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Soojin Yeom - One of the best experts on this subject based on the ideXlab platform.
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production of l ribose from l ribulose by a triple site variant of mannose 6 phosphate Isomerase from geobacillus thermodenitrificans
Applied and Environmental Microbiology, 2012Co-Authors: Yuri Lim, Soojin YeomAbstract:A triple-site variant (W17Q N90A L129F) of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) for l-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/liter l-ribose from 300 g/liter l-ribulose for 60 min, with a volumetric productivity of 213 g liter−1 h−1, which was 4.5-fold higher than that of the wild-type enzyme. The kcat/Km and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of Mannose-6-Phosphate Isomerase, which exhibited the highest values previously reported.
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characterization of a mannose 6 phosphate Isomerase from thermus thermophilus and increased l ribose production by its r142n mutant
Applied and Environmental Microbiology, 2011Co-Authors: Soojin Yeom, Deokkun OhAbstract:Optically pure carbohydrates are important precursors for pharmaceutical, food, and agrochemical products (22). Among carbohydrates, l-enantiomers have been widely used as antiviral nucleoside analogue drugs in the treatment of severe viral diseases due to their potent biological activities and lower toxicity than the corresponding d-nucleosides (3). l-Ribose, a pentose sugar, can be used as a precursor for the synthesis of antiviral drugs, such as l-nucleoside derivatives (2, 5, 14). l-Ribose can be synthesized by chemical methods from l-arabinose (1, 6, 9), l-xylose (13), d-glucose (15), d-galactose (19), d-ribose (26), or d-mannono-1,4-lactone (20). However, chemical synthesis has several disadvantages, including multiple steps, by-product formation, and chemical waste production. Recently, the enzymatic production of l-ribose has been investigated using l-arabinose (7) or l-ribulose (25). l-Ribose has been produced primarily from the cheap sugar l-arabinose because l-ribulose is an expensive sugar. An l-arabinose Isomerase mutant of Escherichia coli (4) and a d-xylose Isomerase mutant of Actinoplanes missouriensis (17) converted l-arabinose to l-ribose by a two-step isomerization reaction with low productivity. A recombinant E. coli strain containing l-arabinose Isomerase and l-ribose Isomerase (7) and purified l-arabinose Isomerase and Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans (24) were used to produce l-ribose from l-arabinose via l-ribulose with high productivity. However, a rate-limiting step in the two enzyme systems is the conversion of l-ribulose to l-ribose using l-ribose Isomerase (7, 12) or Mannose-6-Phosphate Isomerase (23-25). Thus, biotechnological production of l-ribose has been focused on these enzymes. Mannose-6-Phosphate Isomerase from G. thermodenitrificans exhibits the highest activity to date for l-ribose production. Greater efficiency can be attained only through the discovery or synthesis of l-ribose-producing enzymes with higher kcat/Km. Increases in the kcat/Km ratio can be realized by genetic improvements via directed evolution and by structural modification of the determinant residues at or near the active site, based on homology models or the determined structure of the enzymes. In this study, the activities of a recombinant Mannose-6-Phosphate Isomerase from Thermus thermophilus with different metal ions, pHs, and temperatures for l-ribulose isomerization and its substrate specificities for various aldoses and ketoses were characterized. Mutational analyses were performed with predicted active-site residues obtained from homology studies; the R142N mutant was selected as an effective l-ribose producer. The specific activity, kcat/Km, and conversion for l-ribulose using the R142N mutant were determined.
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characterization of a mannose 6 phosphate Isomerase from thermus thermophilus and increased l ribose production by its r142n mutant
Applied and Environmental Microbiology, 2011Co-Authors: Soojin Yeom, Eunsun Seo, Bina Kim, Yeongsu KimAbstract:ABSTRACT An uncharacterized gene from Thermus thermophilus, thought to encode a Mannose-6-Phosphate Isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for l-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of l-ribulose to l-ribose, a potential starting material for many l-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in l-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) for l-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The kcat/Km of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans Mannose-6-Phosphate Isomerase, which exhibited the highest activity to date for the previously reported kcat/Km. The R142N mutant enzyme produced 213 g/liter l-ribose from 300 g/liter l-ribulose for 2 h, with a volumetric productivity of 107 g liter−1 h−1, which was 1.5-fold higher than that of the wild-type enzyme.
