Immobilization Of Pullulanase Research Articles

Immobilization of pullulanase from Bacillus licheniformis on magnetic multi-walled carbon nanotubes for maltooligosaccharide production

Immobilization of pullulanase from Bacillus licheniformis on magnetic multi-walled carbon nanotubes for maltooligosaccharide production

Immobilization of Pullulanase from Local Isolate Bacillus subtilis MF467279 Strain onto Magnetic@TEMPO Cellulose Nanospheres for Hydrolysis of Pullulan

Immobilization of Pullulanase from Local Isolate Bacillus subtilis MF467279 Strain onto Magnetic@TEMPO Cellulose Nanospheres for Hydrolysis of Pullulan

Regulation of the catalytic behavior of pullulanases chelated onto nickel (II)-modified magnetic nanoparticles.

Chelating of pullulanases onto nickel (II)-modified magnetic nanoparticles results in one-step purification and immobilization of pullulanase, and facilitates the commercial application of pullulanase in industrial scale. To improve the catalytic behavior, especially the operational stability, of the nanocatalyst in consecutive batch reactions, we prepared various iminodiacetic acid-modified magnetic nanoparticles differed in surface polarity and spacer length, on which the His6-tagged pullulanases were chelated via nickel ions, and then studied the correlation between the MNPs surface property and the corresponding catalyst behavior. When pullulanases were chelated onto the surface-modified MNPs, the thermostability of all pullulanase derivatives were lower than that of free counterpart, being not relevant to the protein orientation guided by the locality of the His6-tag, but related to the MNPs basal surface polarity and the grafted spacer length. After chelating of pullulanases onto MNPs, there were changes observed in the pH-activity profile and the apparent Michaelis constant toward pullulan. The changing tendencies were mainly dependent on the His6-tagged pullulanase orientation, and the changing extents were tuned by the spacer length. The reusability of pullulanase immobilized by N-terminal His6-tag was higher than that of pullulanase immobilized by C-terminal His6-tag. Moreover, the reusability of the immobilized pullulanase tested increased till grafting polyether amine-400 as spacer-arm, therefore the N-terminal His6-tagged pullulanase chelating MNPs grafted polyether amine-400 gave the best reusability, which retained 60% of initial activity after 18 consecutive cycles with a total reaction time of 9h. Additionally, the correlation analysis of the catalyst behaviors indicated that the reusability was independent from other catalytic properties such as thermostability and substrate affinity. All the results revealed that the catalyst behavior can be mainly controlled by the His6-tagged pullulanase orientation than by the MNPs surface property which can tune the catalyst function.

Covalent Immobilization and Characterization of a Novel Pullulanase from Fontibacillus sp. Strain DSHK 107 onto Florisil® and Nano-silica for Pullulan Hydrolysis.

A novel pullulanase partially purified from Fontibacillus sp. was covalently immobilized on Florisil® and nano-silica through both glutaraldehyde and (3-glycidyloxypropyl)trimethoxysilane spacer arms. The pullulanase immobilized on Florisil® and nano-silica through glutaraldehyde spacer arm showed 85 and 190% activity of its free form, respectively, whereas no activity was observed when it was immobilized on the same supports through (3-glycidyloxypropyl)trimethoxysilane spacer arm. The maximum working pHs of both the immobilized pullulanases on Florisil® and nano-silica through glutaraldehyde spacer arm were determined as 5.0; however, the maximum working pH of the free pullulanase was pH 6.0. The maximum temperatures of all the pullulanase preparations were determined as 35°C. The apparent K m values were 1.49, 1.54, and 0.59mg/mL pullunan, respectively, for the free and immobilized pullulanases on Florisil® and nano-silica. The corresponding apparent V max values were 0.59, 1.53, and 1.57Umgprot.(-1) min.(-1). Thermal stability of pullulanases immobilized on Florisil® and nano-silica was enhanced 6.5- and 15.6-folds, respectively at 35°C and 6.6- and 16.0-folds, respectively, at 50°C. The pullulanases immobilized on Florisil® and nano-silica protected 71 and 90% of their initial activities after 10 reuses.

