229 results on '"Dextrins metabolism"'
Search Results
2. Essential dextrin structure as donor substrate for 4-α-glucanotransferase in glycogen debranching enzyme.
- Author
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Uno R, Makino Y, and Matsubara H
- Subjects
- Substrate Specificity, Glycogen Debranching Enzyme System metabolism, Glycogen Debranching Enzyme System chemistry, Glycogen Debranching Enzyme System genetics, Dextrins metabolism, Dextrins chemistry
- Abstract
Glycogen debranching enzyme is a single polypeptide with distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. To allow phosphorylase to degrade the inner tiers of highly branched glycogen, 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch G-G-G-G-(G-G-G-G↔)G-G- (G: d-glucose, hyphens: α-1,4-linkages; double-headed arrow: α-1,6-linkage) into the G-G-G-G-(G↔)G-G- residue, which is then subjected to amylo-α-1,6-glucosidase to release the remaining G↔ residue. However, while the essential side-chain structure of the 4-α-glucanotransferase donor substrate has been determined to be the G-G-G-G↔ residue (Watanabe, Y., et al. (2008) J. Biochem.143, 435-440), its essential main-chain structure remains to be investigated. In this study, we probed the 4-α-glucanotransferase donor-binding region using novel fluorogenic dextrins Gm-(G4↔)G-Gn-F (F: 1-deoxy-1-[(2-pyridyl)amino]-d-glucitol) and maltohexaose (G6) as the donor and acceptor substrates, respectively. 4-α-Glucanotransferase exhibited maximum activity towards G4-(G4↔)G-F and G4-(G4↔)G-G-F, indicating that recognition of the G4-(G4↔)G-moiety was essential for full enzyme function. Notably, when the 4-α-glucanotransferase activity towards G4-(G4↔)G-G-F was taken as unity, those towards nonbranching dextrins were < 0.001. This indicated that the disproportionation activities towards maltooligosaccharides (Gm) are abnormal behaviours of 4-α-glucanotransferase. Notably, however, these activities have been traditionally measured to identify the 4-α-glucanotransferase mutations causing glycogen storage disease type III. This study provides a basis for more accurate identification., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)
- Published
- 2024
- Full Text
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3. Combined resistant dextrin and low-dose Mg oxide administration increases short-chain fatty acid and lactic acid production by gut microbiota.
- Author
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Sasaki H, Hayashi K, Imamura M, Hirota Y, Hosoki H, Nitta L, Furutani A, and Shibata S
- Subjects
- Animals, Mice, Humans, Dextrins pharmacology, Dextrins metabolism, Magnesium Oxide, Mice, Inbred ICR, Fatty Acids, Volatile metabolism, Dietary Fiber metabolism, Constipation, Gastrointestinal Microbiome
- Abstract
The consumption of resistant dextrin improves constipation, while its fermentation and degradation by the intestinal microbiota produce short-chain fatty acids (SCFA) and lactic acid, which have beneficial effects on host metabolism and immunity. Mg oxide (MgO) is an important mineral that is used to treat constipation. Therefore, resistant dextrin and MgO are often administered together to improve constipation. However, limited information is available regarding the effect of this combination on SCFA and lactic acid production. Crl:CD1(ICR) mice were fed a Mg-free diet with 5% resistant dextrin, followed by oral administration of MgO. We collected the cecum contents and measured SCFA and lactic acid levels. Additionally, the human subjects received resistant dextrin and Mg supplements as part of their habitual diet. The results of this study demonstrate that intestinal microbiota cannot promote SCFA and lactic acid production in the absence of Mg. In a mouse model, low doses of MgO promoted the production of SCFA and lactic acid, whereas high doses decreased their production. In humans, the combined consumption of resistant dextrin and Mg supplements increased the production of SCFA and lactic acid. The production of SCFA and lactic acid from dietary fiber may be augmented by the presence of MgO., Competing Interests: Declaration of competing interest We declare that there are no conflicts of interest related to this study., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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4. Slowly digestible property of highly branched α-limit dextrins produced by 4,6-α-glucanotransferase from Streptococcus thermophilus evaluated in vitro and in vivo.
- Author
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Ryu JJ, Li X, Lee ES, Li D, and Lee BH
- Subjects
- Amylose metabolism, Digestion, Glucose metabolism, Glycogen Debranching Enzyme System chemistry, Humans, Hydrolysis, Molecular Weight, Pancreatic alpha-Amylases metabolism, Starch chemistry, alpha-Glucosidases metabolism, Dextrins chemistry, Dextrins metabolism, Glycogen Debranching Enzyme System metabolism, Streptococcus thermophilus enzymology
- Abstract
Starch molecules are first degraded to slowly digestible α-limit dextrins (α-LDx) and rapidly hydrolyzable linear malto-oligosaccharides (LMOs) by salivary and pancreatic α-amylases. In this study, we designed a slowly digestible highly branched α-LDx with maximized α-1,6 linkages using 4,6-α-glucanotransferase (4,6-αGT), which creates a short length of α-1,4 side chains with increasing branching points. The results showed that a short length of external chains mainly composed of 1-8 glucosyl units was newly synthesized in different amylose contents of corn starches, and the α-1,6 linkage ratio of branched α-LDx after the chromatographical purification was significantly increased from 4.6% to 22.1%. Both in vitro and in vivo studies confirmed that enzymatically modified α-LDx had improved slowly digestible properties and extended glycemic responses. Therefore, 4,6-αGT treatment enhanced the slowly digestible properties of highly branched α-LDx and promises usefulness as a functional ingredient to attenuate postprandial glucose homeostasis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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5. Observation of Cellodextrin Accumulation Resulted from Non-Conventional Secretion of Intracellular β-Glucosidase by Engineered Saccharomyces cerevisiae Fermenting Cellobiose.
- Author
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Lee WH and Jin YS
- Subjects
- Biofuels, Cellulose metabolism, Ethanol metabolism, Fermentation, Glycosylation, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Metabolic Engineering, Qa-SNARE Proteins genetics, Qa-SNARE Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Secretory Pathway genetics, beta-Glucosidase genetics, Cellobiose metabolism, Cellulose analogs & derivatives, Dextrins metabolism, Saccharomyces cerevisiae metabolism, beta-Glucosidase metabolism
- Abstract
Although engineered Saccharomyces cerevisiae fermenting cellobiose is useful for the production of biofuels from cellulosic biomass, cellodextrin accumulation is one of the main problems reducing ethanol yield and productivity in cellobiose fermentation with S. cerevisiae expressing cellodextrin transporter (CDT) and intracellular β-glucosidase (GH1-1). In this study, we investigated the reason for the cellodextrin accumulation and how to alleviate its formation during cellobiose fermentation using engineered S. cerevisiae fermenting cellobiose. From the series of cellobiose fermentation using S. cerevisiae expressing only GH1-1 under several culture conditions, it was discovered that small amounts of GH1-1 were secreted and cellodextrin was generated through trans-glycosylation activity of the secreted GH1-1. As GH1-1 does not have a secretion signal peptide, non-conventional protein secretion might facilitate the secretion of GH1-1. In cellobiose fermentations with S. cerevisiae expressing only GH1-1, knockout of TLG2 gene involved in non-conventional protein secretion pathway significantly delayed cellodextrin formation by reducing the secretion of GH1-1 by more than 50%. However, in cellobiose fermentations with S. cerevisiae expressing both GH1-1 and CDT-1, TLG2 knockout did not show a significant effect on cellodextrin formation, although secretion of GH1-1 was reduced by more than 40%. These results suggest that the development of new intracellular β-glucosidase, not influenced by non-conventional protein secretion, is required for better cellobiose fermentation performances of engineered S. cerevisiae fermenting cellobiose.
- Published
- 2021
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6. Modulation of the fecal microbiome and metabolome by resistant dextrin ameliorates hepatic steatosis and mitochondrial abnormalities in mice.
- Author
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Zhang Z, Chen X, and Cui B
- Subjects
- Alanine Transaminase, Animals, Antioxidants metabolism, Aspartate Aminotransferases metabolism, Bile Acids and Salts metabolism, Clostridiales metabolism, Diet, High-Fat, Disease Models, Animal, Fatty Liver pathology, Firmicutes metabolism, Lipid Metabolism, Liver metabolism, Liver pathology, Male, Mice, Mice, Inbred C57BL, Mitochondria pathology, Non-alcoholic Fatty Liver Disease, Dextrins administration & dosage, Dextrins metabolism, Fatty Liver drug therapy, Feces microbiology, Gastrointestinal Microbiome drug effects, Metabolome, Microbiota drug effects, Mitochondria metabolism
- Abstract
Targeting the gut-liver axis by manipulating the intestinal microbiome is a promising therapy for nonalcoholic fatty liver disease (NAFLD). This study modulated the intestinal microbiota to explore whether resistant dextrin, as a potential prebiotic, could ameliorate high-fat diet (HFD)-induced hepatic steatosis in C57BL/6J mice. After two months of feeding, significant hepatic steatosis with mitochondrial dysfunction was observed in the HFD-fed mice. However, the concentrations of triglycerides and malondialdehyde in liver tissue and the levels of alanine aminotransferase and aspartate aminotransferase in the serum of mice fed an HFD plus resistant dextrin diet (HFID) were significantly decreased compared to the HFD-fed mice. Additionally, hepatic mitochondrial integrity and reactive oxygen species accumulation were improved in HFID-fed mice, ameliorating hepatic steatosis. The fecal microbiome of HFD-fed mice was enriched in Bifidobacterium, Lactobacillus, and Globicatella, while resistant dextrin increased the abundance of Parabacteroides, Blautia, and Dubosiella. Major changes in fecal metabolites were confirmed for HFID-fed mice, including those related to entero-hepatic circulation (i.e., bile acids), tryptophan metabolism (e.g., indole derivatives), and lipid metabolism (e.g., lipoic acid), as well as increased antioxidants including isorhapontigenin. Furthermore, resistant dextrin decreased inflammatory cytokine levels and intestinal permeability and ameliorated intestinal damage. Together, these findings augmented current knowledge on prebiotic treatment for NAFLD.
