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The concept of the α-amylase family and specificity engineering of starch-related enzymes. —Monograph—
VOL. 89, 2000
Abstracts of the Articles Printed in Seibutsu-kogakuKaishi Vol. 78, Nos. 4 & 6 (2000) Liquid Circulation
Reactors. and HIDEHIRO (Department of Material System Engirteering and Applied Life Science, Toyama University, 3190 Gofuku, Toyama 9308555) Seibutsu-kogaku 77: 115-l 19. 2000. An experimental study was performed on the liquid circulation time (t,-) in an internal-loop airlift reactor when the external operating conditions such as the gas flow rate, diameter (Dn) and length (Ln) of the draft tube, and unaerated liquid height (Ho) from the upper end of the draft tube were varied. The findings revelad that tc could be broadly categorized into two different regions with respect to the linear gas velocity (uoa) in the riser. In one region, tc was proportional to uos raised to the power of - l/3 and Ln raised to the power of l/2. In the other region, tc was constant and increased in proportion to Lo. The values of tc in both regions were independent of Ho under the experimental conditions employed. On the basis of the experimental findings, empirical equations predicting tc in the two regions are presented. HIROYUKI KUMAZAWA
Time in Internal-Loop
KAWASAKI,*
TATSUMI
Airlift
The Concept of the a-Amylase Family and Specificity Engineering of Starch-Related Enzymes. -MonographTAKASHI KURIKI (Biochemical Research Laboratory, Ezaki Glico Co. Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502) Seibutsu-kogaku 78: 128-135. 2000. A general concept for an enzyme family-the a-amylase family, including most amylases and related enzymes-is proposed and defined based on structural similarities and common catalytic mechanisms. Primary structural analyses, secondary structural predictions, and three-dimensional structural determinations stimulated this work. However, a series of studies on neopullulanase, which catalyzes both the hydrolysis and transglycosylation at a-1,4and a-1,6-glucosidic linkages by one active center, was the key that opened the door to the concept. On the basis of the proposed aamylase family concept, the substrate specificity and the ratio of the hydrolysis/transglycosylation activity of neopullulanase were altered. The three-dimensional structure of neopullulanase was predicted by homology modeling. The enzyme-substrate structure was constructed by a docking study and a molecular dynamics calculation, Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE II, respectively) have quite different properties; to elucidate the domains that determine the differences, chimeric enzymes consisting of part mBE I and part mBE II were constructed. The results indicated that the N- and C-terminus regions of the enzymes are involved in determining substrate preference, catalytic capacity, and chain-length transfer.
YAMAMOTO,
* Corresponding author. Glucoamylase-Encoding Genes of Aspergillus oryzae. -MonographYOJI HATA and HIROKI ISHIDA (Research Institute, Gekkeikan Sake Co. Ltd., 300 Katahara-cho, Fushimi-ku, Kyoto 612-8361) Seibutsukogaku 78: 120-127. 2000. Aspergillus oryzae, the most commonly used fungus in the Japanese fermentation industry, is of considerable industrial significance due to its ability to secrete copious quantities of hydrolytic enzymes. Some hydrolytic enzymes-especially glucoamylases, proteinases, and phosphatases-important in fermentation processes are produced in much higher amounts in solid-state culture than in submerged culture. However, the mechanism of the solid-state culture of this organism, including its higher enzyme productivity, domains unclear. Here, we describe the cloning and characterization of two glucoamylase-encoding genes, glaA and glaB, and examine their expression in order to clarify the mechanism of higher glucoamylase production in solid-state culture of A. oryzae. Sequence analysis of the two genes revealed that the glucoamylase encoded by glad consists of two functional domains, a catalytic domain at the N-terminus and a starch-binding domain at the C-terminus, as is the case other AspergiNus glucoamylases. However, the glucoamylase encoded by glaB has no C-terminal domain related to raw starch binding activity. The glaB gene is markedly expressed in solid-state culture, but only a little in submerged culture. To elucidate the mechanism of glaB induction in solid-state culture, we analyzed the promoter using the uidA gene (GUS gene) as a reporter gene. In solid-state culture, glaB expression at the transcriptional level is enhanced by low-Aw (water activity), high-temperature, and physical barriers to hyphal extension, as well as by starch. Deletion analysis of the promoter showed that substitution of the 12 bp CC-rich motif from -335 to - 324 (GC box) resulted in significant loss of starch and low-Aw induction. These findings suggest that the GC box is a cis-eiement essential for the high-level expression of glaB in solid-state culture.
