The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished b... more The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished based on the new findings of the catalytic mechanism of glycogen branching enzymes (GBEs) from Vibrio vulnificus, Deinococcus geothermalis, and Escherichia coli. GBEs from D. geothermalis and E. coli used maltononaose and maltotridecaose as the minimum, respectively, while V. vulnificus GBE (VvGBE) catalyzed the surprisingly small substrate, G8, into polysaccharides. Intriguingly, all three GBEs catalyzed α-1,4-transglycosylation at the early reaction stage of transglycosylation. VvGBE successfully converted the smallest substrate (G8) into two highly branched polysaccharides (HBP), in which the big polysaccharide (1.49 × 105 Da) exhibited structural properties similar to glycogen. Both HBPs had similar side chain distribution with a very short average degree of polymerization compared with mussel glycogen. As a molecular biology reagent, these nucleotide-free HBPs significantly increased the mRNA extraction efficiency of mammalian cells. Our results provide a new approach to the synthesis of novel polysaccharides.
The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished b... more The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished based on the new findings of the catalytic mechanism of glycogen branching enzymes (GBEs) from Vibrio vulnificus, Deinococcus geothermalis, and Escherichia coli. GBEs from D. geothermalis and E. coli used maltononaose and maltotridecaose as the minimum, respectively, while V. vulnificus GBE (VvGBE) catalyzed the surprisingly small substrate, G8, into polysaccharides. Intriguingly, all three GBEs catalyzed α-1,4-transglycosylation at the early reaction stage of transglycosylation. VvGBE successfully converted the smallest substrate (G8) into two highly branched polysaccharides (HBP), in which the big polysaccharide (1.49 × 105 Da) exhibited structural properties similar to glycogen. Both HBPs had similar side chain distribution with a very short average degree of polymerization compared with mussel glycogen. As a molecular biology reagent, these nucleotide-free HBPs significantly increased the mRNA extraction efficiency of mammalian cells. Our results provide a new approach to the synthesis of novel polysaccharides.
ABSTRACT Presence of β-amylase in ramie leaf and its anti-staling effect on starch-based foods we... more ABSTRACT Presence of β-amylase in ramie leaf and its anti-staling effect on starch-based foods were assessed. The ammonium sulfate fractionate (80% saturation) of the ramie leaf extracts showed a β-amylase activity, giving maltose (Glc2) as a major product, exclusively, when incubating with maltopentaose (Glc5) or soluble starch at 45°C, pH 6.0. The starch-based food product (rice cake) prepared with freeze-dried ramie leaf enzyme revealed that the linear maltooligosaccharides ranging from Glc2 to Glc6 significantly increased and the shorter branch chains (DP16) decreased in the product. These results demonstrated that maltosyl residue was released from the non-reducing end of the longer branch chains of amylopectin by β-amylase. The ramie leaf-treatment sample significantly reduced the retrogradation rate during 48 h storage at 4°C. As an alternative plant-origin enzyme, the ramie leaf β-amylase has potential for a novel anti-staling additive.
Inotodiol, an oxysterol found only in Chaga mushroom, has received attention from the pharmaceuti... more Inotodiol, an oxysterol found only in Chaga mushroom, has received attention from the pharmaceutical industry due to its strong antioxidant and anti-allergic activities. However, the production of inotodiol is still challenging, and its fundamental properties have yet to be investigated. This study aims to develop an efficient method to produce high-purity inotodiol from Chaga mushroom. Then, pure inotodiol was used to assess its physicochemical properties and biological activities. By optimizing the solvent used for extraction and purification, a new method to produce inotodiol was developed with high purity (>97%) and purification yield (33.6%). Inotodiol exhibited a melting point (192.06 °C) much higher than lanosterol and cholesterol. However, the solubility of inotodiol in organic solvents was notably lower than those of the other two sterols. The difference in the hydroxyl group at C-22 of inotodiol has shown the distinctive physicochemical properties of inotodiol compared ...
Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 sub... more Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120–180 (N-terminal) and 646–676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, β-CD binds more weakly than the linear substrate, possi...
Three bacterial glycogen branching enzymes (GBEs) having different branching characteristics were... more Three bacterial glycogen branching enzymes (GBEs) having different branching characteristics were used to produce clustered amylopectin (CAP), and structure and functional properties of CAPs were intensively analyzed. Branch distributions of three CAPs varied from very short (DPn = 6.65) to medium (DPn = 14.1). Branch distribution showed profound correlation with hydrodynamic diameter, water solubility, digestibility, and effects on mice gut-microbiota. All the CAPs showed nearly no viscosity and retrogradation. The very short-branch CAP exhibited more than 100-fold water-solubility, 3.5-fold lower α-amylase catalytic efficiency, and 27% lower digestibility in small intestine-mimicking condition than amylopectin. Intriguingly, medium branch CAP had 1.8-fold larger hydrodynamic diameter than the very short one. Mice gut-microbiota composition statistically varied after 12-day feeding of the CAPs, but only the medium chain CAP brought clear positive changes on the gut-microbiota. Consequently, slowly digestible starch was successfully synthesized by the single GBE, but the CAP structure affects in vivo functions in complicated manner.
