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Purification and some properties of acid carboxypeptidases from Saké koji
116
Abstract of the Articles Printed in Hakkokogaku Kaishi
[J. Ferment. Technol.,
16.5 g[l.h was obtained at the dilution rate of 0.25 h -1, and I s o a m y l Acetate F o r m a t i o n by OSAMUAKITA, TETSUOHASUO, TOSHITERU but deflocculation and loss of yeast occurred in 4 days OHBA, and N o B u v ~ I Mrv,~u'~o (National Research at this dilution rate, the test could not continue. Institute of Brewing, 2-6-30, Takinogawa, Kita-ku, Heat evolution from fermentation depended on Tokyo 114, Japan) Hakkokogaku 65: 19-26. 1987. productivity; the productivity of 26.5 g[l.h gave The effect of the amino acid composition on 6.08 kcal/l.h. higher alcohols and isoamyl acetate formation by the sakk yeast, (Saccharomycescerevisiae) during fermentation was studied with the use of modified Wickerham's Purification and S o m e P r o p e r t i e s o f Acid Carbomedium containing 17% glucose and the same concen- x y p e p t i d a s e s f r o m Sak~ Koji. SHISEAKIMXKAMI, OSAMU MIURA, TAKATOSHI WATANABE,KEN-ICHI tration of amino acids in saccharified solutions made by WATANABE, KIMIO IWANO, SATOSHI SHIINOKI, mixing of two saccharified solutions made from polished TOYOAKI SHIMADA, and YATARO NUNOKAWA rice and rice-koji at different ratios. (National Research Institute of Brewing, 2-6-30 We found that there was an optimum concenTakinogawa, Kita-ku, Tokyo 114, Japan) Hakkotration of amino acids for maximum isoamyl acetate kogaku 65" 37-43. 1987. formation. Aspergillus oryzae acid carboxypeptidase (peptidylThe activity of alcohol acetyltransferase (AATFase) L-amino-acid hydrolase, EC 3.4.16.1) la, Ib, IIa, and in the yeast increased with increases in the amino acid concentration in the medium. This phenomenon IIb were isolated and purified from sakk koji extract by depended on the differences in the fatty acid compo- ethanol precipitation, batchwise treatment with sition in the yeast with various levels of amino acids. DEAE-Sephadex A-50, DEAE-cellulose ionexchange The ratio of unsaturated fatty acids, which inhibited chromatography, column chromatography with ~oAATFase, to saturated fatty acids in the yeast decreased amino dodecyl agarose, and Sephacryl S-300 or Sephacryl S-200 gel chromatography. with increases in the amino acid concentration. By gel chromatography, the molecular weight was Formation by the yeast of isoamyl alcohol, one substrate of AATFase decreased with increases in the estimated to be about 230,000 for acid carboxypeptidase Ia, 133,000 for Ib, and 59,000 for IIa and IIb. The amino acid concentration. These findings explained why there is an optimum isoelectric points of acid carboxypeptidases Ia, Ib, IIa, amino acid concentration for isoamyl acetate formation. and IIb were 4.60, 4.52, 4.20, and 4.11, respectively. The optimal pH and temperature for the activity At this concentration, both of the amount of isoamyl alcohol formed and the activity of AATFase were of each of these acid carboxypeptidases were 3.0 and 45°C, respectively. Of the metal ions tested, Cu + was fairly high. the most potent inhibitor of enzyme activity. Each enzyme was inhibited by dlisopropyl fluorophosphate, C o n t i n u o u s Ethanol F e r m e n t a t i o n by Beer Yeast. monoiodoacetate, p-chloromercuribenzoate, N-ethylKENp KXDA, SHIGERU MORIMURA, NORIYUKI maleimide, and sodium dodecylsulfate. SHIMA, SHINICHI ASANO, MOTOZUMI YAMADAKI, The hydrolytic action of each enzyme on 31 kinds and TOSHIKI MIYAZAWA (Hitachi Zosen Technical of N-acyl-peptides and amides was examined. When ResearchLaboratory, Inc., 1-3-22 Sakurajima, Konohana- L-AIa and L-His were present at the penultimate ku, Osaka 554, Japan) Hakkokogaku 65: 27-35. position of the carboxyterminus of the peptide, acid 1987. carboxypeptidase Ia and Ib hydrolyzed the carContinuous ethanol fermentation by a flocculating boxyterminal peptide bond much more rapidly than yeast, beer yeast IFO-2018, was tested at 30°C and the other enzymes. pH 4.5 with uncooked molasses