- Published
- 2021
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7. Effects of reaction condition on glycosidic linkage structure, physical-chemical properties and in vitro digestibility of pyrodextrins prepared from native waxy maize starch.
- Author
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Chen J, Xiao J, Wang Z, Cheng H, Zhang Y, Lin B, Qin L, and Bai Y
- Subjects
- Dextrins metabolism, Glucan 1,4-alpha-Glucosidase metabolism, Hydrolysis, Starch chemistry, Zea mays chemistry, alpha-Amylases metabolism, Dextrins chemistry
- Abstract
Glycosidic linkage structure, physical-chemical properties and in vitro digestibility of pyrodextrins prepared using different reaction conditions were characterized. Intensification of reaction condition promoted starch hydrolysis and transglucosidation until the solubility of pyrodextrin reached 100%. New branch points were formed including α-1,2, β-1,2, β-1,4, β-1,6 and α-1,6 linkages. Majority of the branch points was α-1,6 and β-1,6 linkages which in together accounted for more than 70% of the total branches. The degree of branching increased at intensified reaction conditions and plateaued at approximately 24%. Exhaustively hydrolyzing pyrodextrin by α-amylase and amyloglucosidase significantly decreased the degree of α-1,4 but not α-1,6 linkages. The retained α-1,4 and α-1,6 linkages were probably protected from enzyme hydrolysis by the non-starch linkages due to steric hindrance. The resistant starch content was positively correlated with the degree of branching of pyrodextrin. The decreased in vitro digestibility of pyrodextrin was attributed to the formation of new glycosidic linkages., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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8. A re-evaluation of diastatic Saccharomyces cerevisiae strains and their role in brewing.
- Author
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Krogerus K and Gibson B
- Subjects
- Beer microbiology, Dextrins metabolism, Saccharomyces cerevisiae classification, Starch metabolism, Amylases genetics, Amylases metabolism, Fermentation, Saccharomyces cerevisiae enzymology
- Abstract
Diastatic strains of Saccharomyces cerevisiae possess the unique ability to hydrolyze and ferment long-chain oligosaccharides like dextrin and starch. They have long been regarded as important spoilage microbes in beer, but recent studies have inspired a re-evaluation of the significance of the group. Rather than being merely wild-yeast contaminants, they are highly specialized, domesticated yeasts belonging to a major brewing yeast lineage. In fact, many diastatic strains have unknowingly been used as production strains for decades. These yeasts are used in the production of traditional beer styles, like saison, but also show potential for creation of new beers with novel chemical and physical properties. Herein, we review results of the most recent studies and provide a detailed account of the structure, regulation, and functional role of the glucoamylase-encoding STA1 gene in relation to brewing and other fermentation industries. The state of the art in detecting diastatic yeast in the brewery is also summarized. In summary, these latest results highlight that having diastatic S. cerevisiae in your brewery is not necessarily a bad thing. KEY POINTS: •Diastatic S. cerevisiae strains are important spoilage microbes in brewery fermentations. •These strains belong to the 'Beer 2' or 'Mosaic beer' brewing yeast lineage. •Diastatic strains have unknowingly been used as production strains in breweries. •The STA1-encoded glucoamylase enables efficient maltotriose use.
- Published
- 2020
- Full Text
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9. The Lipomyces starkeyi gene Ls120451 encodes a cellobiose transporter that enables cellobiose fermentation in Saccharomyces cerevisiae.
- Author
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de Ruijter JC, Igarashi K, and Penttilä M
- Subjects
- Biological Transport, Biomass, Cellulose analogs & derivatives, Cellulose metabolism, Dextrins metabolism, Ethanol metabolism, Lipomyces growth & development, Lipomyces metabolism, Membrane Transport Proteins metabolism, Penicillium genetics, Cellobiose metabolism, Fermentation, Lipomyces genetics, Membrane Transport Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Processed lignocellulosic biomass is a source of mixed sugars that can be used for microbial fermentation into fuels or higher value products, like chemicals. Previously, the yeast Saccharomyces cerevisiae was engineered to utilize its cellodextrins through the heterologous expression of sugar transporters together with an intracellular expressed β-glucosidase. In this study, we screened a selection of eight (putative) cellodextrin transporters from different yeast and fungal hosts in order to extend the catalogue of available cellobiose transporters for cellobiose fermentation in S. cerevisiae. We confirmed that several in silico predicted cellodextrin transporters from Aspergillus niger were capable of transporting cellobiose with low affinity. In addition, we found a novel cellobiose transporter from the yeast Lipomyces starkeyi, encoded by the gene Ls120451. This transporter allowed efficient growth on cellobiose, while it also grew on glucose and lactose, but not cellotriose nor cellotetraose. We characterized the transporter more in-depth together with the transporter CdtG from Penicillium oxalicum. CdtG showed to be slightly more efficient in cellobiose consumption than Ls120451 at concentrations below 1.0 g/L. Ls120451 was more efficient in cellobiose consumption at higher concentrations and strains expressing this transporter grew slightly slower, but produced up to 30% more ethanol than CdtG., (© FEMS 2020.)
- Published
- 2020
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10. Three-Enzyme Phosphorylase Cascade for Integrated Production of Short-Chain Cellodextrins.
- Author
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Zhong C and Nidetzky B
- Subjects
- Cellulomonas enzymology, Cellulose metabolism, Glucose metabolism, Glucosephosphates metabolism, Phosphates metabolism, Phosphorylases genetics, Sucrose metabolism, Cellulose analogs & derivatives, Dextrins metabolism, Phosphorylases metabolism
- Abstract
Cellodextrins are linear β-1,4-gluco-oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP-controlled, bottom-up synthesis from expedient substrates is desired for their bulk production. Here, a three-enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short-chain (soluble) cellodextrins (DP range 3-6). The cascade reaction involves iterative β-1,4-glucosylation of glucose from α-glucose 1-phosphate (αGlc1-P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1-P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L
-1 is obtained under near-complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)-1 . Through a simple two-step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production., (© 2019 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2020
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11. In-depth study of the changes in properties and molecular structure of cassava starch during resistant dextrin preparation.
- Author
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Trithavisup K, Krusong K, and Tananuwong K
- Subjects
- Calorimetry, Differential Scanning, Dextrins metabolism, Dietary Fiber analysis, Hydrolysis, Manihot chemistry, Molecular Weight, Solubility, Temperature, Thermodynamics, Dextrins chemistry, Manihot metabolism, Starch chemistry
- Abstract
Physical, chemical and thermal properties, as well as molecular structure of cassava-based resistant dextrins prepared under different dextrinization conditions (0.04-0.10% HCl, 100-120 °C, 60-180 min) were determined. Increasing acid concentration, temperature and heating time resulted in the products with darker color, higher solubility, reducing sugar content, total dietary fiber and proportion of high molecular weight fiber fraction. An endothermic peak at 45-70 °C, having enthalpy of 1.66-2.14 J/g, was found from the samples processed under mild conditions (0.04-0.08% HCl, 100 °C, 60 min). However, harsher dextrinization conditions eliminated this endotherm. Dextrinization led to 1000-fold decrease in weight-average molecular weight (M
w ) of the products, comparing to the native starch. Stronger processing conditions yielded the resistant dextrins with slightly higher Mw but composing of shorter branched chains. During dextrinization, hydrolysis was a predominant step, while transglucosidation and repolymerization played key roles in modifying molecular structure and properties, especially dietary fiber content, of resistant dextrins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2019
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12. Different gelatinization characteristics of small and large barley starch granules impact their enzymatic hydrolysis and sugar production during mashing.
- Author
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Langenaeken NA, De Schepper CF, De Schutter DP, and Courtin CM
- Subjects
- Calorimetry, Differential Scanning, Dextrins metabolism, Enzymes metabolism, Hydrolysis, Particle Size, Starch metabolism, Temperature, Viscosity, Hordeum metabolism, Starch chemistry, Sugars metabolism
- Abstract
This study investigates the impact of different gelatinization characteristics of small and large barley starch granules on their enzymatic hydrolysis and sugar production during mashing. Therefore, a barley malt suspension was consecutively incubated at 45, 62, 72 and 78 °C to monitor starch behavior and enzymatic starch hydrolysis and sugar production. The combination of microscopic and rapid visco analyses showed that small starch granules persisted longer in the system and were present non-gelatinized at temperatures higher than 62 °C. HPAEC-PAD analysis showed that 8% of the total amount of starch, predominantly small granules, gelatinized at temperatures between 62 °C and 78 °C. Due to their delayed gelatinization in this system, their enzymatic hydrolysis yielded relatively more dextrins compared to what was observed for large granules. It was concluded that small granules should be taken into account when optimizing enzymatic hydrolysis of barley starch, like in brewing, distilling or bio-ethanol production., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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13. A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains.
