Conversion of cellobiose to glucose using immobilized β-glucosidase reactors

Cellobiose is an intermediate in the enzymatic hydrolysis of cellulose to glucose and acts as an inhibitor for the cellulase enzymes. The conversion of cellobiose to glucose was studied with β-glucosidase adsorbed on Amberlite DP-1, a cation-exchange resin. The best overall pH for adsorption and reactor operation was near 5.0. The K_m values increased with increasing enzyme loading due to competitive inhibition. The maximum practical enzyme loading was about 28 units/g resin. The immobilized enzyme was operated continously in both packed bed and fluidized bed reactors, giving half-lives between 200 and 375 h.     

Monitoring denaturation behaviour and comparative stability of DNA triple helices using oligonucleotide–gold nanoparticle conjugates

Gold nanoparticle labels, combined with UV-visible optical absorption spectroscopic methods, are employed to probe the temperature-dependent solution properties of DNA triple helices. By using oligonucleotide–nanoparticle conjugates to characterize triplex denaturation, for the first time triplex to duplex melting transitions may be sensitively monitored, with minimal signal interference from duplex to single strand melting, for both parallel and antiparallel triple helices. Further, the comparative sequence-dependent stability of DNA triple helices may also be examined using this approach. Specifically, triplex to duplex melting transitions for triplexes formed using oligonucleotides that incorporate 8-aminoguanine derivatives were successfully monitored and stabilization of both parallel and antiparallel triplexes following 8-aminoguanine substitutions is demonstrated.     

The effect of nanoparticle size on endocytosis dynamics depends on membrane–nanoparticle interaction

Molecular simulation studies on the interaction between nanoparticles (NPs) and cell membranes have been limited by small NP size of several nanometres. In this work, by using a simplified lipid model, we study the endocytosis of large NPs with a size being enlarged to 37.5 nm. It is found that the effect of NP size on endocytosis dynamics depends on the membrane–NP interaction. As the interaction strength between NP and lipid changes, different wrapping modes are observed. For the system with weak membrane–NP attraction, the wrapping process is controlled by the membrane bending, and thus large size of NPs (within the range of NP size we studied) would promote the wrapping dynamics. While for the case with strong membrane–NP adhesion, the wrapping process is dominated by lipid diffusion and small NPs show a larger wrapping rate. In this wrapping mode, the membrane–NP adhesion drives small NPs to move towards the membrane as the wrapping process proceeds. For relatively larger NPs, however, the membrane moves towards the NPs instead. We also find that for the second wrapping mode, the rapid wrapping rate, especially with the hydrophobic ligands on the hydrophilic NP would impose significant perturbations on membrane stability, and consequently, membrane pores may be induced during the process of NP endocytosis.     

Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V₀) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219?mM and 116?mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336?h of reaction, the OG yield and the conversion rate of glucose reached 134?mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.     

The antioxidant and antiaggregant effects of the covalent bienzyme superoxide dismutase — Chondroitin sulfate — Catalase conjugate on platelets

A covalent bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate (SOD-CHS-CAT) demonstrated the dose-dependent inhibitory action on induced platelet aggregation both in the presence and the absence of hydrogen peroxide. The antioxidant activity of SOD-CHS-CAT was noted at much lower doses than that of other CAT derivatives. The conjugate SOD-CHS-CAT inhibited platelet aggregation regardless of the nature of inducers; it also prevented platelet spreading on the glass surface. The latter suggests conjugate influence on adhesive properties of platelets. Novel properties of the SOD-CHS-CAT conjugate underlie prospects of its biopharmaceutical applicability as a tool for antioxidant therapy.     

Predicting the performance of immobilized enzyme reactors using reversible Michaelis–Menten kinetics

A general mathematical model is developed in the present work for predicting the steady state performance of immobilized enzyme reactor performing reversible Michaelis - Menten kinetics. The model takes into account the effect of external diffusional limitations, the axial dispersion and the equilibrium constant on reactor performance quantified as relative substrate conversion and yield. The performance of reactor is characterized using the dimensionless parameters of Damkohler number, Stanton number, Peclet number, the equilibrium constant and the dimensionless input substrate concentration. The reactor performance is described for the two extreme cases of plug flow reactor (PFR) and continuous stirred tank reactor (CSTR) in addition to the intermediate case of dispersed plug flow reactor (DPFR). The performance of reactor is compared for the two cases of zero order and reversible first order kinetics.     