media in a two-tower Every enzyme hydrolyzed bradykinin and fermentor system. angiotensin I sequentially from each carboxyterminus. Ethanol productivity was high (22 g//.h) when the When L-Pro was the penultimate amino acid, the ethanol concentration produced was of 74 g/1 with carboxyterminal amino acid newly formed was most Philippine molasses medium containing 16% total readily released by acid carboxypeptidase Ia and sugar. least readily by acid carboxypeptidase IIa and IIb. With Okinawa molasses medium, the concentration of ethanol produced was about 10 g/l less than with other kinds of molasses. We think that the high A d v a n c e d Water T r e a t m e n t by Biogeofilters. ash content of Okinawa molasses inhibited the fermenSUSUMU HASHLMOTO, KF~NJI FURUKAWA, and tation activity of this yeast. Jt.rNICHI MINAMI* (Department of Environmental In a bench-scale fermentation test, productivity of Engineering, Faculty of Engineering, Osaka University,
Alcohols a Yeast.
Vol. 65, 1987]
Abstract of the Articles Printed in Hakkokogaku Kaishi
2-1 Yamada Oka, Suita-shi, Osaka 565, Japan; * Nissin Food Products Company, Ltd., Research Institute, 140-1 Aza Izeki, Oaza-Shimonagari, Rittocho, Kurita-gun, Shiga Pref. 530-30, Japan) H a k k o k o g a k u 65: 45-52. 1987. W e m a d e use of the ability of zeolite to adsorb NH4 +, that of K a n u m a soil to adsorb PO4 s- a n d that of p a k - b u n g (a tropical aquatic vegetable) to absorb N a n d P to develop a biogeofiher suitable for a d v a n c e d wastewater treatment. Wastewater secondary effluent was used as inftuent, a n d the tests of continuous treatm e n t with a c o l u m n biogeofilter were done. By t r e a t m e n t of secondary effluent a n d diluted secondary effluent corresponding in quality to river water at the total organic carbon ( T O C ) loading rate of 8 to 23 m g of T O C / / . d , it was possible to remove of 40% of the T O C , more t h a n 80% of the total nitrogen a n d more t h a n 98% of the total phosphorus. P a k - b u n g planted on a filter bed of zeolite a n d K a n u m a soil grew well with low influent N a n d P concentrations. Sufficient N a n d P were available to the plants because of adsorption of these elements in the filter bed. T h e mass balances of N a n d P in the biogeofilter system showed that a b o u t 70% of N removal a n d 30 to 40% of the P removal were d u e to absorption by pak-bung. P a k - b u n g was useful in the t r e a t m e n t of wastewater by biogeofihration.
Concentration o f D i m e t h y l Sulphide in W i n e after Different Storage Times. --Note--SHOJI GOTO a n d RYuGo TAKAMURO (The Institute
of Enology and Viticulture, Yamanashi University, Kofit 400, Japan) H a k k o k o g a k u 65: 53-57. 1987. Eighty-six samples of wine of the vintages 19491983, m a d e from the seven grape varieties of Koshu, Riesling, C h a r d o n n a y , M u s c a t Bailey A, C a b e r n e t Sauvignon, C a b e r n e t franc, a n d Pinot noir, were tested to find their concentrations of d e m e t h y l sulphide (DMS) by the head space m e t h o d of gas c h r o m a t o graphy. D M S was found in K o s h u wine by G C - M S analysis. D M S was formed d u r i n g bottle aging, a n d the concentration of D M S increased as storage time increased, reaching a m a x i m u m concentration in
117
wines aged 5-10 years, a n d g r a d u a l l y decreasing with further again. D M S in white wines aged one to two years was 0-trace, a n d 3.2-6.0 #g/l for red wines aged one year. T h e m a x i m u m concentrations of D M S in eight vintage wines tested were as follows: 7.6 a n d 14.9 #g/l in K o s h u I aged 9 years a n d K o s h u II aged 10 years, respectively; 20.1, 22.2, a n d 34.0/~g/l in Riesling, C h a r d o n n a y , a n d M u s c a t Bailey A, aged 6 years; 68.4, 63.9, a n d 19.8/~g// in C a b e r n e t Sauvignon, C a b e r n e t franc, a n d Pinot noir aged 5 years. D M S in red wine forms earlier t h a n in white wine, a n d reaches a higher concentration. T h e formation of D M S contributes to the bottle b o u q u e t of white wines.
