Enzyme Binding Capacity Research Articles

Amorphous-crystalline phase transition and intrinsic magnetic property of nickel organic framework for easy immobilization and recycling of β-Galactosidase

This work describes the synthesis of fibrous nickel-based metal organic framework (Ni-ZIF) via simple solvothermal method. The material formed was calcinated at 400, 600, 800 °C to improve its surface area, porosity and enzyme binding capacity. Changes in X-ray diffraction pattern after calcination revealed the Ni-ZIF transitioned from amorphous to crystalline structure. The surface area, pore volume and pore size for Ni-ZIF@600 were found to be 312.15 m2/g, 0.88 cm3/g and 10.28 nm, with an enzyme loading capacity of 593.85 mg/g after 30 h The free (β-Gal-LEH) and immobilized β-Galactosidase were stable at pH 7.5, temperature 50 °C, and yielded 70.70 and 63.95 mM glucose after milk lactose hydrolysis, respectively. The Ni-ZIF@600@β-Gal-LEH exhibited high enzyme retention capacity, maintaining 59.44 % of its original activity after 6-cycles. The enhanced magnetic property, enzyme binding capacity and easy recoverability of the calcinated Ni-ZIF could guarantee its industrial significance as immobilization module for enzyme-mediated catalysis.

Activity, stability, and binding capacity of β-galactosidase immobilized on electrospun nylon-6 fiber membrane

In this research, we explored various immobilized enzyme support materials, including the novel nylon-6 fiber membrane (NFM), and evaluated the increase in surface area and its effect on enzyme binding potential. We also manipulated incubation and reaction conditions and assessed the subsequent effects on activity and stability of β-galactosidase, with comparisons between various solid support materials and free (dissolved) enzyme. Nylon-6 fiber membranes were created by electrospinning and were compared with other materials as solid supports for enzyme binding. The other materials included polyvinylidene fluoride 5-kDa nanofiltration dairy membranes, nylon-6 pellets, and silica glass beads. Scanning electron microscopy revealed the large surface area of NFM, which correlated with greater enzyme activity compared with the relatively flatter surfaces of the other solid support materials. Enzyme activity was measured spectrophotometrically with the color-changing substrate o-nitrophenyl-β-d-galactopyranoside. Compared with the other solid supports, NFM had greater maximum enzyme binding potential. Across pH conditions ranging from 3.5 to 6.0 (including the optimal pH of 4.0-5.0), enzyme activity was maintained on the membrane-immobilized samples, whereas free enzyme did not maintain activity. Altering the storage temperature (4, 22, and 50°C) affected enzyme stability (i.e., the ability of the enzyme to maintain activity over time) of free and polyvinylidene fluoride membrane samples. However, NFM samples maintained stability across the varying storage temperatures. Increasing the immobilization solution enzyme concentration above the maximum enzyme binding capacity had no significant effect on enzyme stability for membrane-immobilized samples; however, both had lower mean stability than free enzyme by approximately 74%. With further development, β-galactosidase immobilized on NFM or other membranes could be used in continuous processing in the dairy industry for a combination of filtration and lactose hydrolysis-creating products that are reduced in lactose and increased in sweetness, with no requirement for "added sugars" on the nutrition label and no enzyme listed as final product ingredient.

Immobilization of \u03b2-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain

Enzyme immobilization is a powerful tool not only as a protective agent against harsh reaction conditions but also for the enhancement of enzyme activity, stability, reusability, and for the improvement of enzyme properties as well. Herein, immobilization of β-glucosidase from Thermotoga maritima (Tm-β-Glu) on magnetic nanoparticles (MNPs) functionalized with chitin (Ch) was investigated. This technology showed a novel thermostable chitin-binding domain (Tt-ChBD), which is more desirable in a wide range of large-scale applications. This exclusive approach was fabricated to improve the Galacto-oligosaccharide (GOS) production from a cheap and abundant by-product such as lactose through a novel green synthesis route. Additionally, SDS-PAGE, enzyme activity kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) revealed that among the immobilization strategies for Thermotoga maritime-β-Glucosidase thermostable chitin-binding domain (Tm-β-Glu-Tt-ChBD) on the attractive substrate; Ch-MNPs had the highest enzyme binding capacity and GOS production ratio when compared to the native enzyme. More interestingly, a magnetic separation technique was successfully employed in recycling the immobilized Tm-β-Glu for repetitive batch-wise GOS without significant loss or reduction of enzyme activity. This immobilization system displayed an operative stability status under various parameters, for instance, temperature, pH, thermal conditions, storage stabilities, and enzyme kinetics when compared with the native enzyme. Conclusively, the GOS yield and residual activity of the immobilized enzyme after the 10th cycles were 31.23% and 66%, respectively. Whereas the GOS yield from native enzyme synthesis was just 25% after 12 h in the first batch. This study recommends applying Tt-ChBD in the immobilization process of Tm-β-Glu on Ch-MNPs to produce a low-cost GOS as a new eco-friendly process besides increasing the biostability and efficiency of the immobilized enzyme.

Incorporating Lignin into Polyethylene Glycol Enhanced Its Performance for Promoting Enzymatic Hydrolysis of Hardwood

Polyethylene glycol (PEG) has been widely applied to improve enzymatic hydrolysis of lignocellulose by reducing enzyme nonproductive binding on lignins. To improve the performance of PEG in enzymatic hydrolysis, lignin-based surfactants were synthesized through lignin graft modification with poly(ethylene glycol) diglycidyl ether in this study. More importantly, effects of incorporating lignin into PEG on its behavior in enzymatic hydrolysis were investigated. Enzymatic hydrolysis of poplar results indicated that lignin-based surfactants exhibited better performance compared to PEG for promoting enzymatic hydrolysis. Moreover, a lower lignin-based surfactant loading was needed to achieve equal enzymatic hydrolysis. To explore the potential mechanism, changes of enzyme distribution and residual enzyme activities in supernatant after adding surfactants were determined. Quartz crystal microbalance with dissipation analysis was used to monitor the interaction between surfactants/enzyme and lignin in real-time. It revealed that the binding rates of lignin-based surfactants on lignin surfaces (1.06–1.32 min–1) were higher than those of PEG (0.43 min–1). Additionally, lignin-based surfactants respectively reduced the enzyme binding rates and maximum enzyme binding capacity on lignin by 36.7–40.9% and 43.3–64.9%, which was more significant than the reduction in enzyme binding rates and maximum enzyme binding capacity with PEG. A better understanding of this beneficial mechanism is a benefit for the design and utilization of surfactant in the lignocellulosic biorefinery process.

Surface structural features control in vitro digestion kinetics of bean starches

Although many studies have investigated the enzyme susceptibility of bean starches, the factors that reduce the lower hydrolysis of bean starch are not well understood. In the present study, we isolated starches from eight commercial bean varieties and established a causal relationship between micro and macro-structure with α-amylase susceptibility. The in vitro starch digestion kinetic profiles were monitored at a fixed enzyme concentration, and the data were fitted into the Logarithm of Slope model to obtain first-order rate coefficients and extent of digestion. Among eight varieties, pinto bean starch showed the lowest digestion rate (0.0013 min−1) and extent (40.7%), whereas red kidney bean starch has the highest rate (0.0042min−1) and extent (69.2%). In order to probe the interdependability of digestion kinetic parameters with starch microstructure, the enzyme binding capacity, molecular size, crystalline structure as well as thermal parameters of eight bean starches were correlated as Pearson's matrix. Whilst, significant correlation was not observed between digestion rate/extent and the structural parameters, the residual enzyme activity after binding with starch granules at non-hydrolyzing condition had significant negative correlation with the digestion rate and extent. Similarly, positive correlation was observed for degree of starch damage and enzyme susceptibility. Thus, it is concluded that for bean starches with similar biological origins, the available sites for initial binding of enzymes predominates the succeeding catalysis rate.