Biochemical and biophysical research communications, Jan 5, 2014
To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amyla... more To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM(-1)s(-1) for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM(-1)s(-1) for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235-located on a wide open groove near subsite +1-is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.
Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were ... more Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in Δ malP (encoding maltodextrin phosphorylase), Δ malQ (encoding amylomaltase), Δ glgA (encoding glycogen synthase), and Δ glgA Δ malP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the Δ malP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the Δ malP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The Δ malQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthes...
ABSTRACT Jackfruit seed starch (JFSS) cultivated in Vietnam had a high amylose content (∼44%). Di... more ABSTRACT Jackfruit seed starch (JFSS) cultivated in Vietnam had a high amylose content (∼44%). Differential scanning calorimetry thermogram of JFSS consisted of two separate endothermic peaks with distinct onset and conclusion temperatures. When JFSS was heated at 70°C, the first endothermic peak disappeared, whereas the second endotherm remained. The partially-gelatinized JFSS still possessed A-type X-ray diffraction pattern. SEM images showed that the rim of the starch granule at the hollow bottom of the bell shape first melted during the partial gelatinization at 70°C. Both native and partially-gelatinized JFSS produced soft and highly elastic gels, but their moduli differed slightly. Additionally, the partially-gelatinized JFSS was successfully used to produce genistin-cycloamylose (CA) and amylose-lysolecithin complexes with and without 4-α-glucanotransferase. Water sorption isotherms of partially gelatinized JFSS showed higher water-holding capacity than that of the native starch.
↵3 To whom correspondence may be addressed. Tel.: 82-42-879-8432; Fax: 82-42-879-8596 ; E-mail: e... more ↵3 To whom correspondence may be addressed. Tel.: 82-42-879-8432; Fax: 82-42-879-8596 ; E-mail: ejwoo{at}kribb.re.kr. ... ↵4 To whom correspondence may be addressed. Tel.: 82-2-781-7528; Fax: 82-2-391-8758; E-mail: parkkh{at}snu.ac.kr. ... Background: Maltogenic amylases that are known to date form dimers to perform hydrolysis. ... Results: The structure of maltogenic amylase from Staphylothermus showed a novel domain at the N terminus associated with the active site. ... Conclusion: Staphylothermus amylase has all of its ...
The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished b... more The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished based on the new findings of the catalytic mechanism of glycogen branching enzymes (GBEs) from Vibrio vulnificus, Deinococcus geothermalis, and Escherichia coli. GBEs from D. geothermalis and E. coli used maltononaose and maltotridecaose as the minimum, respectively, while V. vulnificus GBE (VvGBE) catalyzed the surprisingly small substrate, G8, into polysaccharides. Intriguingly, all three GBEs catalyzed α-1,4-transglycosylation at the early reaction stage of transglycosylation. VvGBE successfully converted the smallest substrate (G8) into two highly branched polysaccharides (HBP), in which the big polysaccharide (1.49 × 105 Da) exhibited structural properties similar to glycogen. Both HBPs had similar side chain distribution with a very short average degree of polymerization compared with mussel glycogen. As a molecular biology reagent, these nucleotide-free HBPs significantly increased the mRNA extraction efficiency of mammalian cells. Our results provide a new approach to the synthesis of novel polysaccharides.
The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished b... more The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished based on the new findings of the catalytic mechanism of glycogen branching enzymes (GBEs) from Vibrio vulnificus, Deinococcus geothermalis, and Escherichia coli. GBEs from D. geothermalis and E. coli used maltononaose and maltotridecaose as the minimum, respectively, while V. vulnificus GBE (VvGBE) catalyzed the surprisingly small substrate, G8, into polysaccharides. Intriguingly, all three GBEs catalyzed α-1,4-transglycosylation at the early reaction stage of transglycosylation. VvGBE successfully converted the smallest substrate (G8) into two highly branched polysaccharides (HBP), in which the big polysaccharide (1.49 × 105 Da) exhibited structural properties similar to glycogen. Both HBPs had similar side chain distribution with a very short average degree of polymerization compared with mussel glycogen. As a molecular biology reagent, these nucleotide-free HBPs significantly increased the mRNA extraction efficiency of mammalian cells. Our results provide a new approach to the synthesis of novel polysaccharides.