- Author
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Krogerus K, Magalhães F, Kuivanen J, and Gibson B
- Subjects
- Beer microbiology, CRISPR-Cas Systems, Dextrins metabolism, Fermentation, Reproducibility of Results, Reverse Genetics, Saccharomyces cerevisiae Proteins genetics, Sequence Deletion, Fungal Proteins genetics, Promoter Regions, Genetic, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Starch metabolism, Trisaccharides metabolism
- Abstract
Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here, we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162-bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent among the 'Beer 2'/'Mosaic Beer' brewing strains. These strains utilize maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of maltotriose through STA1 appears to be the dominant mechanism enabling maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewer's wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer and can be exploited for strain development where maltotriose use is desired.
- Published
- 2019
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14. High level production of flavonoid rhamnosides by metagenome-derived Glycosyltransferase C in Escherichia coli utilizing dextrins of starch as a single carbon source.
- Author
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Ruprecht C, Bönisch F, Ilmberger N, Heyer TV, Haupt ETK, Streit WR, and Rabausch U
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Dextrins metabolism, Escherichia coli genetics, Escherichia coli metabolism, Glycosyltransferases genetics, Glycosyltransferases metabolism, Hesperidin biosynthesis, Metagenome, Microorganisms, Genetically-Modified genetics, Microorganisms, Genetically-Modified metabolism
- Abstract
Flavonoids exert a wide variety of biological functions that are highly attractive for the pharmaceutical and healthcare industries. However, their application is often limited by low water solubility and poor bioavailability, which can generally be relieved through glycosylation. Glycosyltransferase C (GtfC), a metagenome-derived, bacterial glycosyltransferase, was used to produce novel and rare rhamnosides of various flavonoids, including chrysin, diosmetin, biochanin A, and hesperetin. Some of them are to our knowledge firstly described within this work. In our study we deployed a new metabolic engineering approach to increase the rhamnosylation rate in Escherichia coli whole cell biotransformations. The coupling of maltodextrin metabolism to glycosylation was developed in E. coli MG1655 with the model substrate hesperetin. The process proved to be highly dependent on the availability of maltodextrins. Maximal production was achieved by the deletion of the phosphoglucomutase (pgm) and UTP-glucose-1-phosphate uridyltransferase (galU) genes and simultaneous overexpression of the dTDP-rhamnose synthesis genes (rmlABCD) as well as glucan 1,4-alpha-maltohexaosidase for increased maltodextrin degradation next to GtfC in E. coli UHH_CR5-A. These modifications resulted in a 3.2-fold increase of hesperetin rhamnosides compared to E. coli MG1655 expressing GtfC in 24 h batch fermentations. Furthermore, E. coli UHH-CR_5-A was able to produce a final product titer of 2.4 g/L of hesperetin-3'-O-rhamnoside after 48 h. To show the versatility of the engineered E. coli strain, biotransformations of quercetin and kaempferol were performed, leading to production of 4.3 g/L quercitrin and 1.9 g/L afzelin in a 48 h time period, respectively. So far, these are the highest published yields of flavonoid rhamnosylation using a biotransformation approach. These results clearly demonstrate the high potential of the engineered E. coli production host as a platform for the high level biotransformation of flavonoid rhamnosides., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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15. Polymer Masked-Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin-Colistin Conjugates.
- Author
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Varache M, Powell LC, Aarstad OA, Williams TL, Wenzel MN, Thomas DW, and Ferguson EL
- Subjects
- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents metabolism, Biofilms drug effects, Cell Line, Cell Survival drug effects, Chromatography, Gel, Escherichia coli drug effects, Humans, Kidney Tubules, Proximal cytology, Mass Spectrometry, Microbial Sensitivity Tests, Microscopy, Confocal, Molecular Structure, Amylases metabolism, Colistin chemistry, Colistin metabolism, Dextrins chemistry, Dextrins metabolism, Drug Compounding methods, Drug Delivery Systems methods
- Abstract
Polymer masked-unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide's activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin-colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. UPLC-MS revealed that the fully "unmasked" dextrin-colistin conjugate composed of colistin bound to at least one linker, whereas larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully "unmasked" conjugate compared to the partially degraded species (MIC = 0.25 and 2-8 μg/mL, respectively), whereas dextrin conjugation reduced colistin's in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure-antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC-MS to aid the design of biodegradable polymer-antibiotic conjugates.
- Published
- 2019
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16. Enhanced depolymerization and utilization of raw lignocellulosic material by co-cultures of Ruminiclostridium thermocellum with hemicellulose-utilizing partners.
- Author
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Froese A, Schellenberg J, and Sparling R
- Subjects
- Biofuels, Cellulose analogs & derivatives, Cellulose metabolism, Clostridium thermocellum metabolism, Coculture Techniques, DNA, Bacterial genetics, Dextrins metabolism, Hydrolysis, Real-Time Polymerase Chain Reaction, Thermoanaerobacter metabolism, Clostridium thermocellum growth & development, Lignin metabolism, Polysaccharides metabolism, Polysaccharides, Bacterial metabolism, Thermoanaerobacter growth & development
- Abstract
Ruminiclostridium thermocellum is one of the most promising candidates for consolidated bioprocessing (CBP) of low-cost lignocellulosic materials to biofuels but it still shows poor performance in its ability to deconstruct untreated lignocellulosic substrates. One promising approach to increase R. thermocellum's rate of hydrolysis is to co-culture this cellulose-specialist with partners that possess synergistic hydrolysis enzymes and metabolic capabilities. We have created co-cultures of R. thermocellum with two hemicellulose utilizers, Ruminiclostridium stercorarium and Thermoanaerobacter thermohydrosulfuricus, both of which secrete xylanolytic enzymes and utilize the pentose oligo- and monosaccharides that inhibit R. thermocellum's hydrolysis and metabolism. When grown on milled wheat straw, the co-cultures were able to solubilize up to 58% more of the total polysaccharides than the R. thermocellum mono-culture control. Repeated passaging of the co-cultures on wheat straw yielded stable populations with reduced R. thermocellum cell numbers, indicating competition for cellodextrins released from cellulose hydrolysis, although these stabilized co-cultures were still able to outperform the mono-culture controls. Repeated passaging on Avicel cellulose also yielded stable populations. Overall, the observed synergism suggests that co-culturing R. thermocellum with other members is a viable option for increasing the rate and extent of untreated lignocellulose deconstruction by R. thermocellum for CBP purposes.
- Published
- 2019
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17. Characterization of the Transglycosylation Reaction of 4-α-Glucanotransferase (MalQ) and Its Role in Glycogen Breakdown in Escherichia coli.
- Author
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Nguyen DHD, Park SH, Tran PL, Kim JW, Le QT, Boos W, and Park JT
- Subjects
- Cyclodextrins metabolism, Dextrins antagonists & inhibitors, Dextrins metabolism, Escherichia coli genetics, Escherichia coli growth & development, Gene Expression Regulation, Bacterial, Glucans metabolism, Glucose metabolism, Glucosephosphates metabolism, Glucosyltransferases metabolism, Glycogen genetics, Glycogen Debranching Enzyme System genetics, Glycogen Phosphorylase metabolism, Glycosylation, Metabolic Networks and Pathways, Multigene Family, Escherichia coli enzymology, Escherichia coli metabolism, Glycogen metabolism, Glycogen Debranching Enzyme System metabolism
- Abstract
We first confirmed the involvement of MalQ (4-α-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ΔmalQ , showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-β-CD: G4- β-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.
- Published
- 2019
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18. Newly Identified Electrically Coupled Neurons Support Development of the Drosophila Giant Fiber Model Circuit.
- Author
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Kennedy T and Broadie K
- Subjects
- Animals, Animals, Genetically Modified, Dextrins metabolism, Drosophila, Drosophila Proteins genetics, Drosophila Proteins metabolism, Ganglia cytology, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Microscopy, Confocal, Rhodamines metabolism, Electrical Synapses physiology, Ganglia physiology, Nerve Net physiology, Nervous System cytology, Neurons physiology
- Abstract
The Drosophila giant fiber (GF) escape circuit is an extensively studied model for neuron connectivity and function. Researchers have long taken advantage of the simple linear neuronal pathway, which begins at peripheral sensory modalities, travels through the central GF interneuron (GFI) to motor neurons, and terminates on wing/leg muscles. This circuit is more complex than it seems, however, as there exists a complex web of coupled neurons connected to the GFI that widely innervates the thoracic ganglion. Here, we define four new neuron clusters dye coupled to the central GFI, which we name GF coupled (GFC) 1-4. We identify new transgenic Gal4 drivers that express specifically in these neurons, and map both neuronal architecture and synaptic polarity. GFC1-4 share a central site of GFI connectivity, the inframedial bridge, where the neurons each form electrical synapses. Targeted apoptotic ablation of GFC1 reveals a key role for the proper development of the GF circuit, including the maintenance of GFI connectivity with upstream and downstream synaptic partners. GFC1 ablation frequently results in the loss of one GFI, which is always compensated for by contralateral innervation from a branch of the persisting GFI axon. Overall, this work reveals extensively coupled interconnectivity within the GF circuit, and the requirement of coupled neurons for circuit development. Identification of this large population of electrically coupled neurons in this classic model, and the ability to genetically manipulate these electrically synapsed neurons, expands the GF system capabilities for the nuanced, sophisticated circuit dissection necessary for deeper investigations into brain formation.
- Published
- 2018
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19. Controlled release of dextrin-conjugated growth factors to support growth and differentiation of neural stem cells.