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l ribose production from l arabinose by using purified l arabinose Isomerase and mannose 6 phosphate Isomerase from geobacillus thermodenitrificans
Applied and Environmental Microbiology, 2009Co-Authors: Soojin Yeom, Namhee Kim, Changsu ParkAbstract:Two enzymes, l-arabinose Isomerase and Mannose-6-Phosphate Isomerase, from Geobacillus thermodenitrificans produced 118 g/liter l-ribose from 500 g/liter l-arabinose at pH 7.0, 70°C, and 1 mM Co2+ for 3 h, with a conversion yield of 23.6% and a volumetric productivity of 39.3 g liter−1 h−1.
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substrate specificity of a mannose 6 phosphate Isomerase from bacillus subtilis and its application in the production of l ribose
Applied and Environmental Microbiology, 2009Co-Authors: Soojin Yeom, Namhee Kim, Changsu ParkAbstract:The uncharacterized gene previously proposed as a Mannose-6-Phosphate Isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co2+. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by Mannose-6-Phosphate Isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter−1 h−1.
Changsu Park - One of the best experts on this subject based on the ideXlab platform.
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l ribose production from l arabinose by using purified l arabinose Isomerase and mannose 6 phosphate Isomerase from geobacillus thermodenitrificans
Applied and Environmental Microbiology, 2009Co-Authors: Soojin Yeom, Namhee Kim, Changsu ParkAbstract:Two enzymes, l-arabinose Isomerase and Mannose-6-Phosphate Isomerase, from Geobacillus thermodenitrificans produced 118 g/liter l-ribose from 500 g/liter l-arabinose at pH 7.0, 70°C, and 1 mM Co2+ for 3 h, with a conversion yield of 23.6% and a volumetric productivity of 39.3 g liter−1 h−1.
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substrate specificity of a mannose 6 phosphate Isomerase from bacillus subtilis and its application in the production of l ribose
Applied and Environmental Microbiology, 2009Co-Authors: Soojin Yeom, Namhee Kim, Changsu ParkAbstract:The uncharacterized gene previously proposed as a Mannose-6-Phosphate Isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co2+. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by Mannose-6-Phosphate Isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter−1 h−1.
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characterization of a mannose 6 phosphate Isomerase from geobacillus thermodenitrificans that converts monosaccharides
Biotechnology Letters, 2009Co-Authors: Soojin Yeom, Namhee Kim, Ranyoung Yoon, Hyunjung Kwon, Changsu ParkAbstract:A recombinant Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the d- and l-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for d-lyxose isomerization was optimal at pH 7.0, 70°C and 1 mM Co2+. Under these conditions, the kcat and Km values were 74,300 s−1 and 390 mM for d-lyxose and 28,800 s−1 and 470 mM for l-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70°C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of d-lyxose to d-xylulose with a 38% conversion yield after 3 h, and converted l-ribose to l-ribulose with a 29% conversion yield.
Deokkun Oh - One of the best experts on this subject based on the ideXlab platform.
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characterization of a mannose 6 phosphate Isomerase from thermus thermophilus and increased l ribose production by its r142n mutant
Applied and Environmental Microbiology, 2011Co-Authors: Soojin Yeom, Deokkun OhAbstract:Optically pure carbohydrates are important precursors for pharmaceutical, food, and agrochemical products (22). Among carbohydrates, l-enantiomers have been widely used as antiviral nucleoside analogue drugs in the treatment of severe viral diseases due to their potent biological activities and lower toxicity than the corresponding d-nucleosides (3). l-Ribose, a pentose sugar, can be used as a precursor for the synthesis of antiviral drugs, such as l-nucleoside derivatives (2, 5, 14). l-Ribose can be synthesized by chemical methods from l-arabinose (1, 6, 9), l-xylose (13), d-glucose (15), d-galactose (19), d-ribose (26), or d-mannono-1,4-lactone (20). However, chemical synthesis has several disadvantages, including multiple steps, by-product formation, and chemical waste production. Recently, the enzymatic production of l-ribose has been investigated using l-arabinose (7) or l-ribulose (25). l-Ribose has been produced primarily from the cheap sugar l-arabinose because l-ribulose is an expensive sugar. An l-arabinose Isomerase mutant of Escherichia coli (4) and a d-xylose Isomerase mutant of Actinoplanes missouriensis (17) converted l-arabinose to l-ribose by a two-step isomerization reaction with low productivity. A recombinant E. coli strain containing l-arabinose Isomerase and l-ribose Isomerase (7) and purified l-arabinose Isomerase and Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans (24) were used to produce l-ribose from l-arabinose via l-ribulose with high productivity. However, a rate-limiting step in the two enzyme systems is the conversion of l-ribulose to l-ribose using l-ribose Isomerase (7, 12) or Mannose-6-Phosphate Isomerase (23-25). Thus, biotechnological production of l-ribose has been focused on these enzymes. Mannose-6-Phosphate Isomerase from G. thermodenitrificans exhibits the highest activity to date for l-ribose production. Greater efficiency can be attained only through the discovery or synthesis of l-ribose-producing enzymes with higher kcat/Km. Increases in the kcat/Km ratio can be realized by genetic improvements via directed evolution and by structural modification of the determinant residues at or near the active site, based on homology models or the determined structure of the enzymes. In this study, the activities of a recombinant Mannose-6-Phosphate Isomerase from Thermus thermophilus with different metal ions, pHs, and temperatures for l-ribulose isomerization and its substrate specificities for various aldoses and ketoses were characterized. Mutational analyses were performed with predicted active-site residues obtained from homology studies; the R142N mutant was selected as an effective l-ribose producer. The specific activity, kcat/Km, and conversion for l-ribulose using the R142N mutant were determined.
