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Activity of α-glucosidase in sake koji and its role during sake brewing
VOL. 79, 1995
Abstracts of the Articles Printed in Seibutsu-kogaku
Kaishi
Vol. 73, No. 2 (1995)
Effect of Citric Acid on Glycerol Formation by Saccharomyces cerevisiae in Barley Shochu Mash. TOSHIRO OYORI,* KIYOSHI OGAWA, and ~~ASAHIKO SHIMODA (Research LQbOrQiOry, SanwQ Sh#& Co. Ltd., 2231-L YQ~Q~o~o, USQ, Oita 879-04) Seibutsu-kogaku 13: 89-95. 1995. Factors influencing the enhancement of glycerol formation by shochu yeast (SQcchQromyces cerevisiae) in barley shochu mash (BSM) were investigated. Glycerol formation was influenced in the case of 3% of glucose at the beginning of fermentation. However, the glucose concentration was found not to have an important influence on the glycerol concentration in BSM because it remained at less than 1% during fermentation. The citric acid concentration in BSM of all-koji and mash with a koji ratio of 33% were 1% and 0.3% at the beginning of fermentation, and decreased gradually as the fermentation progressed. As the concentration of citric acid in BSM increased from 0.3?4 to l%, the amount of glycerol formed by shochu yeast increased from 5.1 g/l to 5.8 g/I. From these results, the citric acid concentration is thought to influence glycerol formation by shochu yeast in BSM. Glycerol formation also increased with an increase in the citric acid concentration of YEPD-modified medium, because intracellular glycerophosphatase was enhanced by the increase of citric acid concentration. ~____ * Corresponding author.
Activity of a-Glucosidase Brewing.
in Sake Koji and Its Role during Sake
YOSHIHISAMORIMOTO,KATSIJHIKOKITAMOTO,*YOSHITOFUJITA, KATSUYAGOMI, and CHIEKOKUMAGAI(NQtionat Research Institute of Brewing, 2-6-30 TQkiflOgaWQ, Kita-ku, Tokyo 114) Seibutsukogaku 73: 97-104. 1995. Enzyme activities of koji samples from 12 sake breweries were determined, and the relationship between their production conditions was analyzed. u-Glucosidase activity showed a high correlation with glucoamylase and u-amylase, and was especially in the case of the latter. Ginjo koji had higher relative activity of a-glucosidase to (Yamylase, suggesting that a-glucosidase may play an important role in ginjo (high-quality) sake brewing. Addition of purified a-glucosidase to koji enzyme solution increased the glucoamylase activity. Glucose was produced by the addition of both purified a-glucosidase and purified a-amylase to starch solution, but not by the addition of only tr-glucosidase. These results indicate that the conventional method for measuring glucoamylase activity measures not only glucoamylase but also cu-glucosidase activity. The contribution rate of n-glucosidase to glucoamylase was estimated to be lO-20% of the glucoamylase enzyme activity of koji determined by the conventional method. ~___ * Corresponding author.
of Koji Ratio on the Formation of Flavour during Rice Shochu Fermentation.+ -NoteTSUNAHIKOSAIGUSA,*MASAHIROYAMAGAMI, SHIGEMICHI OKAMURA, and MICHIO HARADA(Nagareyama Piant, Kikkoman Corporation, 3-90 NQgQreyama, Chiba 270-01) Seibutsu-kogaku 13: 105-108. 1995. The koji ratio (rice for koji/rice for kake) had a significant influence on the formation of higher alcohols and aromatic esters by yeast (Saccharomyces cerevisiae) during rice shochu fermentation. The concentrations of acetaldehyde, isobutyl alcohol, isoamyl alcohol and P-phenylethyl alcohol in shochu increased with decreasing koji ratio. There was an optimum koji ratio for the formation of isoamyl acetate and P-phenylethyl acetate. The form01 titration value
Effects
403
in matured mash was about 3 ml at the optimum koji ratio. ~~~ * Corresponding author. + Study on the Control of Shochu Flavour (IV). Effects of Aeration and Fermentation Temperature on Stability of Repeated-Batch Fermentation by a Thermotoleraot Flocculating Yeast. -Note-
Aam SHOU,’ SHIGERU MORIMUKA,~and KENII KIDA~* (Chengdu University of Science and Technology, Chengdu, P.O. BOX 610065, Sichuan, PR China’; Department of Applied Chemistry, Fact&y of Engineering, Kumamoto University, 2-39-l Kurokami, KUmQmOtOCity, Kumamoto 8602) Seibutsu-kogaku 73: 109-I 12. 1995. The effects of aeration and fermentation temperature on the stability of repeated-batch fermentation using a thermotolerant flocculating yeast, Saccharomyces cerevisiae KF-7, were investigated. Repeated-batch fermentation tests were carried out with a nonsterilized molasses medium containing 20% (w/v) sugar. Under continuous aeration, condition during ethanol fermentation the ethanol productivities were almost constant, being 8.5, 7.7, and 7.7 g/f. h at 30, 33 and 35°C. respectively, and the conversion ratio of the sugar consumed to the ethanol produced was more than 0.45, a value which corresponds to over 88% (hereafter called the ethanol yield) of the theoretical value of 0.51 at each temperature. However, the number of viable cells decreased with stepwise increases in temperature. At 33°C the aeration time was decreased stepwisely. The productivity and viable cells were kept constant, at about 7.6 g/l. h and 4.0 x lOa cells/ml even when aeration was applied only for the first 5 h of each fermentation. However, under non-aeration conditions, they decreased to 5.1 g/l. h and 1.3 x lo8 cells/ml, respectively, but the fermentation could be repeated stably. At 35°C the fermentation could not be conducted stably when the aeration conditions were changed from continuous aeration to aeration for the first 5 h. During the 80 batches of repeated-batch fermentation, except when aeration was conducted the first 5 h at 35°C the ethanol yield was maintained stably at more than 90%. *
Corresponding
author.