- Author
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Ferguson EL, Naseer S, Powell LC, Hardwicke J, Young FI, Zhu B, Liu Q, Song B, and Thomas DW
- Subjects
- Cell Differentiation, Cell Proliferation, Humans, Cell Culture Techniques methods, Dextrins metabolism, Fibroblast Growth Factor 2 metabolism, Neural Stem Cells metabolism
- Abstract
An essential aspect of stem cell in vitro culture and in vivo therapy is achieving sustained levels of growth factors to support stem cell survival and expansion, while maintaining their multipotency and differentiation potential. This study investigated the ability of dextrin (~74,000 g/mol; 27.8 mol% succinoylation) conjugated to epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF; or FGF-2) (3.9 and 6.7% w/w protein loading, respectively) to support the expansion and differentiation of stem cells in vitro via sustained, controllable growth factor release. Supplementation of mouse neural stem cells (mNSCs) with dextrin-growth factor conjugates led to greater and prolonged proliferation compared to unbound EGF/bFGF controls, with no detectable apoptosis after 7 days of treatment. Immunocytochemical detection of neural precursor (nestin) and differentiation (Olig2, MAP2, GFAP) markers verified that controlled release of dextrin-conjugated growth factors preserves stem cell properties of mNSCs for up to 7 days. These results show the potential of dextrin-growth factor conjugates for localized delivery of bioactive therapeutic agents to support stem cell expansion and differentiation, and as an adjunct to direct neuronal repair., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
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20. Evidence of Dextrin Hydrolyzing Enzymes in Cascade Hops ( Humulus lupulus).
- Author
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Kirkpatrick KR and Shellhammer TH
- Subjects
- Amylases chemistry, Amylases metabolism, Beer microbiology, Chromatography, High Pressure Liquid, Dextrins metabolism, Food Handling, Glucan 1,4-alpha-Glucosidase chemistry, Glucan 1,4-alpha-Glucosidase metabolism, Humulus chemistry, Humulus microbiology, Hydrolysis, Kinetics, Plant Proteins chemistry, Yeasts metabolism, Beer analysis, Humulus enzymology, Plant Proteins metabolism
- Abstract
Dry-hopping, the addition of hops to beer during or after fermentation, is a common practice in brewing to impart hoppy flavor to beer. Previously assumed to be inert ingredients, recent evidence suggests that hops contain biologically active compounds that may also extract into beer and complicate the brewing process by altering the final composition of beer. Experiments described herein provide evidence of microbial and/or plant-derived enzymes associated with hops ( Humulus lupulus) which can impact beer quality by influencing the composition of fermentable and nonfermentable carbohydrates in dry-hopped beer. Fully attenuated and packaged commercial lager beer was dry-hopped at a rate of 10 g hops/L beer with pelletized Cascade hops, dosed with 10
6 cells/mL of ale yeast, and incubated at 20 °C. Real extract of the treated beer declined significantly within several days with a reduction of 1 °P (% w/w) after 5 days and then slowly to a total reduction of approximately 2 °P after 40 days. When fully fermented, this was equivalent to the production of an additional 4.75% (v/v) of CO2 and an additional 1.3% (v/v) of alcohol. The refermentation of beer driven by dry-hopping was attributed to the low but persistent activities of several starch degrading enzymes present in Cascade hops including amyloglucosidase, α-amylase, β-amylase, and limit dextrinase. The effect of hop-derived enzymes on beer was time, temperature, and dose-dependent. Characterizing bioactive enzymes in hops will help hop suppliers and brewers to address the unexpected quality and safety issues surrounding hopping practices in beer.- Published
- 2018
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21. Enhanced cellobiose fermentation by engineered Saccharomyces cerevisiae expressing a mutant cellodextrin facilitator and cellobiose phosphorylase.
- Author
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Kim H, Oh EJ, Lane ST, Lee WH, Cate JHD, and Jin YS
- Subjects
- Cellulose analogs & derivatives, Cellulose metabolism, Dextrins metabolism, Fermentation, Glucosyltransferases metabolism, Membrane Transport Proteins metabolism, Metabolic Engineering, Recombinant Proteins, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Cellobiose chemistry, Glucosyltransferases genetics, Membrane Transport Proteins genetics, Saccharomyces cerevisiae growth & development
- Abstract
To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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22. A new interpretation of sulfate activation of rabbit muscle glycogen phosphorylase.
- Author
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Fujii Y, Makino Y, and Sato M
- Subjects
- Animals, Binding Sites, Dextrins metabolism, Enzyme Activation, Glycogen metabolism, Glycogen Phosphorylase, Muscle Form metabolism, Rabbits, Sulfates metabolism, Dextrins chemistry, Glycogen chemistry, Glycogen Phosphorylase, Muscle Form chemistry, Sulfates chemistry
- Abstract
It is widely known that sulfate ion at high concentration serves like an allosteric activator of glycogen phosphorylase (GP). Based on the crystallographic studies on GP, it has been assumed that the sulfate ion is bound close to the phosphorylatable Ser
14 site of nonactivated GP, causing a conformational change to catalytically-active GP. However, there are also reports that sulfate ion inhibits allosterically-activated GP by preventing the phosphate substrate from attaching to the catalytic site. In the present study, using a high concentration of sulfate ion, significant enhancement of GP activity was observed when macromolecular glycogen was used as substrate but not when smaller maltohexaose was used. In glycogen solution, nonreducing-end glucose residues are localized on the surface of glycogen and are not distributed homogenously in the solution. Using cyclodextrin-immobilized column chromatography, we found that sulfate at high concentration promoted GP-dextrin binding through the dextrin-binding site (DBS) located away from the catalytic site. This result is consistent with the properties of the DBSs found in glycogen-debranching enzyme and β-amylase. Therefore, we propose a new interpretation of the sulfate activation of GP, wherein sulfate ions at high concentration promote glycogen-binding to the DBS directly, and glycogen-binding to the catalytic site indirectly. Our findings were successfully applied to the affinity purification of porcine brain GP.- Published
- 2018
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23. Expression and characterization of the processive exo-β-1,4-cellobiohydrolase SCO6546 from Streptomyces coelicolor A(3).
- Author
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Lee CR, Chi WJ, Lim JH, Dhakshnamoorthy V, and Hong SK
- Subjects
- Cellulose analogs & derivatives, Cellulose metabolism, Cellulose 1,4-beta-Cellobiosidase isolation & purification, Chromatography, Thin Layer, Cloning, Molecular, Dextrins metabolism, Escherichia coli genetics, Gene Expression, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Molecular Weight, Streptomyces coelicolor genetics, Substrate Specificity, Tetroses metabolism, Cellulose 1,4-beta-Cellobiosidase genetics, Cellulose 1,4-beta-Cellobiosidase metabolism, Streptomyces coelicolor enzymology, Streptomyces lividans genetics
- Abstract
The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and β-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50 °C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters k
cat and KM , for cellotetraose at pH 5.0 and 50 °C were 13.3 s-1 and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-β-1,4-cellobiohydrolase (EC 3.2.1.91), acting on nonreducing end of cellulose., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2018
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24. Influence of dextrins on the production of spiramycin and impurity components by Streptomyces ambofaciens.
- Author
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Yao K, Gao S, Wu Y, Zhao Z, Wang W, and Mao Q
- Subjects
- Anti-Bacterial Agents analysis, Dextrins analysis, Drug Contamination, Spiramycin analysis, Anti-Bacterial Agents biosynthesis, Dextrins metabolism, Spiramycin biosynthesis, Streptomyces metabolism
- Abstract
Spiramycin is a 16-membered macrolide antibiotic produced by Streptomyces ambofaciens and used in human medicine for the treatment of various respiratory tract and genital infections. Several impurities were detected in spiramycin-fermentation broth, especially impurities D and F, which decreased the separation-extraction yield and increased production cost. Dextrins, as the main carbon source, influence the accumulation of spiramycin and impurities. In this work, two types of dextrin from vendor Y and Z were compared to study their influences on spiramycin production. Our results showed that final spiramycin production with dextrin Z was enhanced twofold as compared with dextrin Y; however, the content of impurities F and D were higher with dextrin Z relative to dextrin Y. Several parameters (adenosine triphosphate, total sugar, reducing sugar, and reducing sugar to total sugar) were analyzed to reveal differences in the fermentation process. In vitro dextrin hydrolysis by amylase revealed structural differences in the two types of dextrin, and real-time quantitative polymerase chain reaction analyses showed that the transcription of srm7 and srm21 (involved in forosaminyl methylation) was enhanced and potentially related to the reduced formation of impurity F with dextrin Y. Furthermore, the srm20/srm33 ratio, representing flux balance of forosaminyl and mycarosyl, was ~ 1, implying that forosaminyl and mycarosyl biosynthesis were well balanced, resulting in reduced production of impurity D with dextrin Y.
- Published
- 2018
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25. Recombinant Lactococcus lactis for efficient conversion of cellodextrins into L-lactic acid.
- Author
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Gandini C, Tarraran L, Kalemasi D, Pessione E, and Mazzoli R
- Subjects
- Cellulose genetics, Cellulose metabolism, Dextrins genetics, Lactic Acid isolation & purification, Cellulose analogs & derivatives, Dextrins metabolism, Genetic Enhancement methods, Lactic Acid biosynthesis, Lactococcus lactis genetics, Lactococcus lactis metabolism, Recombinant Proteins metabolism, Recombination, Genetic genetics
- Abstract
Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, that is, polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides. Here, we engineered a Lactococcus lactis strain which constitutively secretes a β-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step toward cost-sustainable consolidated bioprocessing of cellulose into optically pure LA., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2017
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26. Do model polymer therapeutics sufficiently diffuse through articular cartilage to be a viable therapeutic route?
