Abstract:Pullulanase from Klebsiella pneumoniae was entrapped into calcium alginate beads. Its activity was estimated by the determination of number-average molar masses using two different methods: a colorimetric assay of reducing ends (REs) and a size-exclusion chromatography/multiangle light scattering/differential refractive index. The second method also provided weight-average molar masses of hydrolyzed pullulan and the quantity of maltotriose (DP3) and its multiples (DP6 and DP9) produced by the enzymatic treatme… Show more
“…The number and weight molar masses were determined by coupling on‐line a size exclusion chromatograph (SEC) with a multiangle light scattering system (MALS) and a differential refractive index detector (DRI). The principle of this method was already described in previous works . The carrier LiNO 3 at 0.1 mol L −1 was filtered through a 0.1 µm filter unit (Millipore, USA), degassed (DGU‐20A3 Shimadzu, Japan), and eluted at a 0.5 mL min −1 flow rate (LC10Ai Shimadzu, Japan).…”
Section: Methodsmentioning
confidence: 99%
“…The principle of this method was already described in previous works. 7 The carrier LiNO 3 at 0.1 mol L 21 was filtered through a 0.1 mm filter unit (Millipore, USA), degassed (DGU-20A3 Shimadzu, Japan), and eluted at a 0.5 mL min 21 flow rate (LC10Ai Shimadzu, Japan). Sample solutions were injected with an automatic injector (SIL-20A Shimadzu, Japan).…”
“…[3][4][5][6] However, it can be classified in two various methods that are physical or chemical retention. The physical retention, largely used in hydrogels systems 7 is based either on steric entrapment or on physical interaction (ionic or hydrophobic). The steric entrapment exploits the size difference between the enzyme and its substrate.…”
The immobilization of pullulanase from Klebsiella pneumoniae by grafting was investigated. Pullulanase was linked after activation of alginate via a covalent bond between the amine groups of the enzyme and the carboxylic acid groups of alginate. The immobilization yield was 60%. The activity of free pullulanase and immobilized pullulanase was followed by the quantification of reducing ends by colorimetric assay and the determination of the molar masses of the hydrolyzed pullulan by SEC/MALS/DRI. Compared to free pullulanase, the kinetics is largely slowed. The evolution of the weight average molar mass of pullulan leading to high production of shorter oligosaccharides during hydrolysis is not the same as that obtained with free enzyme. Immobilized pullulanase retained 75% and 30% of its initial activity after 24 h and 14 days of incubation at 60°C, respectively while free pullulanase lost its activity after 5 h of hydrolysis at the same temperature. The kinetic parameters of immobilized pullulanase were also investigated by isothermal titration calorimetry (ITC). The affinity of immobilized enzyme to its substrate was reduced compared to the free pullulanase due to steric hindrance and chemical links.
“…The number and weight molar masses were determined by coupling on‐line a size exclusion chromatograph (SEC) with a multiangle light scattering system (MALS) and a differential refractive index detector (DRI). The principle of this method was already described in previous works . The carrier LiNO 3 at 0.1 mol L −1 was filtered through a 0.1 µm filter unit (Millipore, USA), degassed (DGU‐20A3 Shimadzu, Japan), and eluted at a 0.5 mL min −1 flow rate (LC10Ai Shimadzu, Japan).…”
Section: Methodsmentioning
confidence: 99%
“…The principle of this method was already described in previous works. 7 The carrier LiNO 3 at 0.1 mol L 21 was filtered through a 0.1 mm filter unit (Millipore, USA), degassed (DGU-20A3 Shimadzu, Japan), and eluted at a 0.5 mL min 21 flow rate (LC10Ai Shimadzu, Japan). Sample solutions were injected with an automatic injector (SIL-20A Shimadzu, Japan).…”
“…[3][4][5][6] However, it can be classified in two various methods that are physical or chemical retention. The physical retention, largely used in hydrogels systems 7 is based either on steric entrapment or on physical interaction (ionic or hydrophobic). The steric entrapment exploits the size difference between the enzyme and its substrate.…”
The immobilization of pullulanase from Klebsiella pneumoniae by grafting was investigated. Pullulanase was linked after activation of alginate via a covalent bond between the amine groups of the enzyme and the carboxylic acid groups of alginate. The immobilization yield was 60%. The activity of free pullulanase and immobilized pullulanase was followed by the quantification of reducing ends by colorimetric assay and the determination of the molar masses of the hydrolyzed pullulan by SEC/MALS/DRI. Compared to free pullulanase, the kinetics is largely slowed. The evolution of the weight average molar mass of pullulan leading to high production of shorter oligosaccharides during hydrolysis is not the same as that obtained with free enzyme. Immobilized pullulanase retained 75% and 30% of its initial activity after 24 h and 14 days of incubation at 60°C, respectively while free pullulanase lost its activity after 5 h of hydrolysis at the same temperature. The kinetic parameters of immobilized pullulanase were also investigated by isothermal titration calorimetry (ITC). The affinity of immobilized enzyme to its substrate was reduced compared to the free pullulanase due to steric hindrance and chemical links.
New biopolymer beads, composite of Mg–AL–La tri-metal oxides and alginate, were synthesized, characterized and tested for their fluoride removal efficiency from wastewater. The maximum adsorption capacity of the adsorbent was 30.9 mg g−1.
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