase (ppm/h/mg protein)
AATase @pm/h/mg protein)
0.20 0.19 0.30 0.17 1.44
4.0 3.8 5.5 3.7 3.5
a CL-70, CL-80 and CL-90 were resistant to i-AmOClAc, while AOFlO was resistant to i-AmOFAc. All mutants were derived from the parent strain HL-69.
Components of sake brewed with various strains Straina CL-70 CL-80 CL-90 AOFlO HL-69
Sake meter +4.0 Alcohol (%) 19.1 Acidity (ml) 2.1 Amino acidity (ml) 2.0 n-Propyl alcohol (ppm) 93.8 i-Amy1 alcohol (ppm) 234 i-Butyl alcohol (ppm) 71.8 i-Amy1 acetate (ppm) 11.3 i-Butyl acetate (ppm) 1.2 Ethyl acetate (ppm) 105 Ethylcaproate(ppm) 1.3
+4.0 19.2 2.2 1.8 95.4 239 68.3 10.5 1.l 101 1.3
+1.8 19.1 2.6 1.9 132 237 71.9 14.2 0.9 123 1.1
+4.0 19.2 2.2 1.9 92.7 235 73.0 11.7 1.3 107 1.1
f4.3 19.2 2.2 1.9 97.3 244 71.3 8.3 0.5 94.6 1.3
KlO f4.0 19.2 2.5 1.7 159 228 68.4 7.1 0.4 99.3 0.9
a KlO is Kyokai no. 10. Other strains are the same as in Table 2.
brewed with mutants, except CL-90, were similar to those of the parent strain HL-69. In the sake brewed with CL-90, the sake meter was slightly lower and the acidity was slightly higher. Mutants resistant to iAmOClAc produced levels of i-AmOAc 1.3 to 1.7-fold greater and levels of i-BuOAc 1.8 to 2.4-fold greater than HL-69, while the contents of isoamyl alcohol and isobutyl alcohol were not different. These results are identical to those for AOFlO, and suggest that the improved i-AmOAc productivity of the mutants was due to their lowered esterase activity (4, 5). The increase in iBuOAc content has been suggested to be a characteristic of these mutants because the esterase hydrolyzed iBuOAc faster than i-AmOAc (12). CL-90 accumulated the highest amount of i-AmOAc, in spite of having the highest esterase activity among these mutants. Only CL-90 produced an increased amount (1.3-fold) of ethyl acetate (Table 3). The activity of AATase, which catalyzes the formation of acetic esters, was determined according to the method of Fujii et al. (13). CL-90 had AATase activity which was 1.6 times greater than the other strains (Table 2). Recently, molecular cloning of the ATFZ gene, which encodes AATase, has been reported (13). Transformants carrying multicopies of ATFI expressed elevated levels of AATase activity and produced significant amounts of ethyl acetate and i-AmOAc. It is believed that the high i-AmOAc productivity of CL-90 was partly due to the increase in AATase activity. AATase is an SH-enzyme containing a relatively high number of cysteine residues (1, 13). i-AmOClAc has a structure very similar to the AATase product i-AmOAc. Based on these observations, the relationship between i-AmOClAc and AATase activity was suggested. Many investigators have reported that yeast possess several isozymes of esterase (2, 3, 7, 8, 11, 12, 14-16). Wohrmann and Lange (7) demonstrated using gel electrophoresis that there were four loci (ESTl-4) for esterase isozymes in wine yeast. Esterase patterns were examined by polyacrylamide gel electrophoresis in order to identify which isozyme was deleted in our mutants. The electrophoresis was performed at 150 V for 2 h. The volume of each sample applied was adjusted to 400 pg of protein. Immediately after the electrophoresis, the gel was stained with 250ppm 1-naphthyl acetate and 400 ppm Fast blue salt B (Merck) in 0.1 M potassium phosphate buffer (pH 7.0) at 30°C for 20min. AOFll and AOF16, which have already been isolated (5), were also examined simultaneously.
NOTES
origin
293
The authors are grateful to Professor Takahito Suzuki of Nara Women’s University for his suggestions concerning monoiodoacetic acid. REFERENCES
cEST1 cEST2
hont FIG. 1. Polyacrylamide gel electrophoresis of esterase. The esterase activity toward I-naphthyl acetate was stained with Fast blue salt B. Lane 1, K10; lane 2, HL-69; lane 3, CL-90; lane 4, CL-80; lane 5, CL-70; lane 6, AOFlO; lane 7, AOF16; lane 8, HL-68; lane 9, AOFll. KlO is sake yeast Kyokai no. 10. HL-68 and HL-69 are haploid strains derived from KlO. CL-70, CL-80 and CL-90 are mutants resistant to i-AmOClAc, while AOFlO, AOFll and AOF are mutants resistant to i-AmOFAc. All mutants had less than 20% of the original esterase activity toward i-AmOAc,exceptAOFll which
wasderivedfrom HL-68 and had 60%. Figure 1 presents the results of activity staining for the esterase toward 1-naphthyl acetate. The strongest band and the band having the smallest mobility appeared to be EST2 and ESTI, respectively (7). AOFlO, AOF16, CL-70 and CL-80 completely lost the EST2 band, while CL-90, which had slightly higher esterase activity toward i-AmOAc than the other four mutants exhibited a faint EST2 band. AOFll, which had 60% of this activity, showed a little weak EST2 band. Therefore, it can be seen that the band intensity of EST2 changed in close relation to the activity of the esterase toward i-AmOAc. This esterase has been considered to correspond to EST2 (8). Mutants isolated by diazo staining of colonies, which had low esterase activity toward i-AmOAc, also lacked the EST2 band (Fukuda, K. et al., Abstr. Annu. Meet., Japan Sot. Biosci. Biotech. Agrochem., p.348, 1993), leading to the conclusion that their mutants and our mutants must have been missing the same EST2 isozyme. Other minor bands which appeared in the gel exhibited no differences between the mutant and parent strains, suggesting that they were not involved in iAmOAc hydrolysis.
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