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Buford L Nichols - One of the best experts on this subject based on the ideXlab platform.
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13c labeled starch breath test in congenital sucrase isomaltase deficiency
Journal of Pediatric Gastroenterology and Nutrition, 2018Co-Authors: Claudia C Robayotorres, Susan S Baker, Bruce R Hamaker, Antone R. Opekun, Marisela Diazsotomayor, Bruno P Chumpitazi, Buford L NicholsAbstract:ABSTRACTBackground and Hypotheses:Human starch digestion is a multienzyme process involving 6 different enzymes: salivary and pancreatic α-amylase; sucrase and isomaltase (from sucrose-isomaltase [SI]), and maltase and Glucoamylase (from maltase-Glucoamylase [MGAM]). Together these enzymes cleave st
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improved starch digestion of sucrase deficient shrews treated with oral Glucoamylase enzyme supplements
Journal of Pediatric Gastroenterology and Nutrition, 2017Co-Authors: Buford L Nichols, Stephen E Avery, Roberto Quezadacalvillo, Shadi B Kilani, Amy Huimei Lin, Douglas G Burrin, Benjamin E Hodges, Shaji K ChackoAbstract:BACKGROUND AND OBJECTIVE Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and Glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-Glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral Glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral Glucoamylase supplements. METHODS Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam Glucoamylase (M20) was supplemented to improve starch digestion. RESULTS After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of Glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. CONCLUSIONS Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant Glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.
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direct starch digestion by sucrase isomaltase and maltase Glucoamylase
Journal of Pediatric Gastroenterology and Nutrition, 2012Co-Authors: Bruce R Hamaker, Buford L NicholsAbstract:1. Jane J, Chen YY, Lee LF, et al. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chem 1999;76:629–37. 2. Quezada-Calvillo R, Robayo-Torres CC, Opekum AR, et al. Contribution of mucosal maltase-Glucoamylase activities to mouse small intestinal starch alpha-glucogenesis. J Nutr 2007;137:1725–33. 3. Nichols BL, Quezada-Calvillo R, Robayo-Torres CC, et al. Mucosal maltase-Glucoamylase plays a crucial role in starch digestion and prandial glucose homeostasis of mice. J Nutr 2009;139:684–90. 4. Sim L, Quezada-Calvillo R, Sterchi EE, et al. Human intestinal maltaseGlucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity. J Mol Biol 2008;375:782–92. 5. Ao Z, Quezada-Calvillo R, Sim L, et al. Evidence of native starch degradation with human small intestinal maltase-Glucoamylase (recombinant). FEBS Lett 2007;581:2381–8.
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congenital maltase Glucoamylase deficiency associated with lactase and sucrase deficiencies
Journal of Pediatric Gastroenterology and Nutrition, 2002Co-Authors: Buford L Nichols, Dagmar Hahn, Wikrom Karnsakul, Ursula Luginbuehl, Stephen E Avery, Dallas M. Swallow, Farook Jahoor, Erwin E. SterchiAbstract:Background: Multiple enzyme deficiencies have been reported in some cases of congenital Glucoamylase, sucrase, or lactase deficiency. Here we describe such a case and the investigations that we have made to determine the cause of this deficiency.Methods and Results: A 2.5 month-old infant, admitted with congenital lactase deficiency, failed to gain weight on a glucose oligomer formula (Nutramigen(R)). Jejunal mucosal biopsy at 4 and 12 months revealed normal histology with decreased maltase-Glucoamylase, sucrase-isomaltase, and lactase-phlorizin hydrolase activities. Testing with a C-13-starch/breath (CO2)-C-13 loading test confirmed proximal starch malabsorption. Sequencing of maltase-Glucoamylase cDNA revealed homozygosity for a nucleotide change (C1673T) in the infant, which causes an amino acid substitution (S542L) 12 amino acids after the N-terminal catalytic aspartic acid. The introduction of this mutation into "wildtype" N-terminus maltase-Glucoamylase cDNA was not associated with obvious loss of maltase-Glucoamylase enzyme activities when expressed in COS 1 cells and this amino-acid change was subsequently found in other people. Sequencing of the promoter region revealed no nucleotide changes. Maltase-Glucoamylase, lactase, and sucrase-isomaltase were each normally synthesized and processed in organ culture.Conclusions: The lack of evidence for a causal nucleotide change in the maltase-Glucoamylase gene in this patient, and the concomitant low levels of lactase and sucrase activity, suggest that the depletion of mucosal maltase-Glucoamylase activity and starch digestion was caused by shared, pleiotropic regulatory factors.