Behavior o f Carbon Dioxide and Its Function Affected to the F e r m e n t a t i o n in S u b m e r g e d Culture. - - M o n o g r a p h - - AYAAKI ISHIZAKI (Depart-
ment of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka-shi 812, Japan) H a k k o k o g a k u 65: 59-69. 1987. In biotechnology, different culture m e t h o d are used to produce novel products, b u t s u b m e r g e d fermentation is still the reliable way to get a large biomass efficiently on a industrial production line. Technology for s u b m e r g e d cultures should be u p g r a d e d as well as m a k i n g cultures more sophisticated. T h e oxygen transfer rate m a y be the best m e a n s to evaluate the scale-up a n d optimization of the culture system in s u b m e r g e d fermentation. Also, carbon dioxide should be taken into account as a factor that directly affects on the fermentation process. O x y g e n , a substrate for aerobic microorganisms, limits the fermentation kinetics, whereas carbon dioxide acts as the acid in the culture liquid a n d has influences p H a n d the acid-base equilibrium of the s u b m e r g e d culture with a m e d i u m of electrolytes. This study deals with the kinetics of carbon dioxide evolution in aeration a n d agitation systems, the behavior of carbon dioxide d u r i n g product formation, a n d its various effects on sophisticated p H - s t a t submerged fermentation.
Abstract of the Articles Printed in Hakkokogaku Kaishi
[J. Ferment. Technol.,
16.5 g[l.h was obtained at the dilution rate of 0.25 h -1, and I s o a m y l Acetate F o r m a t i o n by OSAMUAKITA, TETSUOHASUO, TOSHITERU but deflocculation and loss of yeast occurred in 4 days OHBA, and N o B u v ~ I Mrv,~u'~o (National Research at this dilution rate, the test could not continue. Institute of Brewing, 2-6-30, Takinogawa, Kita-ku, Heat evolution from fermentation depended on Tokyo 114, Japan) Hakkokogaku 65: 19-26. 1987. productivity; the productivity of 26.5 g[l.h gave The effect of the amino acid composition on 6.08 kcal/l.h. higher alcohols and isoamyl acetate formation by the sakk yeast, (Saccharomycescerevisiae) during fermentation was studied with the use of modified Wickerham's Purification and S o m e P r o p e r t i e s o f Acid Carbomedium containing 17% glucose and the same concen- x y p e p t i d a s e s f r o m Sak~ Koji. SHISEAKIMXKAMI, OSAMU MIURA, TAKATOSHI WATANABE,KEN-ICHI tration of amino acids in saccharified solutions made by WATANABE, KIMIO IWANO, SATOSHI SHIINOKI, mixing of two saccharified solutions made from polished TOYOAKI SHIMADA, and YATARO NUNOKAWA rice and rice-koji at different ratios. (National Research Institute of Brewing, 2-6-30 We found that there was an optimum concenTakinogawa, Kita-ku, Tokyo 114, Japan) Hakkotration of amino acids for maximum isoamyl acetate kogaku 65" 37-43. 