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica.

Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Zbasic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 μmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O2-dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports.

Characterization of major metabolites of polymethoxylated flavonoids in Pericarpium Citri Reticulatae using liver microsomes immobilized on magnetic nanoparticles coupled with UPLC/MS\u2013MS

The peels of citrus fruits (Pericarpium Citri Reticulatae, PCR) have long been used in traditional Chinese medicines (TCMs). Polymethoxylated flavonoids (PMFs) were found to be the main components present in PCR extracts, but their metabolism remains unclear which restrain the utilization of this TCM. In the present work, rat liver microsomes were immobilized on magnetic nanoparticles (LMMNPs) for in vitro metabolic study on the whole PMFs of PCR. LMMNPs were characterized by transmission electron microscope and Fourier-transform infrared spectrum. The relative enzyme binding capacity of LMMNPs was estimated to be about 428 μg/mg from thermogravimetric analysis. Incubation of LMMNPs with PMFs produced demethylated metabolites of PMFs, six of which were identified by ultrahigh pressure liquid chromatography–mass spectrometry (UPLC–MS/MS). The 3′-hydroxylated tangeretin (T3) was detected from the metabolites of tangeretin for the first time, which suggested that 4′-demethylated and 3′-hydroxylated derivative of tangeretin (3′-hydroxy-5,6,7,8,4′-pentamethoxyflavone, T4) was not only derived from 4′-demethylated tangeretin (T2) as previously reported, but also from T3. This is the first investigation of the metabolism of the whole PMFs, which may shed light on the mechanism of action of PCR.

Immobilization of cellulase on polyamidoamine dendrimer-grafted silica

► A new type of immobilized cellulase was developed based on PAMAM-grafted silica. ► The enzyme binding capacity increased with rising PAMAM generations. ► The properties of adsorption and crosslinking immobilization methods were compared. ► The prepared media possessed high enzymatic activity and exhibited better stability. ► The protocol can be potentially adapted to support other biomacromolecules. Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups, and is suitable to carry biomolecules due to its affinity and biocompatibility. In this study, PAMAM was grafted onto the surface of silica by microwave irradiation. A novel media was developed through immobilizing cellulase onto the prepared PAMAM-grafted silica by adsorption and crosslinking methods and applied in hydrolysis of carboxymethyl cellulose. The results demonstrate that the enzyme binding capacity and enzymolysis efficiency increased with generations of PAMAM. The properties of the immobilized cellulase-PAMAM-grafted silica were investigated, which possessed high enzymatic activity and exhibited better stability with respect to pH, temperature compared with free enzyme. The optimal immobilization conditions for adsorption and crosslinking method were respectively obtained at 5 and 4 mg ml −1 cellulase for 2 h of immobilization. A high enzymolysis efficiency was achieved by employing pH 4.8 and 5.8 substrate solution at 60 °C for adsorbed and crosslinked cellulase, respectively. After repeated three run cycles, the retained activities were found to be 75% and 82%. The results indicate that the PAMAM has a good performance as a carrier, and can be potentially adapted to support other biomacromolecules.

Preparation by suspension polymerization of porous beads for enzyme immobilization

AbstractPorous beads have been prepared by suspension polymerization of acrylic monomers and divinyl benzene in the presence of a porogen. The porosity varies greatly with the polymer composition, with the amount and type of porogen and with the conditions for the preparation. The enzyme binding capacity and activity has been determined for a lipase. The best results are found for crosslinked poly(methyl methacrylate) beads while other polyacrylates are less effective. Compositions based on poly(methacrylic acid) are not suitable for enzyme binding.