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disaccharidase activities in dyspeptic children biochemical and molecular investigations of maltase Glucoamylase activity
Journal of Pediatric Gastroenterology and Nutrition, 2002Co-Authors: Wikrom Karnsakul, Dagmar Hahn, Ursula Luginbuehl, Erwin E. Sterchi, Stephen E Avery, Dallas M. Swallow, Buford L NicholsAbstract:ABSTRACTBackgroundMaltase-Glucoamylase enzyme plays an important role in starch digestion. Glucoamylase deficiency is reported to cause chronic diarrhea in infants, but its role in dyspeptic children is unknown.MethodsGlucoamylase and other disaccharidase specific activities were assayed from duoden
Akihiko Kondo - One of the best experts on this subject based on the ideXlab platform.
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Efficient co-displaying and artificial ratio control of α-amylase and Glucoamylase on the yeast cell surface by using combinations of different anchoring domains
Applied Microbiology and Biotechnology, 2015Co-Authors: Kentaro Inokuma, Takanobu Yoshida, Jun Ishii, Tomohisa Hasunuma, Akihiko KondoAbstract:Recombinant yeast strains that display heterologous amylolytic enzymes on their cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system are considered as promising biocatalysts for direct ethanol production from starchy materials. For the effective hydrolysis of these materials, the ratio optimization of multienzyme activity displayed on the cell surface is important. In this study, we have presented a ratio control system of multienzymes displayed on the yeast cell surface by using different GPI-anchoring domains. The novel gene cassettes for the cell-surface display of Streptococcus bovis α-amylase and Rhizopus oryzae Glucoamylase were constructed using the Saccharomyces cerevisiae SED1 promoter and two different GPI-anchoring regions derived from Saccharomyces cerevisiae SED1 or SAG1 . These gene cassettes were integrated into the Saccharomyces cerevisiae genome in different combinations. Then, the cell-surface α-amylase and Glucoamylase activities and ethanol productivity of these recombinant strains were evaluated. The combinations of the gene cassettes of these enzymes affected the ratio of cell-surface α-amylase and Glucoamylase activities and ethanol productivity of the recombinant strains. The highest ethanol productivity from raw starch was achieved by the strain harboring one α-amylase gene cassette carrying the SED1 -anchoring region and two Glucoamylase gene cassettes carrying the SED1 -anchoring region (BY-AASS/GASS/GASS). This strain yielded 22.5 ± 0.6 g/L of ethanol from 100 g/L of raw starch in 120 h of fermentation.
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repeated fermentation from raw starch using saccharomyces cerevisiae displaying both Glucoamylase and α amylase
Enzyme and Microbial Technology, 2012Co-Authors: Syunichi Yamakawa, Ryosuke Yamada, Tsutomu Tanaka, Chiaki Ogino, Akihiko KondoAbstract:A diploid yeast strain displaying both α-amylase and Glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both Glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the Glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or Glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.
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Long anchor using Flo1 protein enhances reactivity of cell surface-displayed Glucoamylase to polymer substrates.
Applied microbiology and biotechnology, 2002Co-Authors: N. Sato, Takeshi Matsumoto, Mitsuyoshi Ueda, Atsuo Tanaka, Hideki Fukuda, Akihiko KondoAbstract:We investigated the influence of anchor length on the reactivity to polymer substrate of enzyme displayed on yeast cell surfaces. Using various lengths [42, 102, 146, 318, 428, and 1,326 amino acids (aa)] of the C-terminal region of the Saccharomyces cerevisiae Flo1 protein (Flo1p), which plays a major role in yeast flocculation, six display systems with various anchor lengths were constructed. In these systems, the target protein was displayed on the yeast cell surface under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL). Cell-surface display of Rhizopus oryzae Glucoamylase by these systems was induced and confirmed in all systems by immunofluorescence microscopy and immunoblotting. Flow-cytometer measurement of the fluorescence intensity of immunofluorescence-labeled yeast cells displaying Glucoamylase indicated that Glucoamylase displayed with longer anchors, especially those of 428 and 1,326 aa in length, had higher reactivity to antibodies. The reactivity of starch to displayed Glucoamylase, which was evaluated by plate assay, increased with anchor length, as did the cell growth-rate in starch-containing medium. These results indicate that cell-surface display systems using 428- and 1,326-aa length anchors of Flo1p are effective for the display of enzymes on the outer surface of yeast cells.