Detection of Pb²⁺ at attomole levels by using dynamic light scattering and unmodified gold nanoparticles

Lead ion (Pb²⁺) accumulation in nature can affect the environment and human health severely. Thus, rapid and sensitive detection is of great importance. One-step detection of Pb²⁺ at attomole levels was realized by using dynamic light scattering (DLS) technique coupled with unmodified gold nanoparticles (AuNPs). Pb²⁺-dependent DNAzyme was double-stranded and could not adsorb on the surface of AuNPs, while the substrate strand could be cleaved into ssDNA fragments on addition of Pb²⁺. The ssDNA fragments could adsorb on the surface of AuNPs and prevent them from aggregating in the presence of NaCl. Therefore, the disperse state of AuNPs changed on addition of Pb²⁺ in the presence of DNAzyme and NaCl, which was estimated with an average hydrodynamic diameter by using DLS. Under optimum conditions, the average diameter of the solution decreased linearly with the concentration of Pb²⁺ over the range from 10 to 300 pM, with a detection limit of 6.2 pM. Moreover, satisfactory results were obtained when the proposed method was applied in the detection of Pb²⁺ in water samples.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Detection of protein–ligand interactions by NMR using reductive methylation of lysine residues

We show that reductive methylation of proteins can be used for highly sensitive NMR identification of conformational changes induced by metal- and small molecule binding, as well as protein-protein interactions. Reductive methylation of proteins introduces two (13)C-methyl groups on each lysine in the protein of interest. This method works well even when the lysines are not actively involved in the interaction, due to changes in the microenvironments of lysine residues. Most lysine residues are located on the protein exterior, and the exposed (13)C-methyl groups may exhibit rapid localized motions. These motions could be faster than the tumbling rate of the molecule as a whole. Thus, this technique has great potential in the study of large molecular weight systems which are currently beyond the scope of conventional NMR methods.     

A response surface methodological study on prediction of glucosylation yields of thiamin using immobilized β-glucosidase

Response surface methodology was used to predict the glucosylation yields of thiamin using immobilized β-glucosidase. A Central Composite Rotatable Design (CCRD) of 32 experiments with immobilized β-glucosidase, thiamin, incubation period, buffer concentration and pH, as five independent variables was employed at five levels. A second-order polynomial equation was developed, the regression coefficient values of which exhibited a R² value of 0.74. Contour plots explained the glucosylation behaviour of the enzyme through a reversal in glucosylation at a cross-over point corresponding to 60% (w/w d-glucose) immobilized β-glucosidase and 0.12 mM buffer concentration at pH 6. The highest conversion yield of 58% obtained experimentally compared well with the predicted yield of 52% under optimum conditions of 40% immobilized β-glucosidase, 0.55 mmol thiamin, 96 h incubation period and 0.16 mM buffer concentration at pH 7. Validation experiments carried out at certain random predictive conditions also showed good correspondence between experimental and predictive yields.     

The treatment of chlorophenols with laccase immobilized on sol–gel-derived silica

Laccase, a so-called “blue-copper” oxidase, is able to oxidize a variety of organic compounds. Sol–gel derived silica glasses are frequently adopted as an immobilization method to improve the stability of enzymes and make them reusable. In this study, immobilization conditions were optimized to achieve improved embedding results. The thermal stability, reaction stability and storage stability were improved with the immobilized enzyme when compared to the free enzyme. 2,4-Dichlorophenol (DCP) and 2,4,6-trichlorophenol (TCP) were chosen as model compounds. The treatment of chlorophenols (CPs) by immobilized laccase demonstrated excellent removal and response stability. The affinity of TCP for immobilized laccase was higher than that of DCP. This finding leads to different removal efficiencies under variable conditions (reaction time, initial concentration, dosage of immobilized laccase and removal rate in mixed solution). By fitting the experimental data with the diffusion model of the degradation process, the degradation of CPs by immobilized laccase matches an intraparticle diffusion-controlled model.     

Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed.     