- Author
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Powell A, Caterson B, Hughes C, Paul A, James C, Hopkins S, Mansour O, and Griffiths P
- Subjects
- Animals, Arthritis drug therapy, Arthritis metabolism, Biocompatible Materials therapeutic use, Cattle, Cells, Cultured, Dextrins metabolism, Dextrins therapeutic use, Diffusion Chambers, Culture, Polymers therapeutic use, Synovial Fluid metabolism, Biocompatible Materials metabolism, Cartilage, Articular metabolism, Polymers metabolism
- Abstract
The ability of a polymer therapeutic to access the appropriate subcellular location is crucial to its efficacy and is defined to a large part by the many and complex cellular biological and biochemical barriers such that a construct must traverse. It is shown here that model dextrin conjugates are able to pass through a cartilaginous extracellular matrix into chondrocytes, with little perturbation of the matrix structure, indicating that targeting of potential therapeutics through a cartilaginous extracellular matrix should be proven possible. Rapid chondrocytic targeting of drugs which require intra cellularisation for their activity and uniform extracellular concentrations of drugs with an extracellular target, is thus enabled though polymer conjugation.
- Published
- 2017
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27. HPMA copolymer-phospholipase C and dextrin-phospholipase A2 as model triggers for polymer enzyme liposome therapy (PELT).
- Author
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Ferguson EL, Scomparin A, Hailu H, and Satchi-Fainaro R
- Subjects
- Cell Survival drug effects, Cell Survival physiology, Dextrins administration & dosage, Dose-Response Relationship, Drug, Doxorubicin administration & dosage, Doxorubicin metabolism, Humans, Liposomes, MCF-7 Cells, Methacrylates administration & dosage, Phospholipases A2 administration & dosage, Polyethylene Glycols administration & dosage, Polyethylene Glycols metabolism, Polymers administration & dosage, Type C Phospholipases administration & dosage, Dextrins metabolism, Doxorubicin analogs & derivatives, Methacrylates metabolism, Phospholipases A2 metabolism, Polymers metabolism, Type C Phospholipases metabolism
- Abstract
'Polymer Enzyme Liposome Therapy' (PELT) is a two-step anticancer approach in which a liposomal drug and polymer-phospholipase conjugate are administered sequentially to target the tumour interstitium by the enhanced permeability and retention effect, and trigger rapid, local, drug release. To date, however, the concept has only been described theoretically. We synthesised two polymer conjugates of phospholipase C (PLC) and A2 (PLA2) and evaluated their ability to trigger anthracycline release from the clinically used liposomes, Caelyx
® and DaunoXome® . N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-PLC and a dextrin-PLA2 were synthesised and their enzymatic activity characterised. Doxorubicin release from polyethyleneglycol-coated (PEGylated) Caelyx® was relatively slow (<20%, 60 min), whereas daunomycin was rapidly released from non-PEGylated DaunoXome® (∼87%) by both enzymes. Incubation with dextrin-PLA2 triggered significantly less daunomycin release than HPMA copolymer-PLC, but when dextrin-PLA2 was pre-incubated with α-amylase, the rate of daunomycin release increased. DaunoXome®' s diameter increased in the presence of PLA2, while Caelyx® 's diameter was unaffected by free or conjugated PLA2. Dextrin-PLA2 potentiated the cytotoxicity of DaunoXome® to MCF-7 cells to a greater extent than free PLA2, while combining dextrin-PLA2 with Caelyx® resulted in antagonism, even in the presence of α-amylase, presumably due to steric hindrance by PEG. Our findings suggest that in vivo studies to evaluate PELT combinations should be further evaluated.- Published
- 2017
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28. Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae.
- Author
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Zhang C, Acosta-Sampson L, Yu VY, and Cate JHD
- Subjects
- Cellobiose metabolism, Cellulose metabolism, Neurospora crassa metabolism, Saccharomyces cerevisiae genetics, Dextrins metabolism, Saccharomyces cerevisiae metabolism
- Abstract
The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.
- Published
- 2017
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29. Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity.
- Author
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Wu Y, Mao G, Fan H, Song A, Zhang YP, and Chen H
- Subjects
- Bacteria genetics, Cellulose metabolism, Cloning, Molecular, Gene Expression, Glucose metabolism, Glucosyltransferases genetics, Hydrogen-Ion Concentration, Kinetics, Oligosaccharides metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Temperature, Tetroses metabolism, Bacteria enzymology, Cellobiose metabolism, Cellulose analogs & derivatives, Dextrins metabolism, Glucosyltransferases metabolism
- Abstract
A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k
cat /Km ) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.- Published
- 2017
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30. Systems analysis in Cellvibrio japonicus resolves predicted redundancy of β-glucosidases and determines essential physiological functions.
- Author
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Nelson CE, Rogowski A, Morland C, Wilhide JA, Gilbert HJ, and Gardner JG
- Subjects
- Cellulases metabolism, Cellulose analogs & derivatives, Cellulose metabolism, Dextrins metabolism, Disaccharides metabolism, Enzymes, Escherichia coli genetics, Kinetics, Polysaccharides metabolism, Systems Analysis, Carbohydrate Metabolism physiology, Cellulases physiology, Cellvibrio physiology
- Abstract
Degradation of polysaccharides forms an essential arc in the carbon cycle, provides a percentage of our daily caloric intake, and is a major driver in the renewable chemical industry. Microorganisms proficient at degrading insoluble polysaccharides possess large numbers of carbohydrate active enzymes (CAZymes), many of which have been categorized as functionally redundant. Here we present data that suggests that CAZymes that have overlapping enzymatic activities can have unique, non-overlapping biological functions in the cell. Our comprehensive study to understand cellodextrin utilization in the soil saprophyte Cellvibrio japonicus found that only one of four predicted β-glucosidases is required in a physiological context. Gene deletion analysis indicated that only the cel3B gene product is essential for efficient cellodextrin utilization in C. japonicus and is constitutively expressed at high levels. Interestingly, expression of individual β-glucosidases in Escherichia coli K-12 enabled this non-cellulolytic bacterium to be fully capable of using cellobiose as a sole carbon source. Furthermore, enzyme kinetic studies indicated that the Cel3A enzyme is significantly more active than the Cel3B enzyme on the oligosaccharides but not disaccharides. Our approach for parsing related CAZymes to determine actual physiological roles in the cell can be applied to other polysaccharide-degradation systems., (© 2017 John Wiley & Sons Ltd.)
- Published
- 2017
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31. Disruption of non-anchored cell wall protein NCW-1 promotes cellulase production by increasing cellobiose uptake in Neurospora crassa.
- Author
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Lin L, Chen Y, Li J, Wang S, Sun W, and Tian C
- Subjects
- Cell Wall metabolism, Cellulase genetics, Cellulose analogs & derivatives, Cellulose metabolism, Dextrins metabolism, Fungal Proteins genetics, Gene Expression Regulation, Fungal, Membrane Transport Proteins genetics, Neurospora crassa genetics, beta-Glucosidase metabolism, Cellobiose metabolism, Cellulase metabolism, Fungal Proteins metabolism, Membrane Transport Proteins metabolism, Neurospora crassa enzymology, Signal Transduction
- Abstract
Objectives: To elucidate the mechanism of cellulase signal transduction in filamentous fungi including the components of the cellulase induction pathway., Results: Neurospora crassa ncw-1 encodes a non-anchored cell wall protein. The absence of ncw-1 increased cellulase gene expression and this is not due to relieving carbon catabolite repression mediated by the cre-1 pathway. A mutant lacking genes encoding both three major β-glucosidase enzymes and NCW-1 (Δ3βGΔncw-1) was constructed. Transcriptome analysis of the quadruple mutant demonstrated enhanced expression of cellodextrin transporters after ncw-1 deletion, indicating that ncw-1 affects cellulase expression and production by inhibiting the uptake of the cellodextrin., Conclusions: NCW-1 is a novel component that plays a critical role in the cellulase induction signaling pathway.
- Published
- 2017
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32. Digestion-Resistant Dextrin Derivatives Are Moderately Digested in the Small Intestine and Contribute More to Energy Production Than Predicted from Large-Bowel Fermentation in Rats.
