Reversible immobilization of glucoamylase onto magnetic carbon nanotubes functionalized with dendrimer

Magnetic carbon nanotubes (MCNTs) with necklace-like nanostructures was prepared via hydrothermal method, and hyperbranched poly(amidoamine) (PAMAM) was grafted on the surface of MCNTs on the basis of the Michael addition of methyl acrylate and the amidation of the resulting ester with a large excess of ethylenediamine (EDA), which could achieve generational growth under such uniform stepwise reactions. The terminal –NH₂ groups from the dendritic PAMAM were reacted with differently functionalized groups to form functionalized MCNTs. Subsequently, enzyme was immobilized on the functionalized MCNTs through adsorption, covalent bond, and metal-ion affinity interactions. The immobilization of glucoamylase, hereby chosen as model enzyme, onto the differently functionalized MCNTs is further demonstrated and assessed based on its activity, thermal stability, as well as reusability. Besides ease in recovery by magnetic separation, the immobilized glucoamylase on functionalized MCNTs offers superior stability and reusability, without compromising the substrate specificity of free glucoamylase. Furthermore, the results indicate that the metal-chelate dendrimer offers an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase.     

Reversible immobilization of glucoamylase onto magnetic polystyrene beads with multifunctional groups

A novel and simple process for the surface functionalization of micron-sized monodisperse magnetic polystyrene (PS) microbeads was reported. The polystyrene seed particles were prepared prior to the dispersion polymerization method. Afterwards, series of surface chemical modifications on polystyrene microspheres were conducted, and three end-functional microspheres with carboxyl, imidazolyl and sulphydryl groups were obtained. The functional magnetic polystyrene microspheres were prepared by impregnation and subsequent precipitation of ferric and ferrous ions into the polystyrene particles. Finally, the functional magnetic polystyrene was used for the reversible immobilization of glucoamylase via metal-affinity adsorption. The results indicated that the obtained immobilized glucoamylase presented excellent reusability, applicability, magnetic response and regeneration of supports. The magnetic PS microspheres retained >65% of its initial activity at 65 °C over 6 h; and the lowest residual activity of immobilized glucoamylase prepared by regenerated supports still remained about 50% of the initial activity after the 10th cycles.     

A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

A novel method was developed for the immobilization of glucoamylase from Aspergillus niger. The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K_m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V_max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.     

Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine

Glucoamylase (GA) from Aspergillus niger was immobilized via ionic adsorption onto DEAE-agarose, Q1A-Sepabeads, and Sepabeads EC-EP3 supports coated with polyethyleneimine (PEI). After optimization of the immobilization conditions (pH, polymer size), it was observed that the adsorption strength was much higher in PEI-Sepabeads than in Q1A-Sepabeads or DEAE-supports, requiring very high ionic strength to remove glucoamylase from the PEI-supports (e.g., 1 M NaCl at pH 5.5). Thermal stability and optimal temperature was marginally improved by this immobilization. Recovered activity depended on the substrate used, maltose or starch, except when very low loading was used. The optimization of the loading allowed the preparation of derivatives with 750 IU/g in the hydrolysis of starch, preserving a high percentage of immobilized activity (around 50%).     

A new route for chitosan immobilization onto polyethylene surface

Low-density polyethylene (LDPE) belongs to commodity polymer materials applied in biomedical applications due to its favorable mechanical and chemical properties. The main disadvantage of LDPE in biomedical applications is low resistance to bacterial infections. An antibacterial modification of LDPE appears to be a solution to this problem. In this paper, the chitosan and chitosan/pectin multilayer was immobilized via polyacrylic acid (PAA) brushes grafted on the LDPE surface. The grafting was initiated by a low-temperature plasma treatment of the LDPE surface. Surface and adhesive properties of the samples prepared were investigated by surface analysis techniques. An antibacterial effect was confirmed by inhibition zone measurements of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The chitosan treatment of LDPE led to the highest and most clear inhibition zones (35mm(2) for E. coli and 275mm(2) for S. aureus).     

Reversible immobilization of antibodies on magnetic beads.

