- Labeling and purification of cellulose-binding proteins for high resolution fluorescence applications
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The study of enzymatic reactions through fluorescence spectroscopy requires the use of bright, functional fluorescent molecules. In the case of proteins, labeling with fluorescent dyes has been carried out through covalent reactions with specific amino ac
- Moran-Mirabal, Jose M.,Corgie, Stephane C.,Bolewski, Jacob C.,Smith, Hanna M.,Cipriany, Benjamin R.,Craighead, Harold G.,Walker, Larry P.
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- High Yielding Acid-Catalysed Hydrolysis of Cellulosic Polysaccharides and Native Biomass into Low Molecular Weight Sugars in Mixed Ionic Liquid Systems
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Ionic media comprising 1-butyl-3-methylimidazolium chloride and the acidic deep eutectic solvent choline chloride/oxalic acid as co-solvent-catalyst, very efficiently convert various cellulosic substrates, including native cellulosic biomass, into water-soluble carbohydrates. The optimum reaction systems yield a narrow range of low molecular weight carbohydrates directly from cellulose, lignocellulose, or algal saccharides, in high yields and selectivities up to 98 %. Cellulose possesses significant potential as a renewable platform from which to generate large volumes of green replacements to many petrochemical products. Within this goal, the production of low molecular weight saccharides from cellulosic substances is the key to success. Native cellulose and lignocellulosic feedstocks are less accessible for such transformations and depolymerisation of polysaccharides remains a primary challenge to be overcome. In this study, we identify the catalytic activity associated with selected deep eutectic solvents that favours the hydrolysis of polysaccharides and develop reaction conditions to improve the outcomes of desirable low molecular weight sugars. We successfully apply the chemistry to raw bulk, non-pretreated cellulosic substances.
- Bodachivskyi, Iurii,Kuzhiumparambil, Unnikrishnan,Bradley G. Williams
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p. 1316 - 1324
(2019/11/11)
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- Cloning, expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans
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β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494 bp, encoded a polypeptide of 497 amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57 kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55 °C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.
- Yuan, Ye,Xu, Fenghua,Yao, Jianzhuang,Hu, Yanho,Wang, Jiao,Zhao, Tianjiao,Zhou, Yifa,Gao, Juan
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p. 362 - 371
(2017/11/03)
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- Regioselective glucosylation of inositols catalyzed by Thermoanaerobacter sp. CGTase
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Monoglucosylated products of l-chiro-, d-chiro-, muco-, and allo-inositol were synthesized by regioselective α-d-glucosylation with cyclodextrin glucosyl transferase from Thermoanaerobacter sp. after hydrolysis of by products with Aspergillus niger glucoamylase. While the reactions carried out with d-chiro-, muco-, and allo-inositol resulted in the regioselective formation of monoglucosylated products, two products were obtained in the reaction with l-chiro-inositol. Through the structural characterization of the glucosylated inositols here we demonstrated that the selectivity observed in the glucosylation of several inositols by Thermoanaerobacter sp. CGTase, is analogous to the specificity observed for the glucosylation of β-d-glucopyranose and equivalent glucosides.
- Miranda-Molina, Alfonso,Marquina-Bahena, Silvia,Alvarez, Laura,Lopez-Munguia, Agustin,Castillo, Edmundo
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p. 93 - 101,9
(2020/08/20)
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- Branched alpha-glucan, alpha-glucosyltransferase which forms the glucan, their preparation and uses
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The present invention has objects to provide a glucan useful as water-soluble dietary fiber, its preparation and uses. The present invention solves the above objects by providing a branched α-glucan, which is constructed by glucose molecules and characterized by methylation analysis as follows: (1) Ratio of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol to 2,3,4-trimethyl-1,5,6-triacetyl-glucitol is in the range of 1:0.6 to 1:4;(2) Total content of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol and 2,3,4-trimethyl-1,5,6-triacetyl-glucitol is 60% or higher in the partially methylated glucitol acetates;(3) Content of 2,4,6-trimethyl-1,3,5-triacetyl-glucitol is 0.5% or higher but less than 10% in the partially methylated glucitol acetates; and(4) Content of 2,4-dimethyl-1,3,5,6-tetraacetyl-glucitol is 0.5% or higher in the partially methylated glucitol acetates; a novel α-glucosyltransferase which forms the branched α-glucan, processes for producing them, and their uses.
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Page/Page column 18-19
(2010/06/11)
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- Molecular cloning and functional expression of a new amylosucrase from Alteromonas macleodii
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The presence of amylosucrase in 12 Alteromonas and Pseudoalteromonas strains was examined. Two Alteromonas species (Alteromonas addita KCTC 12195 and Alteromonas macleodii KCTC 2957) possessed genes that had high sequence homology to known amylosucrases. Genomic clones containing the ASase analogs were obtained from A. addita and A. macleodii, and the deduced amino acid sequences of the corresponding genes (aaas and amas, respectively) revealed that they were highly similar to the ASases of Neisseria polysaccharea, Deinococcus radiodurans, and Deinococcus geothermalis. Functional expression of amas in Escherichia coli was successful, and typical ASase activity was detected in purified recombinant AMAS, whereas the purified recombinant AAAS was nonfunctional. Although maximum total activity of AMAS was observed at 45 °C, the ratio of transglycosylation to total activity increased as the temperature decreased from 55 to 25 °C. These results imply that transglycosylation occurs preferentially at lower temperatures while hydrolysis is predominant at higher temperatures.
- Ha, Suk-Jin,Seo, Dong-Ho,Jung, Jong-Hyun,Cha, Jaeho,Kim, Tae-Jip,Kim, Young-Wan,Park, Cheon-Seok
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experimental part
p. 1505 - 1512
(2010/03/01)
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- Pullulanase-Amylase Complex Enzyme from Bacillus subtilis
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A novel pullulanase-amylase complex enzyme, which hydrolyzes pullulan into maltotriose as well as starch into maltose and maltotriose as the main products, was found in the culture filtrate of a strain of Bacillus subtilis newly isolated from soil.The enzyme was purified to almost complete homogeneity by means of calcium phosphate gel adsorption, DEAE-Sepharose column chromatography and Bio-gel A-1.5 m filtration.The optimum pH of the pullulanase activity was observed at around 7.0 to 7.5, with a discernible shoulder around pH 5.0.While the optimum pH of the amylase activity was 6 - 7.The optimum temperatures of the pullulanase and amylase activities were about 60 deg C and about 50 deg C, respectively.The molecular weight was estimated to be about 450,000 by the gel filtration method.The enzyme could be used for the production of glucose from starch with glucoamylase and the production of a new type of syrup containing a relatively high amount of maltotriose, 50 - 55 percent, from starch.
- Takasaki, Yoshiyuki
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