1987. formation. Aspergillus oryzae acid carboxypeptidase (peptidylThe activity of alcohol acetyltransferase (AATFase) L-amino-acid hydrolase, EC 3.4.16.1) la, Ib, IIa, and in the yeast increased with increases in the amino acid concentration in the medium. This phenomenon IIb were isolated and purified from sakk koji extract by depended on the differences in the fatty acid compo- ethanol precipitation, batchwise treatment with sition in the yeast with various levels of amino acids. DEAE-Sephadex A-50, DEAE-cellulose ionexchange The ratio of unsaturated fatty acids, which inhibited chromatography, column chromatography with ~oAATFase, to saturated fatty acids in the yeast decreased amino dodecyl agarose, and Sephacryl S-300 or Sephacryl S-200 gel chromatography. with increases in the amino acid concentration. By gel chromatography, the molecular weight was Formation by the yeast of isoamyl alcohol, one substrate of AATFase decreased with increases in the estimated to be about 230,000 for acid carboxypeptidase Ia, 133,000 for Ib, and 59,000 for IIa and IIb. The amino acid concentration. These findings explained why there is an optimum isoelectric points of acid carboxypeptidases Ia, Ib, IIa, amino acid concentration for isoamyl acetate formation. and IIb were 4.60, 4.52, 4.20, and 4.11, respectively. The optimal pH and temperature for the activity At this concentration, both of the amount of isoamyl alcohol formed and the activity of AATFase were of each of these acid carboxypeptidases were 3.0 and 45°C, respectively. Of the metal ions tested, Cu + was fairly high. the most potent inhibitor of enzyme activity. Each enzyme was inhibited by dlisopropyl fluorophosphate, C o n t i n u o u s Ethanol F e r m e n t a t i o n by Beer Yeast. monoiodoacetate, p-chloromercuribenzoate, N-ethylKENp KXDA, SHIGERU MORIMURA, NORIYUKI maleimide, and sodium dodecylsulfate. SHIMA, SHINICHI ASANO, MOTOZUMI YAMADAKI, The hydrolytic action of each enzyme on 31 kinds and TOSHIKI MIYAZAWA (Hitachi Zosen Technical of N-acyl-peptides and amides was examined. When ResearchLaboratory, Inc., 1-3-22 Sakurajima, Konohana- L-AIa and L-His were present at the penultimate ku, Osaka 554, Japan) Hakkokogaku 65: 27-35. position of the carboxyterminus of the peptide, acid 1987. carboxypeptidase Ia and Ib hydrolyzed the carContinuous ethanol fermentation by a flocculating boxyterminal peptide bond much more rapidly than yeast, beer yeast IFO-2018, was tested at 30°C and the other enzymes. pH 4.5 with uncooked molasses media in a two-tower Every enzyme hydrolyzed bradykinin and fermentor system. angiotensin I sequentially from each carboxyterminus. Ethanol productivity was high (22 g//.h) when the When L-Pro was the penultimate amino acid, the ethanol concentration produced was of 74 g/1 with carboxyterminal amino acid newly formed was most Philippine molasses medium containing 16% total readily released by acid carboxypeptidase Ia and sugar. least readily by acid carboxypeptidase IIa and IIb. With Okinawa molasses medium, the concentration of ethanol produced was about 10 g/l less than with other kinds of molasses. We think that the high A d v a n c e d Water T r e a t m e n t by Biogeofilters. ash content of Okinawa molasses inhibited the fermenSUSUMU HASHLMOTO, KF~NJI FURUKAWA, and tation activity of this yeast. Jt.rNICHI MINAMI* (Department of Environmental In a bench-scale fermentation test, productivity of Engineering, Faculty of Engineering, Osaka University,
Alcohols a Yeast.
