Immobilization of Chaetomium erraticum dextranase (CED) by adsorption on carboxylated multi walled carbon nanotubes (c-MWCNT)
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Abstract In this study, CED was immobilized onto c-MWCNT by adsorption. Optimization of immobilization conditions (immobilization buffer's pH and molarity, c-MWCNT amount, and immobilization time) was resulted in 100% immobilization yield and 114.13% activity yield. Further, characterization of FCED and ICED was also studied. After immobilization, the optimum pH shifted from 5.0 to 6.0, while the optimum temperature (55 °C) did not change. Furthermore, kinetic constants for FCED and ICED were also determined using the Lineweaver-Burk plot. The K m value for both FCED and ICED were 54.35 g / L, while V max values for FCED and ICED were 2.77 μmol reducing sugar / L.mg.min and 3.19 μmol reducing sugar / L.mg.min, respectively. Moreover, there was no reduction in the initial activity of ICED after 20 consecutive uses and 30 days of storage at optimal storage conditions. Finally, 17.15% and 17.53% of the dextran in 10% dextran solution (pH 6.0) were converted to reduced sugars (IMOs and Glucose) in 12 hours using FCED and ICED, respectively. Consequently, it can be concluded that ICED obtained in this study can be effectively used for industrial production of IMOs and for hydrolysis of dextran.Keywords:
Dextranase
Reducing sugar
Dextranase (DN) has generated interest due to its ability to hydrolyze dextran for the synthesis of isomalto-oligosaccharides (prebiotic). Here, DN from Chaetomium erraticum was purified by a combination of tangential ultrafiltration and ion-exchange chromatography, and then the enzyme was characterized. This enzyme has a molecular weight (Mw) of around 59kDa, calculated by SDS-PAGE, and an Mw of 120kDa estimated on zymogram, suggesting that the native form is a dimer. The purified DN exhibited a pH and temperature optimum of 5.2 and 60°C, respectively. The DN-catalyzed hydrolysis of dextran followed Michaelis⿿Menten kinetics with Km and Vmax values estimated as 2.6±0.1% (260μM) and 2280±9μmolmin⿿¹mg⿿¹ protein, respectively. The activation energy was determined as 52.7±0.4kJmol⿿¹, and the thermodynamic parameters (οG, οH and οS) for the hydrolysis of dextran were also determined. Dextranase activity was enhanced by Ca²⁺, and especially Co²⁺ at 1mM, which improved its activity to 124.5±3.0%, whereas Ni²⁺ and Fe³⁺ negatively affected activity. The main end-products of 100kDa dextran hydrolysis by DN were isomaltose, isomaltotriose, and maltopentaose. Finally, it was found that the thermal inactivation mechanism for dextranase can be described by the first-order irreversible mechanism.
Dextranase
Isomaltose
Enzyme Kinetics
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Thermostable dextranase (1,6-α-d-glucan 6-glucanohydrolysase) from a thermophilic anaerobic bacterium strain Rt364, isolated from a New Zealand hot spring, was partially purified from the cell-free supernatant fluid by adsorption onto Sephacryl S-300, a dextran-based chromatographic resin. It was competitively eluted with 2% T10 dextran, dialysed, concentrated and examined by SDS–PAGE. The overall recovery was 47% and the increase in specific activity by this procedure was 25-fold. The Rt364 dextranase had previously been found to have an optimum temperature of 80 °C and hydrolysed both α-1,6 and α-1,4 glucosidic bonds. Sephacryl S-300 adsorption is a simple, useful step with general application for concentrating and purifying bacterial enzymes that hydrolyse dextrans.
Dextranase
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Abstract Hydrolysis of (1 → 6)‐α‐ DL ‐glucopyranan (synthetic DL ‐dextran) by an endo ‐dextranase from a Penicillium species was examined in an acetate buffer solution (pH 5.3) at 37°C. Three samples of different tacticities (isotactic dyad content, 55, 63, and 72%) were employed with a clinical dextran for comparison. Colorimetric determination of the reducing end units of the saccharides produced during hydrolysis showed that the maximum degrees of hydrolysis based on the D‐glucose units, (D.H.) D , for the DL ‐dextrans were 21.4, 27.8, and 33.0% in the order of increasing isotacitic dyad content, whereas the (D.H.) D value for the clinical dextran was 51.9%. A statistical treatment of the enzymatic hydrolysis is proposed to interpret the experimental results.