Analyses and Applications sp. -Monograph-
of Extracellular
Enzymes
from Bacillus
MASAHIRO TAKAGI (Department of Biotechnology, Faculty of Engineering, Osaka University, 2-I Yamadaoka, Suita, Osaka 565) Seibutsu-kogaku 73: 113-125. 1995. Many species of the genus Bacillus produrr a variety ot extracellular enzymes, some of which are of industrial importance. Studies on these extracellular enzymes are significant not only from a practical standpoint, but also a theoretical point of view of better understanding the secretion merhanism and structure-function relationship of enzymes. We have established a host-vector system of Bacillussubtilis with many
kinds of plasmid vectors with different copy numbers. Low copy number-plasmids such are pTB53 and pTB522 (copy number about 8) for B. subtilis were extremely useful. We have cloned and analyzed many extracellular enzyme genes such as proteases, amylases, and penicillinase. Gene cloning contributed for increase of productivity of target enzymes and moreover cloned genes can be used for further studies about enzyme gene expression regulation and strategic improvement of enzyme characteristics through protein engineering. In this report, giving the cases of neutral protease from Bacillus stearothermophilus and penicillinase from Bacihus licheniformis as examples, four subjects, ‘1) Cloning and sequencing of structural genes, 121Analyses of gene expression regulation, ‘it)Strategic alteration of thermostability of extracellular enzymes and a regulatory protein, and
Abstracts of the Articles Printed in Seibutsu-kogaku
Kaishi
Vol. 73, No. 2 (1995)
Effect of Citric Acid on Glycerol Formation by Saccharomyces cerevisiae in Barley Shochu Mash. TOSHIRO OYORI,* KIYOSHI OGAWA, and ~~ASAHIKO SHIMODA (Research LQbOrQiOry, SanwQ Sh#& Co. Ltd., 2231-L YQ~Q~o~o, USQ, Oita 879-04) Seibutsu-kogaku 13: 89-95. 1995. Factors influencing the enhancement of glycerol formation by shochu yeast (SQcchQromyces cerevisiae) in barley shochu mash (BSM) were investigated. Glycerol formation was influenced in the case of 3% of glucose at the beginning of fermentation. However, the glucose concentration was found not to have an important influence on the glycerol concentration in BSM because it remained at less than 1% during fermentation. The citric acid concentration in BSM of all-koji and mash with a koji ratio of 33% were 1% and 0.3% at the beginning of fermentation, and decreased gradually as the fermentation progressed. As the concentration of citric acid in BSM increased from 0.3?4 to l%, the amount of glycerol formed by shochu yeast increased from 5.1 g/l to 5.8 g/I. From these results, the citric acid concentration is thought to influence glycerol formation by shochu yeast in BSM. Glycerol formation also increased with an increase in the citric acid concentration of YEPD-modified medium, because intracellular glycerophosphatase was enhanced by the increase of citric acid concentration. ~____ * Corresponding author.
Activity of a-Glucosidase Brewing.
in Sake Koji and Its Role during Sake
YOSHIHISAMORIMOTO,KATSIJHIKOKITAMOTO,*YOSHITOFUJITA, KATSUYAGOMI, and CHIEKOKUMAGAI(NQtionat Research Institute of Brewing, 2-6-30 TQkiflOgaWQ, Kita-ku, Tokyo 114) Seibutsukogaku 73: 97-104. 1995. Enzyme activities of koji samples from 12 sake breweries were determined, and the relationship between their production conditions was analyzed. u-Glucosidase activity showed a high correlation with glucoamylase and u-amylase, and was especially in the case of the latter. Ginjo koji had higher relative activity of a-glucosidase to (Yamylase, suggesting that a-glucosidase may play an important role in ginjo (high-quality) sake brewing. Addition of purified a-glucosidase to koji enzyme solution increased the glucoamylase activity. Glucose was produced by the addition of both purified a-glucosidase and purified a-amylase to starch solution, but not by the addition of only tr-glucosidase. These results indicate that the conventional method for measuring glucoamylase activity measures not only glucoamylase but also cu-glucosidase activity. The contribution rate of n-glucosidase to glucoamylase was estimated to be lO-20% of the glucoamylase enzyme activity of koji determined by the conventional method. ~___ * Corresponding author.