- Author
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Kondo T, Handa K, Genda T, Hino S, Hamaguchi N, and Morita T
- Subjects
- Animal Feed analysis, Animals, Dextrins metabolism, Diet veterinary, Energy Metabolism, Feces chemistry, Fermentation, Male, Rats, Dextrins chemistry, Dietary Fiber metabolism, Digestion physiology, Intestine, Large physiology, Intestine, Small physiology
- Abstract
Background: Digestion-resistant dextrin derivatives (DRDDs), including resistant maltodextrin (RM), polydextrose, and resistant glucan (RG), have been developed as low-energy foods. However, data on the resistance of DRDDs to small-intestinal digestion are scarce. Objective: We sought to determine the site and extent of DRDD breakdown in the rat intestine and to predict its energy contributions. Methods: In vitro small-intestinal resistance of DRDDs was evaluated by the AOAC method for dietary fiber measurement and by artificial digestion with the use of pancreatic α-amylase and brush-boarder membrane vesicles. In vivo small-intestinal resistance of DRDDs was determined from the feces of male ileorectostomized Sprague-Dawley rats fed a control diet or a diet containing one of the DRDDs at 50 g/kg for 9 d (period 1) and then for 10 d (period 2), during which they received 1 g neomycin/L in their drinking water. Separately, male Sprague-Dawley rats were fed the same diets for 4 wk, and the whole-gut recoveries of DRDDs were determined from feces at days 8-10. Results: Small-intestinal resistances determined in vitro by artificial digestion (RM: 70%; polydextrose: 67%; RG: 69%) were lower than those measured by the AOAC method (RM: 92%; polydextrose: 80%; RG: 82%). In the ileorectostomized rats, fecal dry-matter excretions were consistently greater in the DRDDs than in the control. The small-intestinal resistances of the DRDDs were 68%, 58%, and 62% in period 1 and 66%, 61%, and 67% during period 2 for RM, polydextrose, and RG, respectively. The resistances did not differ among the DRDDs at either time. In the normal rats, food intakes and body weight gains did not differ among the groups. The whole-gut recovery of RM (13%) was lower than that of polydextrose (33%) and RG (29%), which did not differ. Conclusions: DRDDs were more digestible in the rat small intestine than the AOAC method. The energy contribution from small-intestine digestibility, not just large-bowel fermentability, must be considered in determining the energy contribution of DRDDs. Whether humans respond similarly needs to be tested., (© 2017 American Society for Nutrition.)
- Published
- 2017
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33. Expression and characterization of a glucose-tolerant β-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii.
- Author
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Zhang C, Wang X, Zhang W, Zhao Y, and Lu X
- Subjects
- Cellobiose biosynthesis, Cellulose analogs & derivatives, Cytophaga metabolism, Dextrins metabolism, Escherichia coli genetics, Glucose metabolism, Protein Engineering, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Tetroses metabolism, Cellulose metabolism, Cytophaga enzymology, Escherichia coli metabolism, beta-Glucosidase genetics, beta-Glucosidase metabolism
- Abstract
Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and β-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a β-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most β-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K
m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10-2 , 5.6 × 10-2 , and 5.3 × 10-2 mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and β-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.- Published
- 2017
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34. Glycan Phosphorylases in Multi-Enzyme Synthetic Processes.
- Author
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Pergolizzi G, Kuhaudomlarp S, Kalita E, and Field RA
- Subjects
- Biofuels supply & distribution, Carbohydrate Conformation, Cellulose chemistry, Cellulose metabolism, Dextrins metabolism, Disaccharides metabolism, Escherichia coli enzymology, Escherichia coli genetics, Glucans metabolism, Glycosylation, Kinetics, Phosphorylases metabolism, Plants enzymology, Plants genetics, Starch chemistry, Starch metabolism, Substrate Specificity, Cellulose analogs & derivatives, Chemistry Techniques, Synthetic, Dextrins chemistry, Disaccharides chemistry, Glucans chemistry, Phosphorylases chemistry
- Abstract
Glycoside phosphorylases catalyse the reversible synthesis of glycosidic bonds by glycosylation with concomitant release of inorganic phosphate. The equilibrium position of such reactions can render them of limited synthetic utility, unless coupled with a secondary enzymatic step where the reaction lies heavily in favour of product. This article surveys recent works on the combined use of glycan phosphorylases with other enzymes to achieve synthetically useful processes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)
- Published
- 2017
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- View/download PDF
35. Sensitive, nonradioactive assay of phosphorylase kinase through measurement of enhanced phosphorylase activity towards fluorogenic dextrin.
- Author
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Miyagawa D, Makino Y, and Sato M
- Subjects
- Animals, Chromatography, High Pressure Liquid, Oligosaccharides metabolism, Phosphorylation, Rabbits, Sensitivity and Specificity, Spectrometry, Fluorescence, Dextrins metabolism, Enzyme Assays methods, Glycogen metabolism, Glycogen Phosphorylase, Muscle Form metabolism, Phosphorylase Kinase metabolism
- Abstract
Glycogen phosphorylase (GP) exists in two interconvertible forms, GPa (phosphorylated form, high activity) and GPb (nonphosphorylated form, low activity). Phosphorylase kinase (PhK) catalyses the phosphorylation of GPb and plays a key role in the cascade system for regulating glycogen metabolism. In this study, we developed a highly sensitive and nonradioactive assay for PhK activity by measuring the enhanced GP activity towards a pyridylaminated maltohexaose. The enhanced GP activity (ΔA) was calculated by the following formula: ΔA = A(+) - A(0), where A(+) and A(0) represent the GP activities of the PhK-treated and PhK-nontreated samples, respectively. Using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, the product of GP activity could be isolated and quantified at 10 fmol. This method does not require the use of any radioactive compounds and only 1 µg of GPb per sample was needed to obtain A(+) and A(0) values. The remarkable reduction in GPb concentration enabled us to discuss an interesting new role for glycogen in PhK activity., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)
- Published
- 2016
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36. Isomaltodextrin, a highly branched α-glucan, increases rat colonic H₂ production as well as indigestible dextrin.
- Author
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Nishimura N, Tanabe H, and Yamamoto T
- Subjects
- Animals, Colon metabolism, Hydrogen, Male, Rats, Rats, Sprague-Dawley, Colon drug effects, Dextrins metabolism, Polysaccharides pharmacology
- Abstract
Colonic hydrogen (H2) protects against inflammation-induced oxidative stress. We examined the effect of a new highly branched α-glucan, isomaltodextrin (IMD), on colonic H2 production in rats. Rats were fed a 16.7% IMD, 8.8% indigestible dextrin (ID), or 10.4% high amylose cornstarch diet (Expt. 1), were fed diets containing 3.3-16.7% IMD (Expt. 2), or were fed diets containing 16.7% IMD or 5.2% fructooligosaccharide (FOS) (Expt. 3), for 14 days. Compared with the control group, feeding IMD or other α-glucans dose dependently and significantly increased H2 excretion and portal H2 concentration. The ability of IMD to increase H2 production was not inferior to that of FOS. The cecal Firmicutes/Bacteroidetes ratio in the IMD group was 5-14% of that in the control group. The cecal abundance of bifidobacteria was significantly greater in the IMD group than in the control group. Taken together, IMD, as well as other α-glucans, significantly increased colonic H2 production in a dose-dependent manner.
- Published
- 2016
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37. Antigenotoxic activity of lactic acid bacteria, prebiotics, and products of their fermentation against selected mutagens.
- Author
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Nowak A, Śliżewska K, and Otlewska A
- Subjects
- Caco-2 Cells, Chromatography, High Pressure Liquid, Comet Assay, DNA Repair, Dextrins metabolism, Fatty Acids metabolism, Feces chemistry, Feces microbiology, Female, Humans, Intestinal Mucosa metabolism, Intestines microbiology, Inulin metabolism, Young Adult, Bifidobacterium metabolism, DNA Damage, Fermentation, Hydrogen Peroxide toxicity, Intestines drug effects, Lactic Acid metabolism, Lactobacillus metabolism, Mutagens toxicity, Prebiotics
- Abstract
Dietary components such as lactic acid bacteria (LAB) and prebiotics can modulate the intestinal microbiota and are thought to be involved in the reduction of colorectal cancer risk. The presented study measured, using the comet assay, the antigenotoxic activity of both probiotic and non-probiotic LAB, as well as some prebiotics and the end-products of their fermentation, against fecal water (FW). The production of short chain fatty acids by the bacteria was quantified using HPLC. Seven out of the ten tested viable strains significantly decreased DNA damage induced by FW. The most effective of them were Lactobacillus mucosae 0988 and Bifidobacterium animalis ssp. lactis Bb-12, leading to a 76% and 80% decrease in genotoxicity, respectively. The end-products of fermentation of seven prebiotics by Lactobacillus casei DN 114-001 exhibited the strongest antigenotoxic activity against FW, with fermented inulin reducing genotoxicity by 75%. Among the tested bacteria, this strain produced the highest amounts of butyrate in the process of prebiotic fermentation, and especially from resistant dextrin (4.09 μM/mL). Fermented resistant dextrin improved DNA repair by 78% in cells pre-treated with 6.8 μM methylnitronitrosoguanidine (MNNG). Fermented inulin induced stronger DNA repair in cells pre-treated with mutagens (FW, 25 μM hydrogen peroxide, or MNNG) than non-fermented inulin, and the efficiency of DNA repair after 120 min of incubation decreased by 71%, 50% and 70%, respectively. The different degrees of genotoxicity inhibition observed for the various combinations of bacteria and prebiotics suggest that this effect may be attributable to carbohydrate type, SCFA yield, and the ratio of the end-products of prebiotic fermentation., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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38. Development and physiological characterization of cellobiose-consuming Yarrowia lipolytica.
- Author
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Lane S, Zhang S, Wei N, Rao C, and Jin YS
- Subjects
- Biofuels microbiology, Cellulose analogs & derivatives, Cellulose metabolism, Citric Acid metabolism, Dextrins metabolism, Fermentation, Gene Expression, Lignin metabolism, Neurospora crassa enzymology, Neurospora crassa genetics, Yarrowia enzymology, Yarrowia genetics, beta-Glucosidase metabolism, Cellobiose metabolism, Metabolic Engineering methods, Yarrowia metabolism
- Abstract
Yarrowia lipolytica is a promising production host for a wide range of molecules, but limited sugar consumption abilities prevent utilization of an abundant source of renewable feedstocks. In this study we created a Y. lipolytica strain capable of utilizing cellobiose as a sole carbon source by using endogenous promoters to express the cellodextrin transporter cdt-1 and intracellular β-glucosidase gh1-1 from Neurospora crassa. The engineered strain was also capable of simultaneous co-consumption of glucose and cellobiose. Although cellobiose was consumed slower than glucose when engineered strains were cultured with excess nitrogen, culturing with limited nitrogen led to cellobiose consumption rates comparable to those of glucose. Under limited nitrogen conditions, the engineered strain produced citric acid as a major product and we observed greater citric acid yields from cellobiose (0.37 g/g) than glucose (0.28 g/g). Culturing with a sole carbon source of either glucose or cellobiose induced additional differences on cell physiology and metabolism and a link is suggested to evasion of glucose-sensing mechanisms through intracellular creation and consumption of glucose. We ultimately applied this cellobiose-utilization system to produce citric acid from bioconversion of crystalline cellulose through simultaneous saccharification and fermentation (SSF)., (© 2014 Wiley Periodicals, Inc.)