ABSTRACT Presence of β-amylase in ramie leaf and its anti-staling effect on starch-based foods we... more ABSTRACT Presence of β-amylase in ramie leaf and its anti-staling effect on starch-based foods were assessed. The ammonium sulfate fractionate (80% saturation) of the ramie leaf extracts showed a β-amylase activity, giving maltose (Glc2) as a major product, exclusively, when incubating with maltopentaose (Glc5) or soluble starch at 45°C, pH 6.0. The starch-based food product (rice cake) prepared with freeze-dried ramie leaf enzyme revealed that the linear maltooligosaccharides ranging from Glc2 to Glc6 significantly increased and the shorter branch chains (DP16) decreased in the product. These results demonstrated that maltosyl residue was released from the non-reducing end of the longer branch chains of amylopectin by β-amylase. The ramie leaf-treatment sample significantly reduced the retrogradation rate during 48 h storage at 4°C. As an alternative plant-origin enzyme, the ramie leaf β-amylase has potential for a novel anti-staling additive.
Inotodiol, an oxysterol found only in Chaga mushroom, has received attention from the pharmaceuti... more Inotodiol, an oxysterol found only in Chaga mushroom, has received attention from the pharmaceutical industry due to its strong antioxidant and anti-allergic activities. However, the production of inotodiol is still challenging, and its fundamental properties have yet to be investigated. This study aims to develop an efficient method to produce high-purity inotodiol from Chaga mushroom. Then, pure inotodiol was used to assess its physicochemical properties and biological activities. By optimizing the solvent used for extraction and purification, a new method to produce inotodiol was developed with high purity (>97%) and purification yield (33.6%). Inotodiol exhibited a melting point (192.06 °C) much higher than lanosterol and cholesterol. However, the solubility of inotodiol in organic solvents was notably lower than those of the other two sterols. The difference in the hydroxyl group at C-22 of inotodiol has shown the distinctive physicochemical properties of inotodiol compared ...
Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 sub... more Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120–180 (N-terminal) and 646–676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, β-CD binds more weakly than the linear substrate, possi...
Three bacterial glycogen branching enzymes (GBEs) having different branching characteristics were... more Three bacterial glycogen branching enzymes (GBEs) having different branching characteristics were used to produce clustered amylopectin (CAP), and structure and functional properties of CAPs were intensively analyzed. Branch distributions of three CAPs varied from very short (DPn = 6.65) to medium (DPn = 14.1). Branch distribution showed profound correlation with hydrodynamic diameter, water solubility, digestibility, and effects on mice gut-microbiota. All the CAPs showed nearly no viscosity and retrogradation. The very short-branch CAP exhibited more than 100-fold water-solubility, 3.5-fold lower α-amylase catalytic efficiency, and 27% lower digestibility in small intestine-mimicking condition than amylopectin. Intriguingly, medium branch CAP had 1.8-fold larger hydrodynamic diameter than the very short one. Mice gut-microbiota composition statistically varied after 12-day feeding of the CAPs, but only the medium chain CAP brought clear positive changes on the gut-microbiota. Consequently, slowly digestible starch was successfully synthesized by the single GBE, but the CAP structure affects in vivo functions in complicated manner.
Biochemical and biophysical research communications, Jan 5, 2014
To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amyla... more To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM(-1)s(-1) for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM(-1)s(-1) for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235-located on a wide open groove near subsite +1-is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.
Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were ... more Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in Δ malP (encoding maltodextrin phosphorylase), Δ malQ (encoding amylomaltase), Δ glgA (encoding glycogen synthase), and Δ glgA Δ malP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the Δ malP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the Δ malP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The Δ malQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthes...
ABSTRACT Jackfruit seed starch (JFSS) cultivated in Vietnam had a high amylose content (∼44%). Di... more ABSTRACT Jackfruit seed starch (JFSS) cultivated in Vietnam had a high amylose content (∼44%). Differential scanning calorimetry thermogram of JFSS consisted of two separate endothermic peaks with distinct onset and conclusion temperatures. When JFSS was heated at 70°C, the first endothermic peak disappeared, whereas the second endotherm remained. The partially-gelatinized JFSS still possessed A-type X-ray diffraction pattern. SEM images showed that the rim of the starch granule at the hollow bottom of the bell shape first melted during the partial gelatinization at 70°C. Both native and partially-gelatinized JFSS produced soft and highly elastic gels, but their moduli differed slightly. Additionally, the partially-gelatinized JFSS was successfully used to produce genistin-cycloamylose (CA) and amylose-lysolecithin complexes with and without 4-α-glucanotransferase. Water sorption isotherms of partially gelatinized JFSS showed higher water-holding capacity than that of the native starch.
↵3 To whom correspondence may be addressed. Tel.: 82-42-879-8432; Fax: 82-42-879-8596 ; E-mail: e... more ↵3 To whom correspondence may be addressed. Tel.: 82-42-879-8432; Fax: 82-42-879-8596 ; E-mail: ejwoo{at}kribb.re.kr. ... ↵4 To whom correspondence may be addressed. Tel.: 82-2-781-7528; Fax: 82-2-391-8758; E-mail: parkkh{at}snu.ac.kr. ... Background: Maltogenic amylases that are known to date form dimers to perform hydrolysis. ... Results: The structure of maltogenic amylase from Staphylothermus showed a novel domain at the N terminus associated with the active site. ... Conclusion: Staphylothermus amylase has all of its ...
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