A streptavidin-biotin system was utilized to prepare an antibody-polyadenylic acid conjugate which was subsequently attached to commercially available magnetic beads, Dynabeads oligo(dT)25. Biotinylated polyadenylic acid was combined with streptavidin and the resulting polyadenylic acid-streptavidin was conjugated with an antibody-biotin derivative. The immobilized antibody-polyadenylic acid conjugate was separated from the reaction mixture by hybridization with complementary oligonucleotide immobilized on the surface of Dynabeads oligo(dT)25. The immobilized antibody-polyadenylic acid can be released from the carrier, utilizing low-ionic-strength buffers. The system is intended to be utilized in cell sorting, using immobilized antibodies against cell surface antigens. Dissociation of antibody-containing conjugate from magnetic beads is essential for the isolation of viable cells via positive cell sorting.     

Reversible immobilization of catalase on fibrous polymer grafted and metal chelated chitosan membrane

Poly(itaconic acid) grafted and/or Fe(III) ions incorporated chitosan membranes were used for reversible immobilization of catalase (from bovine liver) via adsorption. The influences of pH and initial catalase concentration on the immobilization capacities of the CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membranes have been investigated in a batch system. Maximum catalase adsorption onto CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membrane were found to be 6.3 and 37.8 mg/g polymer at pH 5.0 and 6.5, respectively. The CH-g-poly(IA)-Fe(III) membrane with high catalase adsorption capacity was used in the rest of the study. The K_m value for immobilized catalase on CH-g-poly(IA)-Fe(III) (25.8 mM) was higher about 1.6-fold than that of free enzyme (13.5 mM). Optimum operational temperature was observed at 40 °C, a 5 °C higher than that of the free enzyme and was significantly broader. The optimum operational pH was same for both free and immobilized catalase (pH 7.0). Thermal stability was found to increase with immobilization. Free catalase lost all its activity within 20 days whereas immobilized catalase lost 23% of its activity during the same incubation period. It was observed that the same support enzyme can be repeatedly used for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity. In addition, the CH-g-poly(IA)-Fe(III) membrane prepared in this work showed promising potential for various biotechnological applications.     

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of “starch” dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

产糖化酶黑曲霉固定化方法比较的研究

采用海藻酸钙凝胶电埋法、以沸石、多孔聚酯等材料为固定化载体的吸附法固定黑曲霉（Aspergillus niger AS3.4309)菌丝细胞,以游离菌丝体作为对照,进行发酵产糖化酶的比较,结果表明：以聚酯泡沫作为固定化载体吸附固定化菌丝细胞产糖化酶活力最高。在产糖化酶的发酵过程中,与游离菌丝体细胞相比,固定化黑曲霉持续产酶时间有一定程度的延长。     

Evaluation of man-tailored cellulose-based carriers in glucoamylase immobilization

Covalent immobilization of glucoamylase on the cellulose-based carrier Granocel was optimized by changing the anchor groups and the methods of activation/immobilization. Binding of the enzyme was via its primary amino groups. It was shown that using carbodiimide and divinyl sulfone for the activation of -COOH and -OH groups on the carrier resulted in the preparations with very low activity. A third method, using pentaethylenehexamine with glutaraldehyde, led to the attachment through a long spacer arm and to the preparations with the highest activity. Further optimization of the carrier's structure consisted of changing pore diameters and amount of functional groups on the carrier surface. The highest activity of bound glucoamylase was obtained by linking the protein via glutaraldehyde on NH(2)-Granocel having high pore size and high number of functional groups. The immobilized enzyme was stable throughout extended storage and possessed higher thermal stability.     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

Stabilization of d-amino acid oxidase from Rhodosporidium toruloides by immobilization onto magnetic nanoparticles

d-Amino acid oxidase from Rhodosporidium toruloides was immobilized onto glutaraldehyde-activated magnetic nanoparticles. Approximately four enzyme molecules were attached to one magnetic nanoparticle when the weight ratio of the enzyme to the support was 0.12. After immobilization, the T _m was increased from 45°C of the free form to 55°C. In the presence of 20 mM H₂O₂, the immobilized form retained 93% of its activity after 5 h while the free form was completely inactivated after 3.5 h.     

Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose

Cellulose fibres from bagasse were oxidized by sodium periodate in sulphuric acid media at positions 2 and 3 of the anhydroglucose unit to produce dialdehyde cellulose. The aldehyde groups of the dialdehyde cellulose were able to react with amino groups of a glucoamylase to form covalent bonds and result in a dialdehyde cellulose immobilized enzyme. The optimum pH of this immobilized enzyme and free enzyme were in the range of 3.0–5.0 and 3.5–5.0, respectively. The optimum temperature for both the free and immobilized enzymes was 60–65 °C. The relative remaining activity of the immobilized enzyme was 36% and its stability was very good, since it could be reused for over 30 cycles. Its activity decreased from the first to the seventh reuse cycles, due to the slow detachment of non-covalently bound enzyme. However, activity tended to stabilize after the seventh cycle of reuse, indicating very stable covalent binding between the enzyme and dialdehyde cellulose.     

Reversible immobilization of chemically modified pullulanase

Summary Pullulanase was provided with up to nine de novo thiol groups through a two-step procedure without substantially affecting its enzymatic activity. The chemically modified enzyme was immobilized via disulfide bond formation on two kinds of thiolreactive gels (pyridyldisulfide- and thiolsulfonate-substituted agarose). Thiolation of pullulanase improved both the immobilization yield and the apparent specific activity of the derivatives.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

铜金属螯合载体固定糖化酶

[目的]制备出含Cu2+的琼脂糖-IDA螯合载体及对其固定糖化酶工艺条件进行优化.[方法]利用金属螯合配体(IDA-Cu2+)与蛋白质表面供电子氨基酸相互作用的原理制备载体,采用紫外分光光度法测定不同影响因素下固定化糖化酶的酶活.[结果]Cu2+的加入量和固定化过程的酸度比给酶量对固定化糖化酶的活性影响还要大,在给酶量80 mg/g载体、1.0× 10-2 mol Cu2+/g载体、pH 4.6和固定化4h的固定化条件下,固定化酶活为252.1 U/g,重复使用5次后酶活为首次固定化酶活的65.1％.[结论]该Cu2+-IDA-金属螯合琼脂糖可用于淀粉水解糖化酶的优良固定化载体材料.     

Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes

The present study describes the preparation of poly(HEMA-co-GMA) reactive membranes that were grafted with polyethylenimine (PEI) following UV photo-polymerization. The immobilization of tyrosinase was carried out via multi-point ionic interactions based on ---NH₂ groups of PEI and Cu(II) ions. Tyrosinase is a copper-dependent enzyme, which should show a binding affinity for the chelated Cu(II) ions on the membrane surfaces. The tyrosinase immobilization was positively correlated with the input enzyme amount in the immobilization medium. The maximum tyrosinase immobilization capacities of the poly(HEMA-co-GMA)–PEI and poly(HEMA-co-GMA)–PEI–Cu(II) membranes were 19.3 and 24.6 mg/m², respectively. The enzyme activity when assessed at various pH and temperatures gave broader range for immobilized preparations when compared to free enzyme. The poly(HEMA-co-GMA)–PEI–Cu(II) tyrosinase membranes retained 82% of their initial activity at the end of 120 h of continuous reaction. Moreover, upon storage for 3 months the activity of the immobilized membranes retained 46% of their initial levels. After deactivation of the enzyme, the poly(HEMA-co-GMA)–PEI membrane was easily regenerated, re-chelated with the Cu(II) ions and reloaded with the enzyme for repeated use. The mild immobilization conditions, easy and rapid membrane preparation, one-step enzyme adsorption at substantially higher levels and membrane reusability are the beneficial properties of such systems and offers promising potential in several biochemical processes.     

Activity and stability of glucoamylase preparations in different methods of immobilization

Catalytic activity and stability of glucoamylases immobilized by different methods (adsorption, covalent binding) are studied comparatively. The highest stability is shown to be obtained under covalent binding. The binding efficiency and immobilized glucoamylase properties depend on the nature of insoluble carrier and a purification degree of the enzyme preparations. The choice of the cross-linking agent promoting a binding between the enzyme and the carrier is very significant. The activity and stability of immobilized glucoamylases obtained when using different cross-linking agents rise in such a sequence: 2,4-toluylenediisocyanate, cyanurochloride, glutaric dialdehyde, gossypol. Catalytic properties and stability are determined for soluble and immobilized glucoamylase forms from different sources.