Effect of chitosan molecular weight on the formation of chitosan–pullulanase soluble complexes and their application in the immobilization of pullulanase onto Fe3O4–κ-carrageenan nanoparticles

The interactions between pullulanase and chitosans of different molecular weights (Mw) were comprehensively studied, and their applications in pullulanase immobilization onto Fe3O4–κ-carrageenan nanoparticles upon chitosan–pullulanase complexation were also evaluated. Chitosan (CS) complexation with pullulanase was found to be dependent on pH and chitosan Mw. The critical pH of structure-forming events during complexation shifted significantly (p<0.05) to a lower pH with a low Mw chitosan (50kDa) compared to other chitosan types. Binding constants for the chitosan–pullulanase interaction increased in the following order: CS-500<CS-400<CS-50<CS-200. The binding induced alterations in the protein secondary structure, which may affect the enzymatic properties of immobilized pullulanase. Pullulanase immobilized upon CS-50 complexation exhibited the most desirable enzymatic properties. These results indicated that the complexation behavior was mainly dependent on chitosan Mw. This study presents a technique for the production of immobilized pullulanase upon complexation that exhibits potential for applications in continuous syrup production.

Covalent immobilization of pullulanase on alginate and study of its hydrolysis of pullulan.

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 method for the immobilization of pullulanase onto hybrid magnetic (Fe3O4-κ-carrageenan) nanoparticles by electrostatic coupling with pullulanase/chitosan complex.

We present a simple method to immobilize pullulanase onto hybrid magnetic (Fe3O4-κ-carrageenan) nanoparticles, involving the in situ synthesis of magnetic carrageenan nanoparticles and the formation of pullulanase/chitosan complex. The complex behavior of pullulanase with chitosan as a function of pH and protein-polysaccharide ratio was studied by turbidimetric titration. Then, the as-prepared immobilized enzymes were characterized by vibrating-sample magnetometer, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffractometer, and thermogravimetric analysis. It was found that the activity retention of immobilized pullulanase and amount of enzyme loaded reached 95.5% and 96.3 mg/g, respectively, under optimal conditions. The immobilized enzyme exhibited great operational stability (retaining approximately 61% residual activity after ten consecutive reuses), demonstrating that enzyme leakage during the catalysis reaction was efficiently reduced. Furthermore, the activity of immobilized pullulanase was significantly (p < 0.01) higher than that of free pullulanase in a low pH range (pH < 3.0) and temperature over 60 °C, and the immobilized enzymes retained 45% of their initial activity after 5 h at 60 °C, compared to 21% for the free enzyme. These results indicated that immobilized pullulanase was efficient in terms of catalytic activity and can be applied to continuous starch processing applications in the food industry.

Immobilization of pullulanase onto activated magnetic chitosan/Fe3O4 nanoparticles prepared by in situ mineralization and effect of surface functional groups on the stability

Abstract Magnetic chitosan/Fe 3 O 4 nanoparticles prepared by in situ mineralization of Fe 3 O 4 in chitosan hydrogel were first modified by 3-aminopropyltriethoxysilane (3-APTES) and then tested for the possibility of being used as carriers for the immobilization of pullulanase. It was found that the immobilized enzyme showed high activity retention (>70%) and the affinity between enzyme and substrate was improved. However, compared to the enzyme immobilized by electrostatic adsorption (87% of its initial activity lost after 10 consecutive reuses), the enzyme immobilized by crosslinking was more stable and there was less loss of activity. The improvement of activity retention and stability was further studied from the view of surface amino concentration of nanoparticles at the function of the amount of 3-APTES added, and crosslinker concentration in the activation process. The highest activity retention (89%) and operation stability (61% residual activity after 10 consecutive reuses) were obtained under the condition of 1.6 amino groups per nm 2 and 15% glutaraldehyde (v/v, dimmer of glutaraldehyde). Therefore, the magnetic chitosan/Fe 3 O 4 nanoparticle prepared by in situ mineralization was proved to be a suitable carrier. In addition, immobilized pullulanase exhibited good resistance to metal ions and detergents.