The effect of cyclic anaerobic–aerobic conditions on biodegradation of azo dyes

The effect of cyclic anaerobic–aerobic conditions on the biodegradative capability of the mixed microbial culture for the azo dye Remazol Brilliant Violet 5R (RBV-5R) was investigated in the sequencing batch reactor (SBR) fed with a synthetic textile wastewater. The SBR had a 12-h cycle time with anaerobic–aerobic periods of 3/9, 6/6 and 9/3 h. General SBR performance was assessed by measurement of catabolic enzymes (catechol 2,3-dioxygenase, azo reductase), chemical oxygen demand (COD), color and amount of aromatic amines. In this study, under steady-state conditions, the anaerobic period of the cyclic SBR was found to allow the reductive decolorization of azo dye. Longer anaerobic periods resulted in higher color removal efficiencies, approximately 71% for the 3-h, 87% for 6-h and 92% for the 9-h duration. Total COD removal efficiencies were over 84% under each of the cyclic conditions and increased as the length of the anaerobic period was increased; however, the highest color removal rate was attained for the cycle with the shortest anaerobic period of 3 h. During the decolorization of RBV-5R, two sulfonated aromatic amines (benzene based and naphthalene based) were formed. Additionally, anaerobic azo reductase enzyme was found to be positively affected with the increasing duration of the anaerobic period; however; it was vice versa for the aerobic catechol 2,3-dioxygenase (C23DO) enzyme.     

Sensitive electrochemical immunosensor for α-synuclein based on dual signal amplification using PAMAM dendrimer-encapsulated Au and enhanced gold nanoparticle labels

A novel electrochemical immunosensor for sensitive detection of α-synuclein (α-SYN), a very important neuronal protein, has been developed based on dual signal amplification strategy. Herein, G4-polyamidoamine dendrimer-encapsulated Au nanoparticles (PAMAM-Au nanocomposites) were covalently bound on the poly-o-aminobenzoic acid (poly-o-ABA), which was initially electropolymerized on the electrode surface to perform abundant carboxyl groups. The formed immunosensor platform, PAMAM-Au, was proved to provide numerous amino groups to allow highly dense immobilization of antigen, and facilitate the improvement of electrochemical responses as well. Subsequently, the enhanced gold nanoparticle labels ({HRP-Ab(2)-GNPs}) were fabricated by immobilizing horseradish peroxidase-secondary antibody (HRP-Ab(2)) on the surface of gold nanoparticles (GNPs). After an immunoassay process, the {HRP-Ab(2)-GNPs} labels were introduced onto the electrode surface, and produced an electrocatalytic response by reduction of hydrogen peroxide (H(2)O(2)) in the presence of enzymatically oxidized thionine. On the basis of the dual signal amplification of PAMAM-Au and {HRP-Ab(2)-GNPs} labels, the designed immunosensor displayed an excellent analytical performance with high sensitivity and stability. This developed strategy was successfully proved as a simple, cost-effective method, and could be easily extended to other protein analysis schemes.     

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.     

Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

The use of oligonucleotide–protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide–protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.     

Synthesis of geranyl propionate in a solvent-free medium using Rhizomucor miehei lipase covalently immobilized on chitosan–graphene oxide beads

The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan–graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box–Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76?±?0.02% of recovered protein had yielded 7.2?±?0.04?mg?g^?1 and 211?±?0.3%?U?g^?1 of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20?hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70?rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.     

The continuous hydrolysis of geniposide to genipin using immobilized β-glucosidase on calcium alginate gel

Summary The reaction velocity of immobilized -glucosidase was approximated by the first-order reaction kinetics. A plug flow reactor was used for continuous hydrolysis of geniposide with this immobilized enzyme. The activity of this immobilized enzyme was retained 100% for 600 h. The amount of genipin formed by using the immobilized enzyme was 17 fold that formed using the native enzyme without reuse. Using immobilized enzyme, purity and yield of genipin, which is a hydrolyzate of geniposide, was improved comparing with the native enzyme.     

Analytical basis for the determination of the lacunar–canalicular permeability of bone using cyclic loading

An analytical model for the determination of the permeability in the lacunar-canalicular porosity of bone using cyclic loading is described in this contribution. The objective of the analysis presented is to relate the lacunar-canalicular permeability to a particular phase angle that is measurable when the bone is subjected to infinitesimal cyclic strain. The phase angle of interest is the lag angle between the applied strain and the resultant stress. Cyclic strain causes the interstitial fluid to move. This movement is essential for the viability of osteocytes and is believed to play a major role in the bone mechanotransduction mechanism. However, certain bone fluid flow properties, notably the permeability of the lacunar-canalicular porosity, are still not accurately determined. In this paper, formulas for the phase angle as a function of permeability for infinitesimal cyclic strain are presented and mathematical expressions for the storage modulus, loss modulus, and loss tangent are obtained. An accurate determination of the PLC permeability will improve our ability to understand mechanotransduction and mechanosensory mechanisms, which are fundamental to the understanding of how to treat osteoporosis, how to cope with microgravity in long-term manned space flights, and how to increase the longevity of prostheses that are implanted in bone tissue.     

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.