Yeongsu Kim - One of the best experts on this subject based on the ideXlab platform.
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characterization of a mannose 6 phosphate Isomerase from thermus thermophilus and increased l ribose production by its r142n mutant
Applied and Environmental Microbiology, 2011Co-Authors: Soojin Yeom, Eunsun Seo, Bina Kim, Yeongsu KimAbstract:ABSTRACT An uncharacterized gene from Thermus thermophilus, thought to encode a Mannose-6-Phosphate Isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for l-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of l-ribulose to l-ribose, a potential starting material for many l-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in l-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) for l-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The kcat/Km of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans Mannose-6-Phosphate Isomerase, which exhibited the highest activity to date for the previously reported kcat/Km. The R142N mutant enzyme produced 213 g/liter l-ribose from 300 g/liter l-ribulose for 2 h, with a volumetric productivity of 107 g liter−1 h−1, which was 1.5-fold higher than that of the wild-type enzyme.
Delin Duan - One of the best experts on this subject based on the ideXlab platform.
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transcriptome sequencing of saccharina japonica sporophytes during whole developmental periods reveals regulatory networks underlying alginate and mannitol biosynthesis
BMC Genomics, 2019Co-Authors: Zhanru Shao, Pengyan Zhang, Zhihang Chen, Xiuliang Wang, Delin DuanAbstract:Alginate is an important cell wall component and mannitol is a soluble storage carbon substance in the brown seaweed Saccharina japonica. Their contents vary with kelp developmental periods and harvesting time. Alginate and mannitol regulatory networks and molecular mechanisms are largely unknown. With WGCNA and trend analysis of 20,940 known genes and 4264 new genes produced from transcriptome sequencing of 30 kelp samples from different stages and tissues, we deduced that ribosomal proteins, light harvesting complex proteins and “imm upregulated 3” gene family are closely associated with the meristematic growth and kelp maturity. Moreover, 134 and 6 genes directly involved in the alginate and mannitol metabolism were identified, respectively. Mannose-6-Phosphate Isomerase (MPI2), phosphomannomutase (PMM1), GDP-mannose 6-dehydrogenase (GMD3) and mannuronate C5-epimerase (MC5E70 and MC5E122) are closely related with the high content of alginate in the distal blade. Mannitol accumulation in the basal blade might be ascribed to high expression of mannitol-1-phosphate dehydrogenase (M1PDH1) and mannitol-1-phosphatase (M1Pase) (in biosynthesis direction) and low expression of mannitol-2-dehydrogenase (M2DH) and Fructokinase (FK) (in degradation direction). Oxidative phosphorylation and photosynthesis provide ATP and NADH for mannitol metabolism whereas glycosylated cycle and tricarboxylic acid (TCA) cycle produce GTP for alginate biosynthesis. RNA/protein synthesis and transportation might affect alginate complex polymerization and secretion processes. Cryptochrome (CRY-DASH), xanthophyll cycle, photosynthesis and carbon fixation influence the production of intermediate metabolite of fructose-6-phosphate, contributing to high content of mannitol in the basal blade. The network of co-responsive DNA synthesis, repair and proteolysis are presumed to be involved in alginate polymerization and secretion, while upstream light-responsive reactions are important for mannitol accumulation in meristem of kelp. Our transcriptome analysis provides new insights into the transcriptional regulatory networks underlying the biosynthesis of alginate and mannitol during S. japonica developments.