Breeding of Aromatic Yeast by Hybridization of Haploid Strains Having Alcohol Tolerance. -NoteNAOTAKA KUROSE,* TADAO Asmo, Suon TARUMI, and SADAO KAWAIUTA (Alcoholic Beverages and Seasoning & Food Research Laboratories, Takara Shuzo Co. Ltd., 3-4-l Seta, Otsu, Shiga 520-2193) Seibutsu-kogaku 78: 191-194. 2000. Thirty-two haploid strains derived from the sake yeast Kyokai no. 701 (K-701) were efficiently isolated by ethanol (40 v/v%) treatment of sporulated cells and the subsequent culturing of red colonies on agar medium containing eosin Y and amaranth. To obtain diploid strains producing high amounts of isoamyl acetate, which imparts a fruit-like flavor, 6 haploid strains (mating type a: 3 strains; mating type a: 3 strains) with tolerance to 18 v/v% ethanol were first selected and hybridized with one another. Of the diploidal hybrids obtained, 3 alcohol-tolerant strains (8-30-2, 10-30-2, and 10-41-3) were used for laboratory-scale sake brewing tests. All of the hybrid strains had a high survival rate in the late period of the sake mash, and produced isoamyl acetate in amounts ranging from 2.0 to 2.6 times, and ethyl caproate from 2.0 to 2.7 times, the amounts produced by strain K-701 in sake. * Corresponding author.
* Corresponding author.
617
Abstracts of the Articles Printed in Seibutsu-kogakuKaishi Vol. 78, Nos. 4 & 6 (2000) Liquid Circulation
Reactors. and HIDEHIRO (Department of Material System Engirteering and Applied Life Science, Toyama University, 3190 Gofuku, Toyama 9308555) Seibutsu-kogaku 77: 115-l 19. 2000. An experimental study was performed on the liquid circulation time (t,-) in an internal-loop airlift reactor when the external operating conditions such as the gas flow rate, diameter (Dn) and length (Ln) of the draft tube, and unaerated liquid height (Ho) from the upper end of the draft tube were varied. The findings revelad that tc could be broadly categorized into two different regions with respect to the linear gas velocity (uoa) in the riser. In one region, tc was proportional to uos raised to the power of - l/3 and Ln raised to the power of l/2. In the other region, tc was constant and increased in proportion to Lo. The values of tc in both regions were independent of Ho under the experimental conditions employed. On the basis of the experimental findings, empirical equations predicting tc in the two regions are presented. HIROYUKI KUMAZAWA
Time in Internal-Loop
KAWASAKI,*
TATSUMI
Airlift
The Concept of the a-Amylase Family and Specificity Engineering of Starch-Related Enzymes. -MonographTAKASHI KURIKI (Biochemical Research Laboratory, Ezaki Glico Co. Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502) Seibutsu-kogaku 78: 128-135. 2000. A general concept for an enzyme family-the a-amylase family, including most amylases and related enzymes-is proposed and defined based on structural similarities and common catalytic mechanisms. Primary structural analyses, secondary structural predictions, and three-dimensional structural determinations stimulated this work. However, a series of studies on neopullulanase, which catalyzes both the hydrolysis and transglycosylation at a-1,4and a-1,6-glucosidic linkages by one active center, was the key that opened the door to the concept. On the basis of the proposed aamylase family concept, the substrate specificity and the ratio of the hydrolysis/transglycosylation activity of neopullulanase were altered. The three-dimensional structure of neopullulanase was predicted by homology modeling. The enzyme-substrate structure was constructed by a docking study and a molecular dynamics calculation, Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE II, respectively) have quite different properties; to elucidate the domains that determine the differences, chimeric enzymes consisting of part mBE I and part mBE II were constructed. The results indicated that the N- and C-terminus regions of the enzymes are involved in determining substrate preference, catalytic capacity, and chain-length transfer.