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efficient ethanol production from starch through development of novel flocculent yeast strains displaying Glucoamylase and co displaying or secreting α amylase
Journal of Molecular Catalysis B-enzymatic, 2002Co-Authors: Hisayori Shigechi, Ken Uyama, Yasuya Fujita, Takeshi Matsumoto, Mitsuyoshi Ueda, Atsuo Tanaka, Hideki Fukuda, Akihiko KondoAbstract:To develop novel yeasts with high starch-to-ethanol productivity, we constructed two cell-surface-engineered flocculent Saccharomyces cerevisiae strains; one co-displaying Glucoamylase and α-amylase on the cell surface and the other displaying Glucoamylase and secreting α-amylase into the culture medium. With starch as the carbon source, both yeast strains grew faster under aerobic conditions than strains displaying only Glucoamylase. In fed-batch fermentation of ethanol, these recombinant yeasts co-expressing sequential amylolytic enzymes also showed higher starch decomposition and ethanol production abilities than yeast cells displaying only Glucoamylase, with the concentration of ethanol produced reaching 60 g/l after approximately 100 h fermentation under anaerobic conditions. Both co-display and secretion of α-amylase are thus effective in improving ethanol production from starchy materials in Glucoamylase-displaying yeast cells.
Erwin E. Sterchi - One of the best experts on this subject based on the ideXlab platform.
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disaccharidase activities in dyspeptic children biochemical and molecular investigations of maltase Glucoamylase activity
Journal of Pediatric Gastroenterology and Nutrition, 2002Co-Authors: Wikrom Karnsakul, Dagmar Hahn, Ursula Luginbuehl, Erwin E. Sterchi, Stephen E Avery, Dallas M. Swallow, Buford L NicholsAbstract:ABSTRACTBackgroundMaltase-Glucoamylase enzyme plays an important role in starch digestion. Glucoamylase deficiency is reported to cause chronic diarrhea in infants, but its role in dyspeptic children is unknown.MethodsGlucoamylase and other disaccharidase specific activities were assayed from duoden
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congenital maltase Glucoamylase deficiency associated with lactase and sucrase deficiencies
Journal of Pediatric Gastroenterology and Nutrition, 2002Co-Authors: Buford L Nichols, Dagmar Hahn, Wikrom Karnsakul, Ursula Luginbuehl, Stephen E Avery, Dallas M. Swallow, Farook Jahoor, Erwin E. SterchiAbstract:Background: Multiple enzyme deficiencies have been reported in some cases of congenital Glucoamylase, sucrase, or lactase deficiency. Here we describe such a case and the investigations that we have made to determine the cause of this deficiency.Methods and Results: A 2.5 month-old infant, admitted with congenital lactase deficiency, failed to gain weight on a glucose oligomer formula (Nutramigen(R)). Jejunal mucosal biopsy at 4 and 12 months revealed normal histology with decreased maltase-Glucoamylase, sucrase-isomaltase, and lactase-phlorizin hydrolase activities. Testing with a C-13-starch/breath (CO2)-C-13 loading test confirmed proximal starch malabsorption. Sequencing of maltase-Glucoamylase cDNA revealed homozygosity for a nucleotide change (C1673T) in the infant, which causes an amino acid substitution (S542L) 12 amino acids after the N-terminal catalytic aspartic acid. The introduction of this mutation into "wildtype" N-terminus maltase-Glucoamylase cDNA was not associated with obvious loss of maltase-Glucoamylase enzyme activities when expressed in COS 1 cells and this amino-acid change was subsequently found in other people. Sequencing of the promoter region revealed no nucleotide changes. Maltase-Glucoamylase, lactase, and sucrase-isomaltase were each normally synthesized and processed in organ culture.Conclusions: The lack of evidence for a causal nucleotide change in the maltase-Glucoamylase gene in this patient, and the concomitant low levels of lactase and sucrase activity, suggest that the depletion of mucosal maltase-Glucoamylase activity and starch digestion was caused by shared, pleiotropic regulatory factors.