of Koji Ratio on the Formation of Flavour during Rice Shochu Fermentation.+ -NoteTSUNAHIKOSAIGUSA,*MASAHIROYAMAGAMI, SHIGEMICHI OKAMURA, and MICHIO HARADA(Nagareyama Piant, Kikkoman Corporation, 3-90 NQgQreyama, Chiba 270-01) Seibutsu-kogaku 13: 105-108. 1995. The koji ratio (rice for koji/rice for kake) had a significant influence on the formation of higher alcohols and aromatic esters by yeast (Saccharomyces cerevisiae) during rice shochu fermentation. The concentrations of acetaldehyde, isobutyl alcohol, isoamyl alcohol and P-phenylethyl alcohol in shochu increased with decreasing koji ratio. There was an optimum koji ratio for the formation of isoamyl acetate and P-phenylethyl acetate. The form01 titration value
Effects
403
in matured mash was about 3 ml at the optimum koji ratio. ~~~ * Corresponding author. + Study on the Control of Shochu Flavour (IV). Effects of Aeration and Fermentation Temperature on Stability of Repeated-Batch Fermentation by a Thermotoleraot Flocculating Yeast. -Note-
Aam SHOU,’ SHIGERU MORIMUKA,~and KENII KIDA~* (Chengdu University of Science and Technology, Chengdu, P.O. BOX 610065, Sichuan, PR China’; Department of Applied Chemistry, Fact&y of Engineering, Kumamoto University, 2-39-l Kurokami, KUmQmOtOCity, Kumamoto 8602) Seibutsu-kogaku 73: 109-I 12. 1995. The effects of aeration and fermentation temperature on the stability of repeated-batch fermentation using a thermotolerant flocculating yeast, Saccharomyces cerevisiae KF-7, were investigated. Repeated-batch fermentation tests were carried out with a nonsterilized molasses medium containing 20% (w/v) sugar. Under continuous aeration, condition during ethanol fermentation the ethanol productivities were almost constant, being 8.5, 7.7, and 7.7 g/f. h at 30, 33 and 35°C. respectively, and the conversion ratio of the sugar consumed to the ethanol produced was more than 0.45, a value which corresponds to over 88% (hereafter called the ethanol yield) of the theoretical value of 0.51 at each temperature. However, the number of viable cells decreased with stepwise increases in temperature. At 33°C the aeration time was decreased stepwisely. The productivity and viable cells were kept constant, at about 7.6 g/l. h and 4.0 x lOa cells/ml even when aeration was applied only for the first 5 h of each fermentation. However, under non-aeration conditions, they decreased to 5.1 g/l. h and 1.3 x lo8 cells/ml, respectively, but the fermentation could be repeated stably. At 35°C the fermentation could not be conducted stably when the aeration conditions were changed from continuous aeration to aeration for the first 5 h. During the 80 batches of repeated-batch fermentation, except when aeration was conducted the first 5 h at 35°C the ethanol yield was maintained stably at more than 90%. *
Corresponding
author.
Analyses and Applications sp. -Monograph-
of Extracellular
Enzymes
from Bacillus
MASAHIRO TAKAGI (Department of Biotechnology, Faculty of Engineering, Osaka University, 2-I Yamadaoka, Suita, Osaka 565) Seibutsu-kogaku 73: 113-125. 1995. Many species of the genus Bacillus produrr a variety ot extracellular enzymes, some of which are of industrial importance. Studies on these extracellular enzymes are significant not only from a practical standpoint, but also a theoretical point of view of better understanding the secretion merhanism and structure-function relationship of enzymes. We have established a host-vector system of Bacillussubtilis with many
kinds of plasmid vectors with different copy numbers. Low copy number-plasmids such are pTB53 and pTB522 (copy number about 8) for B. subtilis were extremely useful. We have cloned and analyzed many extracellular enzyme genes such as proteases, amylases, and penicillinase. Gene cloning contributed for increase of productivity of target enzymes and moreover cloned genes can be used for further studies about enzyme gene expression regulation and strategic improvement of enzyme characteristics through protein engineering. In this report, giving the cases of neutral protease from Bacillus stearothermophilus and penicillinase from Bacihus licheniformis as examples, four subjects, ‘1) Cloning and sequencing of structural genes, 121Analyses of gene expression regulation, ‘it)Strategic alteration of thermostability of extracellular enzymes and a regulatory protein, and