YAMAMOTO,
* Corresponding author. Glucoamylase-Encoding Genes of Aspergillus oryzae. -MonographYOJI HATA and HIROKI ISHIDA (Research Institute, Gekkeikan Sake Co. Ltd., 300 Katahara-cho, Fushimi-ku, Kyoto 612-8361) Seibutsukogaku 78: 120-127. 2000. Aspergillus oryzae, the most commonly used fungus in the Japanese fermentation industry, is of considerable industrial significance due to its ability to secrete copious quantities of hydrolytic enzymes. Some hydrolytic enzymes-especially glucoamylases, proteinases, and phosphatases-important in fermentation processes are produced in much higher amounts in solid-state culture than in submerged culture. However, the mechanism of the solid-state culture of this organism, including its higher enzyme productivity, domains unclear. Here, we describe the cloning and characterization of two glucoamylase-encoding genes, glaA and glaB, and examine their expression in order to clarify the mechanism of higher glucoamylase production in solid-state culture of A. oryzae. Sequence analysis of the two genes revealed that the glucoamylase encoded by glad consists of two functional domains, a catalytic domain at the N-terminus and a starch-binding domain at the C-terminus, as is the case other AspergiNus glucoamylases. However, the glucoamylase encoded by glaB has no C-terminal domain related to raw starch binding activity. The glaB gene is markedly expressed in solid-state culture, but only a little in submerged culture. To elucidate the mechanism of glaB induction in solid-state culture, we analyzed the promoter using the uidA gene (GUS gene) as a reporter gene. In solid-state culture, glaB expression at the transcriptional level is enhanced by low-Aw (water activity), high-temperature, and physical barriers to hyphal extension, as well as by starch. Deletion analysis of the promoter showed that substitution of the 12 bp CC-rich motif from -335 to - 324 (GC box) resulted in significant loss of starch and low-Aw induction. These findings suggest that the GC box is a cis-eiement essential for the high-level expression of glaB in solid-state culture.
Breeding of Aromatic Yeast by Hybridization of Haploid Strains Having Alcohol Tolerance. -NoteNAOTAKA KUROSE,* TADAO Asmo, Suon TARUMI, and SADAO KAWAIUTA (Alcoholic Beverages and Seasoning & Food Research Laboratories, Takara Shuzo Co. Ltd., 3-4-l Seta, Otsu, Shiga 520-2193) Seibutsu-kogaku 78: 191-194. 2000. Thirty-two haploid strains derived from the sake yeast Kyokai no. 701 (K-701) were efficiently isolated by ethanol (40 v/v%) treatment of sporulated cells and the subsequent culturing of red colonies on agar medium containing eosin Y and amaranth. To obtain diploid strains producing high amounts of isoamyl acetate, which imparts a fruit-like flavor, 6 haploid strains (mating type a: 3 strains; mating type a: 3 strains) with tolerance to 18 v/v% ethanol were first selected and hybridized with one another. Of the diploidal hybrids obtained, 3 alcohol-tolerant strains (8-30-2, 10-30-2, and 10-41-3) were used for laboratory-scale sake brewing tests. All of the hybrid strains had a high survival rate in the late period of the sake mash, and produced isoamyl acetate in amounts ranging from 2.0 to 2.6 times, and ethyl caproate from 2.0 to 2.7 times, the amounts produced by strain K-701 in sake. * Corresponding author.
* Corresponding author.
617