Vol. 65, 1987]
Abstract of the Articles Printed in Hakkokogaku Kaishi
2-1 Yamada Oka, Suita-shi, Osaka 565, Japan; * Nissin Food Products Company, Ltd., Research Institute, 140-1 Aza Izeki, Oaza-Shimonagari, Rittocho, Kurita-gun, Shiga Pref. 530-30, Japan) H a k k o k o g a k u 65: 45-52. 1987. W e m a d e use of the ability of zeolite to adsorb NH4 +, that of K a n u m a soil to adsorb PO4 s- a n d that of p a k - b u n g (a tropical aquatic vegetable) to absorb N a n d P to develop a biogeofiher suitable for a d v a n c e d wastewater treatment. Wastewater secondary effluent was used as inftuent, a n d the tests of continuous treatm e n t with a c o l u m n biogeofilter were done. By t r e a t m e n t of secondary effluent a n d diluted secondary effluent corresponding in quality to river water at the total organic carbon ( T O C ) loading rate of 8 to 23 m g of T O C / / . d , it was possible to remove of 40% of the T O C , more t h a n 80% of the total nitrogen a n d more t h a n 98% of the total phosphorus. P a k - b u n g planted on a filter bed of zeolite a n d K a n u m a soil grew well with low influent N a n d P concentrations. Sufficient N a n d P were available to the plants because of adsorption of these elements in the filter bed. T h e mass balances of N a n d P in the biogeofilter system showed that a b o u t 70% of N removal a n d 30 to 40% of the P removal were d u e to absorption by pak-bung. P a k - b u n g was useful in the t r e a t m e n t of wastewater by biogeofihration.
Concentration o f D i m e t h y l Sulphide in W i n e after Different Storage Times. --Note--SHOJI GOTO a n d RYuGo TAKAMURO (The Institute
of Enology and Viticulture, Yamanashi University, Kofit 400, Japan) H a k k o k o g a k u 65: 53-57. 1987. Eighty-six samples of wine of the vintages 19491983, m a d e from the seven grape varieties of Koshu, Riesling, C h a r d o n n a y , M u s c a t Bailey A, C a b e r n e t Sauvignon, C a b e r n e t franc, a n d Pinot noir, were tested to find their concentrations of d e m e t h y l sulphide (DMS) by the head space m e t h o d of gas c h r o m a t o graphy. D M S was found in K o s h u wine by G C - M S analysis. D M S was formed d u r i n g bottle aging, a n d the concentration of D M S increased as storage time increased, reaching a m a x i m u m concentration in
117
wines aged 5-10 years, a n d g r a d u a l l y decreasing with further again. D M S in white wines aged one to two years was 0-trace, a n d 3.2-6.0 #g/l for red wines aged one year. T h e m a x i m u m concentrations of D M S in eight vintage wines tested were as follows: 7.6 a n d 14.9 #g/l in K o s h u I aged 9 years a n d K o s h u II aged 10 years, respectively; 20.1, 22.2, a n d 34.0/~g/l in Riesling, C h a r d o n n a y , a n d M u s c a t Bailey A, aged 6 years; 68.4, 63.9, a n d 19.8/~g// in C a b e r n e t Sauvignon, C a b e r n e t franc, a n d Pinot noir aged 5 years. D M S in red wine forms earlier t h a n in white wine, a n d reaches a higher concentration. T h e formation of D M S contributes to the bottle b o u q u e t of white wines.
Behavior o f Carbon Dioxide and Its Function Affected to the F e r m e n t a t i o n in S u b m e r g e d Culture. - - M o n o g r a p h - - AYAAKI ISHIZAKI (Depart-
ment of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka-shi 812, Japan) H a k k o k o g a k u 65: 59-69. 1987. In biotechnology, different culture m e t h o d are used to produce novel products, b u t s u b m e r g e d fermentation is still the reliable way to get a large biomass efficiently on a industrial production line. Technology for s u b m e r g e d cultures should be u p g r a d e d as well as m a k i n g cultures more sophisticated. T h e oxygen transfer rate m a y be the best m e a n s to evaluate the scale-up a n d optimization of the culture system in s u b m e r g e d fermentation. Also, carbon dioxide should be taken into account as a factor that directly affects on the fermentation process. O x y g e n , a substrate for aerobic microorganisms, limits the fermentation kinetics, whereas carbon dioxide acts as the acid in the culture liquid a n d has influences p H a n d the acid-base equilibrium of the s u b m e r g e d culture with a m e d i u m of electrolytes. This study deals with the kinetics of carbon dioxide evolution in aeration a n d agitation systems, the behavior of carbon dioxide d u r i n g product formation, a n d its various effects on sophisticated p H - s t a t submerged fermentation.