Site-directed mutagenesis of β-galactosidase ( E. coli) reveals that tyr-503 is essential for activity

By using the technique of site-directed mutagenesis we have succeeded in replacing tyr-503 of beta-galactosidase (E. coli) with a phe. A study of the kinetic and stability properties of this mutant enzyme (F-503 beta-galactosidase) showed that the loss in activity upon this change is due to the loss of a catalytic group (rather than a detrimental change in the enzyme's overall structure or a change in the enzyme's binding capacity). This confirms previous suggestions that this tyr residue is involved in catalysis.

Isolation and characterization of 3-hydroxyacyl coenzyme A dehydrogenase-binding protein from pig heart inner mitochondrial membrane.

3-Hydroxyacyl coenzyme A (CoA) dehydrogenase-binding protein was solubilized from inner mitochondrial membrane by using taurodeoxycholate at high ionic strength. The binding protein was isolated from the suspension using 3-hydroxyacyl-CoA dehydrogenase affinity chromatography. The protein eluted from the affinity column had a molecular weight of approximately 150,000, as determined by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protein is a dimer consisting of 69,000 and 71,000 molecular weight subunits. The enzyme binding capacity of this protein was tested with a polyethylene glycol precipitation method: 0.5 mg of enzyme could be precipitated together with 1 mg of binding protein, showing that 1 mol of binding protein binds 1 mol of enzyme. This protein had no affinity toward malic dehydrogenase, citrate synthase, and fumarase. The approximately 2-fold increase in the 3-hydroxyacyl-CoA dehydrogenase activity when it was measured in the presence of the binding protein is additional evidence of enzyme-binding protein interaction. When incorporated into liposomes, the binding protein retained its ability to bind 3-hydroxyacyl-CoA dehydrogenase, but did not bind malic dehydrogenase, citrate synthase, and fumarase. These results suggest that the protein isolated by us has a specific function in anchoring a beta-oxidation enzyme to the matrix surface of the mitochondrial membrane.

Immobilization of Enzymes on Polychlorotrifluoroethylene Particles Packed in Small Columns

Abstract The use of polychlorotrifluoroethylene (PCTFE) particles packed in 5–10 cm × 0.4 cm ID columns for the immobilization of enzymes by hydrophobic adsorption is investigated. Enzyme binding capacity of PCTFE is about 0.2 mg/gm of polymer. PCTFE-immobilized lactate dehydrogenase, alcohol dehydrogenase, and urease-glutamate dehydrogenase are demonstrated to be useful for the determinations of pyruvate, ethanol, and urea, respectively. Immobilized enzyme lifetimes are generally about 1–2 months.

Sodium Dodecyl Sulfate Inactivation of Bovine Liver Glutamate Dehydrogenase

Abstract Inactivation of bovine liver glutamate dehydrogenase by the anionic detergent, sodium dodecyl sulfate, occurs by two different mechanisms, depending upon detergent concentrations: (a) uncompetitive inhibition and (b) denaturation. Uncompetitive inhibition kinetic data with sodium dodecyl sulfate (0 to 0.1 mm) indicate detergent combination with enzyme-NADP+-glutamate complex. Use of a structural analogue of glutamate indicated that sodium dodecyl sulfate did not interfere with binding of substrate to free enzyme. Dialysis of 0.1 mm sodium dodecyl sulfate-treated solutions of enzyme, enzyme-NADP+ complex, and enzyme-glutamate complex completely reversed inhibition, whereas dialysis of enzyme-NADP+-glutamate-detergent complex did not restore activity. A modified Hill plot of enzyme Vm values versus detergent concentrations showed that cooperativity is involved in uncompetitive inhibition by sodium dodecyl sulfate. Denaturation of glutamate dehydrogenase occurs upon treatment with higher concentrations of sodium dodecyl sulfate, as evidenced by loss of protein fluorescence, loss of enzyme binding capacity for NADH and NADPH, and protein dissociation. Surface active or micellar properties of sodium dodecyl sulfate may be involved in denaturation, since surface tension measurements may have indicated mixed protein-detergent micelle formation at 0.12 mm sodium dodecyl sulfate.