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Tsueiyun Fang - One of the best experts on this subject based on the ideXlab platform.
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Production of trehalose from Maltooligosaccharide via MTSase and MTHase.
2013Co-Authors: Yu-ping Wang, Tsueiyun Fang, Thy-hou LinAbstract:In the first step, MTSase turns the α-1,4-linked terminal disaccharide of Maltooligosaccharide into the α-1,1-linked terminal disaccharide. In the second step, MTHase cleaves the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose and produces a trehalose.
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identification of the essential catalytic residues and selectivity related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Journal of Agricultural and Food Chemistry, 2008Co-Authors: Tsueiyun Fang, Tongyuan Shih, Wenchi Tseng, Meiying WangAbstract:Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of Maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased ...
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expression purification and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Journal of Agricultural and Food Chemistry, 2006Co-Authors: Tsueiyun Fang, Tongyuan Shih, Wenchi Tseng, Xingguang HungAbstract:The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the Maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 °C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45−85 °C. The kcat values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4−7 were 193, 1030, 1190, and 1230 s-1, respect...
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characterization of the trehalosyl dextrin forming enzyme from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Extremophiles, 2004Co-Authors: Tsueiyun Fang, Xingguang Hung, Tongyuan Shih, Wenchi TsengAbstract:The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the α-1,4-glucosidic linkage at the reducing end to an α-1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75°C. The enzyme was stable in a pH range of 4.5–11, and the activity remained unchanged after a 2-h incubation at 80°C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than Maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP Maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2–14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.
Wenchi Tseng - One of the best experts on this subject based on the ideXlab platform.
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identification of the essential catalytic residues and selectivity related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Journal of Agricultural and Food Chemistry, 2008Co-Authors: Tsueiyun Fang, Tongyuan Shih, Wenchi Tseng, Meiying WangAbstract:Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of Maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased ...
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expression purification and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Journal of Agricultural and Food Chemistry, 2006Co-Authors: Tsueiyun Fang, Tongyuan Shih, Wenchi Tseng, Xingguang HungAbstract:The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the Maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 °C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45−85 °C. The kcat values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4−7 were 193, 1030, 1190, and 1230 s-1, respect...
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characterization of the trehalosyl dextrin forming enzyme from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Extremophiles, 2004Co-Authors: Tsueiyun Fang, Xingguang Hung, Tongyuan Shih, Wenchi TsengAbstract:The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the α-1,4-glucosidic linkage at the reducing end to an α-1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75°C. The enzyme was stable in a pH range of 4.5–11, and the activity remained unchanged after a 2-h incubation at 80°C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than Maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP Maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2–14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.
Xingguang Hung - One of the best experts on this subject based on the ideXlab platform.
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expression purification and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Journal of Agricultural and Food Chemistry, 2006Co-Authors: Tsueiyun Fang, Tongyuan Shih, Wenchi Tseng, Xingguang HungAbstract:The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the Maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 °C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45−85 °C. The kcat values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4−7 were 193, 1030, 1190, and 1230 s-1, respect...
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characterization of the trehalosyl dextrin forming enzyme from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Extremophiles, 2004Co-Authors: Tsueiyun Fang, Xingguang Hung, Tongyuan Shih, Wenchi TsengAbstract:The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the α-1,4-glucosidic linkage at the reducing end to an α-1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75°C. The enzyme was stable in a pH range of 4.5–11, and the activity remained unchanged after a 2-h incubation at 80°C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than Maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP Maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2–14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.
Shigetaka Okada - One of the best experts on this subject based on the ideXlab platform.
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thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of Maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with Maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
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thermus aquaticus atcc 33923 amylomaltase gene cloning and expression and enzyme characterization production of cycloamylose
Applied and Environmental Microbiology, 1999Co-Authors: Yoshinobu Terada, Takeshi Takaha, Kazutoshi Fujii, Shigetaka OkadaAbstract:The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of Maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with Maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.
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disproportionating enzyme 4 alpha glucanotransferase ec 2 4 1 25 of potato purification molecular cloning and potential role in starch metabolism
Journal of Biological Chemistry, 1993Co-Authors: Takeshi Takaha, Michiyo Yanase, Shigetaka Okada, Steven M SmithAbstract:Disproportionating enzyme (D-enzyme, 4-alpha-glucanotransferase; EC 2.4.1.25) has been purified to homogeneity from potato tubers and its activity characterized. The enzyme catalyzes the transfer of Maltooligosaccharides from one 1,4-alpha-D-glucan molecule to another, or to glucose. Maltooligosaccharides are effective donor molecules, but short chain amylose and amylopectin may also function as donors. Enzyme activity is not affected by inorganic phosphate, 3-phosphoglycerate, or hexose phosphates. A cDNA clone encoding the enzyme was isolated using oligonucleotide probes derived from partial peptide sequences of the purified enzyme. The identity of the cDNA clone was confirmed by expression in Escherichia coli resulting in D-enzyme activity. The amino acid sequence deduced from the cDNA shows significant homology with a 4-alpha-glucanotransferase from Streptococcus. The deduced sequence indicates the presence of an amino-terminal plastid transit peptide of 52 amino acid residues and a mature polypeptide of 524 residues. D-enzyme mRNA is present in leaves, stems, roots, and stolons but is most abundant in developing and mature tubers. The amount of mRNA in leaves increases in response to light and to sucrose added to the medium. These results are discussed in terms of the function of D-enzyme in potato starch metabolism.
Jorge Quillaguaman - One of the best experts on this subject based on the ideXlab platform.
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poly beta hydroxybutyrate production by a moderate halophile halomonas boliviensis lc1 using starch hydrolysate as substrate
Journal of Applied Microbiology, 2005Co-Authors: Jorge Quillaguaman, Suhaila Hashim, F Bento, Bo Mattiasson, Rajni HattikaulAbstract:Aim: The objective of the present work was to enable the use of starch hydrolysate, generated by the action of a recombinant Maltooligosaccharide forming amylase from Bacillus halodurans LBK 34, as the carbon source for the production of poly-beta- hydroxybutyrate ( PHB) by Halomonas boliviensis LC1. Methods and Results: In this work, different amounts of the alpha- amylase ( Amy 34) were utilized for starch hydrolysis, resulting in the production of mixtures of Maltooligosaccharides ( G1 - G6) at varying ratios. The highest PHB accumulation ( 56 wt%) by H. boliviensis cultivated in shake flasks ( with agitation at 160 rev min(-1)) was obtained when 6.4 U ml(-1) of the amylase was used for starch hydrolysis. When H. boliviensis cells were grown in a fermentor with no oxygen limitation the accumulation of PHB was decreased to 35 wt%. Although some improvements in PHB accumulation and cell mass concentration were reached by the addition of peptone and phosphate, respectively, major enhancements were attained when oxygen limitation was induced in the fermentor. Conclusions: Halomonas boliviensis uses preferentially maltose for PHB formation from starch hydrolysate. It is also able to hydrolyse higher sugars if no other simpler carbon source is available but with a significantly lower polymer yield. Furthermore, H. boliviensis is able to adjust its metabolism to oxygen limitation, most probably by directing the excess NAD( P) H to PHB accumulation. Significance and Impact of the Study: There have been no reports related to PHB production amongst the members of the genus Halomonas. The use of a Maltooligosaccharide forming alpha- amylase, which is active at a temperature and pH close to that required for growth of H. boliviensis, and the versatility of this bacterium in the selection of the carbon source may provide an attractive alternative for the utilization of starch- derived raw materials. (Less)