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human small intestinal maltase Glucoamylase cdna cloning homology to sucrase isomaltase
Journal of Biological Chemistry, 1998Co-Authors: Buford L Nichols, Andrea Quaroni, Joyce A Eldering, Dagmar Hahn, Stephen E Avery, Erwin E. SterchiAbstract:Abstract It has been hypothesized that human mucosal Glucoamylase (EC 3.2.1.20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal α-amylase activity is reduced because of immaturity or malnutrition and that maltase-Glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing. As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-Glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments. Human maltase-Glucoamylase was purified by immunoisolation and partially sequenced. Maltase-Glucoamylase cDNA was amplified from human intestinal RNA using degenerate and gene-specific primers with the reverse transcription-polymerase chain reaction. The 6,513-base pair cDNA contains an open reading frame that encodes a 1,857-amino acid protein (molecular mass 209,702 Da). Maltase-Glucoamylase has two catalytic sites identical to those of sucrase-isomaltase, but the proteins are only 59% homologous. Both are members of glycosyl hydrolase family 31, which has a variety of substrate specificities. Our findings suggest that divergences in the carbohydrate binding sequences must determine the substrate specificities for the four different enzyme activities that share a conserved catalytic site.
Stephen E Avery - One of the best experts on this subject based on the ideXlab platform.
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improved starch digestion of sucrase deficient shrews treated with oral Glucoamylase enzyme supplements
Journal of Pediatric Gastroenterology and Nutrition, 2017Co-Authors: Buford L Nichols, Stephen E Avery, Roberto Quezadacalvillo, Shadi B Kilani, Amy Huimei Lin, Douglas G Burrin, Benjamin E Hodges, Shaji K ChackoAbstract:BACKGROUND AND OBJECTIVE Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and Glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-Glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral Glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral Glucoamylase supplements. METHODS Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam Glucoamylase (M20) was supplemented to improve starch digestion. RESULTS After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of Glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. CONCLUSIONS Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant Glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.
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congenital maltase Glucoamylase deficiency associated with lactase and sucrase deficiencies
Journal of Pediatric Gastroenterology and Nutrition, 2002Co-Authors: Buford L Nichols, Dagmar Hahn, Wikrom Karnsakul, Ursula Luginbuehl, Stephen E Avery, Dallas M. Swallow, Farook Jahoor, Erwin E. SterchiAbstract:Background: Multiple enzyme deficiencies have been reported in some cases of congenital Glucoamylase, sucrase, or lactase deficiency. Here we describe such a case and the investigations that we have made to determine the cause of this deficiency.Methods and Results: A 2.5 month-old infant, admitted with congenital lactase deficiency, failed to gain weight on a glucose oligomer formula (Nutramigen(R)). Jejunal mucosal biopsy at 4 and 12 months revealed normal histology with decreased maltase-Glucoamylase, sucrase-isomaltase, and lactase-phlorizin hydrolase activities. Testing with a C-13-starch/breath (CO2)-C-13 loading test confirmed proximal starch malabsorption. Sequencing of maltase-Glucoamylase cDNA revealed homozygosity for a nucleotide change (C1673T) in the infant, which causes an amino acid substitution (S542L) 12 amino acids after the N-terminal catalytic aspartic acid. The introduction of this mutation into "wildtype" N-terminus maltase-Glucoamylase cDNA was not associated with obvious loss of maltase-Glucoamylase enzyme activities when expressed in COS 1 cells and this amino-acid change was subsequently found in other people. Sequencing of the promoter region revealed no nucleotide changes. Maltase-Glucoamylase, lactase, and sucrase-isomaltase were each normally synthesized and processed in organ culture.Conclusions: The lack of evidence for a causal nucleotide change in the maltase-Glucoamylase gene in this patient, and the concomitant low levels of lactase and sucrase activity, suggest that the depletion of mucosal maltase-Glucoamylase activity and starch digestion was caused by shared, pleiotropic regulatory factors.