Dextranase
Enzymatic Hydrolysis
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Dextrans are undesirable compounds, synthesized by Leuconostoc mesenteroide from sucrose, increasing the viscosity of the sugar flow and reducing industrial recovery, resulting significant losses to the sugar industries. The use of dextranase enzyme is the most efficient method for hydrolyzing the dextrans at sugar mills. A preparation of dextranase enzyme namely “Enzydex” developed by Catalysts Biotechnologies Pvt. Ltd which was shown significant reduction at very low concentration 3-5ppm during six plant trials in India and in Philippines. During plant trials, the 38-78% dextran reductions were observed at various stages of sugar process.
Dextranase
Reducing sugar
Sugar production
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The most serious processing problem can clear arise from the presence of dextran gum. The presence of dextran in sugar processing leads to less of sucrose and creates problems to sugar producers by increasing viscosity, lowing sugar yield, increasing molasses purities slowing filtration. The application of dextran enzyme to reduce dextran from raw juice was more efficient and economic than adding it to clear juice and syrup. Sixty percent of dextran removal was achieved when dextranase applied at concentration of 20u/100ml raw juice and 30min incubation. The dextran reduction and reached 65% by the use of 30u under some condition in clear juice, the percentage of dextran reduction reached 25,27 and 45% when dextranase enzyme used at 30u /100mol after 10,20 and 30 min of the incubation respectively. The use of the application of dextranase enzyme to reduce dextran from raw juice was more efficient and economic than adding it to clear juice and syrup.
Sixty percent of the dextran was removed when using dextranase applications at a concentration of 20u/100ml of raw juice over 30 minutes.
Dextranase
Reducing sugar
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[Objective] The aim was to realize efficient utilization of residues of bamboo processing and study its acid hydrolysis technology.[Method] The influence factors of acid hydrolysis of bamboo powder were studied through single-factor test.With yield of total reducing sugar as index,the optimum reaction condition for reducing sugar production by hydrolyzing bamboo powder with dilute sulfuric acid was obtained through analysis.[Result] The results from single-factor tests showed that when material-liquid ratio was 1∶5-1∶7,the yield rate of reducing sugar was increased along with the increase of thick acid volume;when material-liquid ratio was 1∶5 and 1∶8,the yield rates of reducing sugar were 44.0% and 41.6% resp.;when the 1st hydrolysis temperature was 60 ℃,the yield rate of reducing sugar was highest,being 65.3%;when sulfuric acid concn.was 15%-30%,the yield rate of reducing sugar changed little;the yield rate of reducing sugar was reduced along with extending of hydrolysis time;when the 2nd hydrolysis time was 1 h,the yield rate of reducing sugar was highest,being 66.3%.The results from orthogonal test showed that the influence of the 1st hydrolysis temperature was most and that of the 2nd hydrolysis time and bamboo powder/thick sulfuric acid ratio was least;the optimum reaction condition was as follows:the(W/V)ratio of bamboo powder to thick acid was 1∶6,the 1st hydrolysis temperature was 50 ℃,the 2nd hydrolysis temperature was 100 ℃,the concn.of dilute sulfuric acid was 20% and the hydrolysis time was 1 h.[Conclusion] This study provided references for developing and utilizing residues of bamboo processing.
Reducing sugar
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Dextranase
Isomaltose
Enzyme Kinetics
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Dextranase
Isomaltose
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Dextranase
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The hazard of dextran in sugar manufacture has been highly concerned around the world. The application of dextranase is the most effective method to breakdown dextran. The optimization of dextranase to hydrolyze dextran in sugarcane juice was carried out by using response surface methodology. A Box-Behnken factorial designed with four factors and three levels, was used to determine the influence of enzyme dosage(U/mL), pH, temperature(℃) and reaction time(min), and regression model was built. The optimum operational conditions were as follows: enzyme dosage 0.05 U/mL, pH value 5.3, reaction time 15 min and temperature 51 ℃. In this condition, dextranase can hydrolyze all of 800~900 mg/(kg·?Bx) dextran in sugarcane juice.
Dextranase
Reducing sugar
Box–Behnken design
Enzymatic Hydrolysis
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