- Published
- 2015
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39. Genetic diversity and QTL mapping of thermostability of limit dextrinase in barley.
- Author
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Wang X, Zhang X, Cai S, Ye L, Zhou M, Chen Z, Zhang G, and Dai F
- Subjects
- Chromosome Mapping, Dextrins metabolism, Enzyme Stability, Glycoside Hydrolases metabolism, Hordeum classification, Hordeum genetics, Microsatellite Repeats, Plant Proteins metabolism, Genetic Variation, Glycoside Hydrolases chemistry, Glycoside Hydrolases genetics, Hordeum enzymology, Plant Proteins chemistry, Plant Proteins genetics, Quantitative Trait Loci
- Abstract
Limit dextrinase (LD) is an essential amylolytic enzyme for the complete degradation of starch, and it is closely associated with malt quality. A survey of 51 cultivated barley and 40 Tibetan wild barley genotypes showed a wide genetic diversity of LD activity and LD thermostability. Compared with cultivated barley, Tibetan wild barley showed lower LD activity and higher LD thermostability. A doubled haploid population composed of 496 DArT and 28 microsatellite markers was used for mapping Quantitative Trait Loci (QTLs). Parental line Yerong showed low LD activity and high LD thermostability, but Franklin exhibited high LD activity and low LD thermostability. Three QTLs associated with thermostable LD were identified. The major QTL is close to the LD gene on chromosome 7H. The two minor QTLs colocalized with previously reported QTLs determining malt-extract and diastatic power on chromosomes 1H and 2H, respectively. These QTLs may be useful for a better understanding of the genetic control of LD activity and LD thermostability in barley.
- Published
- 2015
- Full Text
- View/download PDF
40. The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.
- Author
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Cai P, Wang B, Ji J, Jiang Y, Wan L, Tian C, and Ma Y
- Subjects
- Biofuels, Cellobiose metabolism, Cellulase genetics, Cellulose analogs & derivatives, Cellulose chemistry, Cellulose metabolism, Dextrins chemistry, Dextrins metabolism, Energy Metabolism, Fungal Proteins chemistry, Gene Expression Regulation, Fungal, Neurospora crassa chemistry, Cellulase metabolism, Fungal Proteins biosynthesis, Fungal Proteins genetics, Gene Expression Profiling, Membrane Transport Proteins genetics, Neurospora crassa metabolism
- Abstract
Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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41. Novel pH-responsive dextrin nanogels for doxorubicin delivery to cancer cells with reduced cytotoxicity to cardiomyocytes and stem cells.
- Author
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Manchun S, Cheewatanakornkool K, Dass CR, and Sriamornsak P
- Subjects
- Animals, Cell Line, Tumor, Cell Survival drug effects, Cell Survival physiology, Dextrins administration & dosage, Doxorubicin administration & dosage, Hydrogen-Ion Concentration, Mice, Myocytes, Cardiac drug effects, Nanogels, Polyethylene Glycols administration & dosage, Polyethyleneimine administration & dosage, Rats, Stem Cells drug effects, Dextrins metabolism, Doxorubicin metabolism, Drug Delivery Systems methods, Myocytes, Cardiac metabolism, Polyethylene Glycols metabolism, Polyethyleneimine metabolism, Stem Cells metabolism
- Abstract
The aim of this study was to develop pH-responsive dextrin nanogels (DNGs) capable of triggered intracellular DOX release at the lower pH of cancer cells. DNGs were prepared by an emulsion cross-linking method using glyoxal as cross-linker to create an acid-labile bond. A higher molecular weight of dextrin with increasing mole ratio of dextrin to glyoxal decreased the average diameter of DNGs. DNGs showed slightly negative surface charge and pH-responsive behavior. The in vitro drug release was slow at pH 7.4 and increased with decreasing pH (pH 5>6.8). The cytotoxicity of DOX-loaded DNGs in mesenchymal stem cells and cardiomyocytes was lower than that of free DOX. Moreover, DOX-loaded DNGs were efficiently internalized by tumor cells with rapid release of DOX into the nucleus. Thus, DOX-loaded DNGs were successful for intracellular targeted anti-tumor drug delivery and reducing side-effects to non-tumor cells such as cardiomyocytes and stem cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
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42. Construction of dextrin and isomaltose-assimilating brewer's yeasts for production of low-carbohydrate beer.
- Author
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Park JY, Lee JY, Choi SH, Ko HM, Kim IC, Lee HB, and Bai S
- Subjects
- Chromatography, High Pressure Liquid, Glucan 1,4-alpha-Glucosidase genetics, Hydrolysis, Plasmids metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Time Factors, Transformation, Genetic, Beer microbiology, Dextrins metabolism, Isomaltose metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer's bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer's yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both α-1,4- and α-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.
- Published
- 2014
- Full Text
- View/download PDF
43. Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii.
- Author
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Zhang C, Wang Y, Li Z, Zhou X, Zhang W, Zhao Y, and Lu X
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Cellulase genetics, Cellulose analogs & derivatives, Cellulose metabolism, Cytophaga chemistry, Cytophaga genetics, Dextrins metabolism, Enzyme Stability, Kinetics, Substrate Specificity, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Cellulase chemistry, Cellulase metabolism, Cytophaga enzymology
- Abstract
Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl β-D-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.
- Published
- 2014
- Full Text
- View/download PDF
44. Directed evolution of a cellodextrin transporter for improved biofuel production under anaerobic conditions in Saccharomyces cerevisiae.
- Author
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Lian J, Li Y, HamediRad M, and Zhao H
- Subjects
- Cellobiose metabolism, Cellulose metabolism, Ethanol metabolism, Fermentation, Membrane Transport Proteins metabolism, Metabolic Engineering methods, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Biofuels microbiology, Cellulose analogs & derivatives, Dextrins metabolism, Directed Molecular Evolution methods, Membrane Transport Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
Introduction of a cellobiose utilization pathway consisting of a cellodextrin transporter and a β-glucosidase into Saccharomyces cerevisiae enables co-fermentation of cellobiose and xylose. Cellodextrin transporter 1 (CDT1) from Neurospora crassa has been established as an effective transporter for the engineered cellobiose utilization pathways. However, cellodextrin transporter 2 (CDT2) from the same species is a facilitator and has the potential to be more efficient than CDT1 under anaerobic conditions due to its energetic benefits. Currently, CDT2 has a very low activity and is considered rate-limiting in cellobiose fermentation. Here, we report the directed evolution of CDT2 with an increased cellobiose uptake activity, which results in improved cellobiose fermentation under anaerobic conditions. After three rounds of directed evolution, the cellobiose uptake activity of CDT2 was increased by 2.2-fold, which resulted from both increased specific activity and transporter expression level. Using high cell density fermentation under anaerobic conditions, the evolved mutant conferred 4.0- and 4.4-fold increase in the cellobiose consumption rate and ethanol productivity, respectively. In addition, although the cellobiose uptake activity was still lower than that of CDT1, the engineered CDT2 showed significantly improved cellobiose consumption and ethanol production under anaerobic conditions, representing the energetic benefits of a sugar facilitator for anaerobic cellobiose fermentation. This study demonstrated that anaerobic biofuel production could be significantly improved via directed evolution of a sugar transporter protein in yeast., (© 2014 Wiley Periodicals, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
45. Biotransformation of phytosterols under aerobic conditions.
- Author
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Dykstra CM, Giles HD, Banerjee S, and Pavlostathis SG
- Subjects
- Biodegradation, Environmental, Biotransformation, Carbon metabolism, Cholesterol metabolism, Dextrins metabolism, Ethanol metabolism, Water Pollutants, Chemical metabolism, Bacteria, Aerobic metabolism, Biotechnology methods, Cholesterol analogs & derivatives, Phytosterols metabolism, Sitosterols metabolism, Stigmasterol metabolism
- Abstract
Phytosterols are plant-derived sterols present in pulp and paper wastewater and have been implicated in the endocrine disruption of aquatic species. Bioassays were performed to assess the effect of an additional carbon source and/or solubilizing agent on the aerobic biotransformation of a mixture of three common phytosterols (β-sitosterol, stigmasterol and campesterol). The aerobic biotransformation of the phytosterol mixture by a mixed culture developed from a pulp and paper wastewater treatment system was examined under three separate conditions: with phytosterols as the sole added carbon source, with phytosterols and dextrin as an additional carbon source, and with phytosterols added with ethanol as an additional carbon source and solubilizing agent. Significant phytosterol removal was not observed in assays set up with phytosterol powder, either with or without an additional carbon source. In contrast, all three phytosterols were aerobically degraded when added as a dissolved solution in ethanol. Thus, under the experimental conditions of this study, the bioavailability of phytosterols was limited without the presence of a solubilizing agent. The total phytosterol removal rate was linear for the first six days before re-spiking, with a rate of 0.47 mg/L-d (R(2) = 0.998). After the second spiking, the total phytosterol removal rate was linear for seven days, with a rate of 0.32 mg/L-d (R(2) = 0.968). Following the 7th day, the phytosterol removal rate markedly accelerated, suggesting two different mechanisms are involved in phytosterol biotransformation, more likely related to the production of enzyme(s) involved in phytosterol degradation, induced under different cell growth conditions. β-sitosterol was preferentially degraded, as compared to stigmasterol and campesterol, although all three phytosterols fell below detection limits by the 24th day of incubation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
46. Evidence of a critical role for cellodextrin transporte 2 (CDT-2) in both cellulose and hemicellulose degradation and utilization in Neurospora crassa.