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disaccharidase activities in dyspeptic children biochemical and molecular investigations of maltase Glucoamylase activity
Journal of Pediatric Gastroenterology and Nutrition, 2002Co-Authors: Wikrom Karnsakul, Dagmar Hahn, Ursula Luginbuehl, Erwin E. Sterchi, Stephen E Avery, Dallas M. Swallow, Buford L NicholsAbstract:ABSTRACTBackgroundMaltase-Glucoamylase enzyme plays an important role in starch digestion. Glucoamylase deficiency is reported to cause chronic diarrhea in infants, but its role in dyspeptic children is unknown.MethodsGlucoamylase and other disaccharidase specific activities were assayed from duoden
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human small intestinal maltase Glucoamylase cdna cloning homology to sucrase isomaltase
Journal of Biological Chemistry, 1998Co-Authors: Buford L Nichols, Andrea Quaroni, Joyce A Eldering, Dagmar Hahn, Stephen E Avery, Erwin E. SterchiAbstract:Abstract It has been hypothesized that human mucosal Glucoamylase (EC 3.2.1.20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal α-amylase activity is reduced because of immaturity or malnutrition and that maltase-Glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing. As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-Glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments. Human maltase-Glucoamylase was purified by immunoisolation and partially sequenced. Maltase-Glucoamylase cDNA was amplified from human intestinal RNA using degenerate and gene-specific primers with the reverse transcription-polymerase chain reaction. The 6,513-base pair cDNA contains an open reading frame that encodes a 1,857-amino acid protein (molecular mass 209,702 Da). Maltase-Glucoamylase has two catalytic sites identical to those of sucrase-isomaltase, but the proteins are only 59% homologous. Both are members of glycosyl hydrolase family 31, which has a variety of substrate specificities. Our findings suggest that divergences in the carbohydrate binding sequences must determine the substrate specificities for the four different enzyme activities that share a conserved catalytic site.
Yoshiyuki Sakano - One of the best experts on this subject based on the ideXlab platform.
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site directed mutagenesis of tryptophan 622 of thermoactinomyces vulgaris r 47 Glucoamylase
Journal of applied glycoscience, 2005Co-Authors: Kazuhiro Ichikawa, Takashi Tonozuka, Atsushi Nishikawa, Masahiro Mizuno, Yoshiyuki SakanoAbstract:In Aspergillus awamori Glucoamylase, the optimal pH has been reported to increase to maintain activity by a mutation of Ser411 which forms a hydrogen-bond with a catalytic base (Fang and Ford, Protein Eng., 11, 383-388 (1998)). Most Glucoamylases have either Ser or Gly at this position, whereas only Thermoactinomyces vulgaris R-47 Glucoamylase (TGA) and two putative Glucoamylases have Trp. We focused on Trp622 in TGA and examined the pH optima of five mutants, W622C, W622D, W622G, W622H and W622S. The pH optima of these mutants were 6.2-6.8, which was identical to or slightly lower than that of the wild-type enzyme. However, the activities of these mutants at pH optima decreased to 4.3-52% of that of wild-type enzyme. From these results and information on the crystal structures of Glucoamylases, Trp622 in TGA is suggested to be an important residue for substrate binding rather than for determination of optimal pH.
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site directed mutagenesis of tryptophan 622 of thermoactinomyces vulgaris r 47 Glucoamylase
Journal of applied glycoscience, 2005Co-Authors: Kazuhiro Ichikawa, Takashi Tonozuka, Atsushi Nishikawa, Masahiro Mizuno, Yoshiyuki SakanoAbstract:In Aspergillus awamori Glucoamylase, the optimal pH has been reported to increase to maintain activity by a mutation of Ser411 which forms a hydrogen-bond with a catalytic base (Fang and Ford, Protein Eng., 11, 383-388 (1998)). Most Glucoamylases have either Ser or Gly at this position, whereas only Thermoactinomyces vulgaris R-47 Glucoamylase (TGA) and two putative Glucoamylases have Trp. We focused on Trp622 in TGA and examined the pH optima of five mutants, W622C, W622D, W622G, W622H and W622S. The pH optima of these mutants were 6.2-6.8, which was identical to or slightly lower than that of the wild-type enzyme. However, the activities of these mutants at pH optima decreased to 4.3-52% of that of wild-type enzyme. From these results and information on the crystal structures of Glucoamylases, Trp622 in TGA is suggested to be an important residue for substrate binding rather than for determination of optimal pH.