A novel method for pullulanase immobilized onto magnetic chitosan/Fe3O4 composite nanoparticles by in situ preparation and evaluation of the enzyme stability

A novel immobilization of pullulanase has been developed by using magnetic chitosan/Fe3O4 nanoparticles (CS-MNPs). The CS-MNPs were synthesized by in situ mineralization of Fe3O4 in chitosan hydrogel and characterized by transmission electron microscopy, vibrating sample magnetometer, Fourier transform infrared spectroscopy, thermogravimetric analyzer and X-ray diffractometer. The resultant material (CS-MNPs) was used as support for the covalent immobilization of pullulanase with glutaraldehyde as cross-linker. In the process of pullulanase immobilization, both the amount (g) of immobilized pullulanase and the activity retention(%) increased with enzyme concentration increasing (15–60μg/ml), and the optimum activity retention (93.5%) was obtained at pH 3.5. In enzymatic properties, the immobilized enzyme exhibited a broadened pH profiles and higher thermal stability than the free enzyme. The optimum pH of immobilized pullulanase (3.5) was lower than that of the free enzyme (4.4), and the immobilized enzymes could retain about 84% of their initial activity after incubated for 5h at 60°C in contrast with 58% of free enzyme. Furthermore, the immobilized pullulanase retained more than 56% of its initial activity after eight consecutive reuses. These results prove that the CS-MNPs are an effective support for the immobilization of pullulanase.

Photochemical preparation of magnetic chitosan beads for immobilization of pullulanase

Magnetic chitosan beads were prepared via photochemical polymerization in Fe 3O 4 magnetite aqueous suspension, and characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), photocorrelation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The FT-IR result indicated that the magnetic chitosan beads were synthesized successfully under UV irradiation in aqueous system. The magnetic chitosan beads were of regularly spherical shape, had a mean diameter of 86 nm, exhibited superparamagnetic property and the saturation magnetization was 52.6 emu/g. Pullulanase was covalently immobilized on magnetic chitosan beads by cross-linking with glutaraldehyde. The maximum quantity of pullulanase immobilized onto 1.0 mg magnetic chitosan beads was 180 μg. The optimum temperature of immobilized pullulanase was 50° C, which was identical to that of free enzyme, and the immobilized pullulanase exhibited a higher relative activity than that of free pullulanase over 50° C. The optimal pH for immobilized pullulanase was 5.0, which was lower than that of the free pullulanase (pH 5.5), and the immobilization resulted in stabilization of enzyme over a broader pH range. The kinetic constant value ( Km) of immobilized pullulanase was three times higher than that of free pullulanase. However, the thermal and operational stabilities of immobilized pullulanase were improved greatly.

Immobilization and stabilization of pullulanase from Klebsiella pneumoniae by a multipoint attachment method using activated agar gel supports

The immobilization and stabilization of pullulanase from Klebsiella pneumoniae were investigated. Pullulanases immobilized on activated agar gel (glyoxyl-agar gel) via multipoint attachment between amino groups of pullulanase and aldehyde groups on the gel surface were significantly stable compared with free pullulanase and pullulanase adsorbed on chitosan beads. The immobilization yield, thermal stability and activity of immobilized pullulanase greatly depended on the surface density of aldehyde groups on the support gel. The optimum temperature and pH of immobilized pullulanases prepared by the multipoint attachment method using activated agar gels were 50 °C and 6.0, respectively. These values were the same as those of free pullulanase. Pullulanase immobilized on agar gel activated with 4.8 M glycidol retained 60% of its initial activity after a 250-h incubation at 45 °C, while free pullulanase completely lost its activity after a 72-h incubation at the same temperature.

Pullulanase immobilization on natural and synthetic polymers

AbstractThis work describes the immobilization of pullulanase onto two different polymers; agarose activated with epichlorohydrin and trichlorotriazine and casein activated with epichlorohydrin, in addition to a synthetic copolymer, butylacrylate–acrylic acid (BuA/AAc). Immobilization by covalent binding yields stable enzyme activity. The operational stability of the free and immobilized enzymes showed that the enzyme immobilized by a crosslinking technique using glutaric dialdehyde (GA) showed poor durability and the relative activity decreased sharply due to leakage after repeated washing, while the enzymes immobilized by covalent bonds resulted in a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4–6 months showed that the free enzyme lost most of its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzymes to doses of γ‐radiation (0.5–10 Mrad) resulted in complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to γ‐radiation up to a dose of 5 Mrad. Nuclear Magnetic Resonance (1H NMR) and FTIR measurements were carried out to confirm the structure of the polymer as well as the immobilization process of the enzyme onto the polymeric carrier. The unique biochemical characteristics, mode of action and utility of the environmentally compatible pullulanase in starch conversion are well known. Using pullulanase with β‐amylase in starch saccharification processes can increase maltose yield by 20–25%.© 2001 Society of Chemical Industry

Thermal stabilization of amylolytic enzymes by covalent coupling to soluble polysaccharides

The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application.