- Author
-
Cai P, Gu R, Wang B, Li J, Wan L, Tian C, and Ma Y
- Subjects
- Biomarkers metabolism, Cell Membrane metabolism, Cellulase genetics, Cellulase metabolism, Glycoside Hydrolases genetics, High-Throughput Nucleotide Sequencing, Membrane Transport Proteins genetics, Neurospora crassa genetics, Neurospora crassa growth & development, Oligonucleotide Array Sequence Analysis, RNA, Messenger genetics, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Cellulose analogs & derivatives, Cellulose metabolism, Dextrins metabolism, Gene Expression Profiling, Membrane Transport Proteins metabolism, Neurospora crassa metabolism, Polysaccharides metabolism
- Abstract
CDT-1 and CDT-2 are two cellodextrin transporters discovered in the filamentous fungus Neurospora crassa. Previous studies focused on characterizing the role of these transporters in only a few conditions, including cellulose degradation, and the function of these two transporters is not yet completely understood. In this study, we show that deletion of cdt-2, but not cdt-1, results in growth defects not only on Avicel but also on xylan. cdt-2 can be highly induced by xylan, and this mutant has a xylodextrin consumption defect. Transcriptomic analysis of the cdt-2 deletion strain on Avicel and xylan showed that major cellulase and hemicellulase genes were significantly down-regulated in the cdt-2 deletion strain and artificial over expression of cdt-2 in N. crassa increased cellulase and hemicellulase production. Together, these data clearly show that CDT-2 plays a critical role in hemicellulose sensing and utilization. This is the first time a sugar transporter has been assigned a function in the hemicellulose degradation pathway. Furthermore, we found that the transcription factor XLR-1 is the major regulator of cdt-2, while cdt-1 is primarily regulated by CLR-1. These results deepen our understanding of the functions of both cellodextrin transporters, particularly for CDT-2. Our study also provides novel insight into the mechanisms for hemicellulose sensing and utilization in N. crassa, and may be applicable to other cellulolytic filamentous fungi.
- Published
- 2014
- Full Text
- View/download PDF
47. Analysis of cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation.
- Author
-
Kim H, Lee WH, Galazka JM, Cate JH, and Jin YS
- Subjects
- Cellulose metabolism, Fermentation, Membrane Transport Proteins genetics, Neurospora crassa genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Cellobiose metabolism, Cellulose analogs & derivatives, Dextrins metabolism, Membrane Transport Proteins metabolism, Neurospora crassa enzymology
- Abstract
Saccharomyces cerevisiae can be engineered to ferment cellodextrins produced by cellulases as a product of cellulose hydrolysis. Direct fermentation of cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. To facilitate cellodextrin utilization by S. cerevisiae, a fungal cellodextrin-utilizing pathway from Neurospora crassa consisting of a cellodextrin transporter and a cellodextrin hydrolase has been introduced into S. cerevisiae. Two cellodextrin transporters (CDT-1 and CDT-2) were previously identified in N. crassa, but their kinetic properties and efficiency for cellobiose fermentation have not been studied in detail. In this study, CDT-1 and CDT-2, which are hypothesized to transport cellodextrin with distinct mechanisms, were introduced into S. cerevisiae along with an intracellular β-glucosidase (GH1-1). Cellobiose transport assays with the resulting strains indicated that CDT-1 is a proton symporter while CDT-2 is a simple facilitator. A strain expressing CDT-1 and GH1-1 (DCDT-1G) showed faster cellobiose fermentation than the strain expressing CDT-2 and GH1-1 (DCDT-2G) under various culture conditions with different medium compositions and aeration levels. While CDT-2 is expected to have energetic benefits, the expression levels and kinetic properties of CDT-1 in S. cerevisiae appears to be optimum for cellobiose fermentation. These results suggest CDT-1 is a more effective cellobiose transporter than CDT-2 for engineering S. cerevisiae to ferment cellobiose.
- Published
- 2014
- Full Text
- View/download PDF
48. Evidence for transceptor function of cellodextrin transporters in Neurospora crassa.
- Author
-
Znameroski EA, Li X, Tsai JC, Galazka JM, Glass NL, and Cate JH
- Subjects
- Biological Transport physiology, Cellulase genetics, Cellulase metabolism, Gene Expression Regulation, Fungal, Membrane Transport Proteins genetics, Mutagenesis, Site-Directed, Neurospora crassa genetics, Neurospora crassa growth & development, beta-Glucosidase genetics, beta-Glucosidase metabolism, Biofuels microbiology, Cellobiose metabolism, Cellulose analogs & derivatives, Cellulose metabolism, Dextrins metabolism, Membrane Transport Proteins metabolism, Neurospora crassa metabolism
- Abstract
Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as "transceptors" with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.
- Published
- 2014
- Full Text
- View/download PDF
49. Cellodextrin transporters play important roles in cellulase induction in the cellulolytic fungus Penicillium oxalicum.
- Author
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Li J, Liu G, Chen M, Li Z, Qin Y, and Qu Y
- Subjects
- Cellobiose metabolism, Cellulose metabolism, Gene Deletion, Gene Expression Profiling, Penicillium growth & development, Transcription, Genetic, Cellulase biosynthesis, Cellulose analogs & derivatives, Dextrins metabolism, Gene Expression Regulation, Fungal drug effects, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Penicillium genetics, Penicillium metabolism
- Abstract
Cellodextrin transporters (cellodextrin permeases) have been identified in fungi in recent years. However, the functions of these transporters in cellulose utilization and cellulase expression have not been well studied. In this study, three cellodextrin transporters, namely, CdtC, CdtD, and CdtG, in the cellulolytic fungus Penicillium oxalicum (formally was classified as P. decumbens) were identified, and their functions were analyzed. The deletion of a single cellodextrin transporter gene slightly decreased cellobiose consumption, but no observable effect on cellulase expression was observed, which was attributed to the overlapping activity of isozymes. Further simultaneous deletion of cdtC and cdtD resulted in significantly decreased cellobiose consumption and poor growth on cellulose. The extracellular activity and transcription level of cellulases in the mutant without cdtC and cdtD were significantly lower than those in the wild-type strain when grown on cellulose. This result provides direct evidence of the crucial function of cellodextrin transporters in the induction of cellulase expression by insoluble cellulose.
- Published
- 2013
- Full Text
- View/download PDF
50. In vitro fermentation of NUTRIOSE(®) FB06, a wheat dextrin soluble fibre, in a continuous culture human colonic model system.
- Author
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Hobden MR, Martin-Morales A, Guérin-Deremaux L, Wils D, Costabile A, Walton GE, Rowland I, Kennedy OB, and Gibson GR
- Subjects
- Acetates metabolism, Bacterial Typing Techniques, Butyrates metabolism, Chromatography, Gas, Clostridium isolation & purification, Dietary Fiber metabolism, Fatty Acids, Volatile biosynthesis, Humans, In Situ Hybridization, Fluorescence, Models, Anatomic, Propionates metabolism, RNA, Ribosomal, 16S classification, Tissue Culture Techniques, Clostridium metabolism, Colon microbiology, Dextrins metabolism, Microbiota physiology, RNA, Ribosomal, 16S genetics, Triticum chemistry
- Abstract
Wheat dextrin soluble fibre may have metabolic and health benefits, potentially acting via mechanisms governed by the selective modulation of the human gut microbiota. Our aim was to examine the impact of wheat dextrin on the composition and metabolic activity of the gut microbiota. We used a validated in vitro three-stage continuous culture human colonic model (gut model) system comprised of vessels simulating anatomical regions of the human colon. To mimic human ingestion, 7 g of wheat dextrin (NUTRIOSE(®) FB06) was administered to three gut models, twice daily at 10.00 and 15.00, for a total of 18 days. Samples were collected and analysed for microbial composition and organic acid concentrations by 16S rRNA-based fluorescence in situ hybridisation and gas chromatography approaches, respectively. Wheat dextrin mediated a significant increase in total bacteria in vessels simulating the transverse and distal colon, and a significant increase in key butyrate-producing bacteria Clostridium cluster XIVa and Roseburia genus in all vessels of the gut model. The production of principal short-chain fatty acids, acetate, propionate and butyrate, which have been purported to have protective, trophic and metabolic host benefits, were increased. Specifically, wheat dextrin fermentation had a significant butyrogenic effect in all vessels of the gut model and significantly increased production of acetate (vessels 2 and 3) and propionate (vessel 3), simulating the transverse and distal regions of the human colon, respectively. In conclusion, wheat dextrin NUTRIOSE(®) FB06 is selectively fermented in vitro by Clostridium cluster XIVa and Roseburia genus and beneficially alters the metabolic profile of the human gut microbiota.
- Published
- 2013
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