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crystallization and preliminary x ray analysis of thermoactinomyces vulgaris r 47 maltooligosaccharide metabolizing enzyme homologous to Glucoamylase
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: Kazuhiro Ichikawa, Takashi Tonozuka, Shigehiro Kamitori, Atsushi Nishikawa, Masahiro Mizuno, Yoshihiro Tanabe, Yoshiyuki SakanoAbstract:A maltooligosaccharide-metabolizing enzyme from Thermoactinomyces vulgaris R-47 (TGA) homologous to Glucoamylase degrades maltooligosaccharides more efficiently than starch, unlike fungal Glucoamylases. TGA was crystallized and the state of the protein in solution was analyzed by gel-filtration chromatography. Diffraction data were collected to 3.31 A resolution. The TGA crystal belongs to the orthorhombic space group P212121 or P21212, with unit-cell parameters a = 110.2, b = 317.6, c = 144.9 A, and is expected to contain five to eight TGA molecules per asymmetric unit. Gel-filtration and native PAGE analyses indicated that TGA exists as a dimer in solution. This is the first report of the crystallization of an oligomeric Glucoamylase.
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crystallization and preliminary x ray analysis of thermoactinomyces vulgaris r 47 maltooligosaccharide metabolizing enzyme homologous to Glucoamylase
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: Kazuhiro Ichikawa, Takashi Tonozuka, Shigehiro Kamitori, Atsushi Nishikawa, Masahiro Mizuno, Yoshihiro Tanabe, Yoshiyuki SakanoAbstract:A maltooligosaccharide-metabolizing enzyme from Thermoactinomyces vulgaris R-47 (TGA) homologous to Glucoamylase degrades maltooligosaccharides more efficiently than starch, unlike fungal Glucoamylases. TGA was crystallized and the state of the protein in solution was analyzed by gel-filtration chromatography. Diffraction data were collected to 3.31 A resolution. The TGA crystal belongs to the orthorhombic space group P212121 or P21212, with unit-cell parameters a = 110.2, b = 317.6, c = 144.9 A, and is expected to contain five to eight TGA molecules per asymmetric unit. Gel-filtration and native PAGE analyses indicated that TGA exists as a dimer in solution. This is the first report of the crystallization of an oligomeric Glucoamylase.
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bacterial and archaeal enzymes homologous to Glucoamylase characterization and subsite affinities of a Glucoamylase from thermoactinomyces vulgaris r 47
2005Co-Authors: Kazuhiro Ichikawa, Takashi Tonozuka, Atsushi Nishikawa, Rie Uotsutomita, Masahiro Mizuno, Yoshiyuki SakanoAbstract:We have studied several bacterial and archaeal glycoside hydrolase (GH) family 15 enzymes, and in this review, mainly focus on a Glucoamylase from thermophilic bacterium, Thermoactinomyces vulgaris R-47 (TGA). The primary structure of TGA resembled archaeal GH family 15 enzymes, but homologies with fungal Glucoamylases were low. Although TGA is an exo-hydrolase that releases β-D-glucose from the non-reducing ends of substrates, as do fungal Glucoamylases, this enzyme hydrolyzed maltooligosaccharides more efficiently than starch, unlike fungal Glucoamylases. Subsite affinities of TGA showed that the A1+A2 value was highly positive whereas A4—A6 values were negative and little affinity was detected at subsites 3 and 7, which is different from those of not only fungal Glucoamylases, but also a bacterial, Clostridium sp. G0005, Glucoamylase. Thus, TGA is a novel metabolizing enzyme specific for small oligosaccharides, and it is likely that the difference in substrate specificities of GH family 15 enzymes is associated with the difference in their physiological roles.