Enzyme Adsorption Research Articles

Biodegradation of poly(3-hydroxybutyrate)-derived polymers with different 4-hydroxybutyrate fractions by a novel depolymerase from Paecilomycessp. 1407

A poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)]-degrading strain was isolated and identified as Paecilomyces sp. A novel P(3HB-co-4HB) depolymerase with a molecular weight of 55 kDa was purified by column chromatography from the culture supernatant of the strain. The purified depolymerase showed optimum activity at pH 6.0 and 30 °C, and it was stabilized at pH 4.0–7.0 and below 40 °C. The depolymerase degraded poly(3-hydroxybutyrate) (PHB), poly(hydroxybutyrate-co-hydroxyvalerate), and several short-chain-length esters other than P(3HB-co-4HB) but showed no remarkable activity on poly(ε-caprolactone) and poly(L-lactic acid). The main enzymatic products comprised hydroxybutyrate monomers, including 3HB and 4HB. The enzymatic erosion rate on the films increased markedly with increasing fraction of 4HB units. Enzymatic hydrolysis processes of PHB and P(3HB-co-4HB) were systematically analyzed in three aspects: enzyme adsorption, single-polymer-chain dissociation, and chemical linkage breakdown. Results indicate that 4HB insertion caused no effect on the degradation of solid-phase PHB-derived material at the molecular chain level but affected enzyme adsorption and crystallinity of the materials. Hindering single-chain stripping by crystallinity dominantly affects the degradation behavior of the polymer.

Microstructural changes in the cell wall and enzyme adsorption properties of lignocellulosic biomass subjected to thermochemical pretreatment

In this study, yellow poplar, as a raw material, was subjected to pretreatment with oxalic and sulfuric acid. We then investigated the characteristics of lignin degradation and enzyme adsorption in terms of microstructural changes, depending on the pretreatment conditions. Statistical analysis revealed that the glucan and lignin content of the sulfuric acid- and oxalic acid-pretreated biomass did not differ under the same pretreatment conditions, irrespective of the catalyst used. In contrast, difference in the degradation characteristics of lignin in the cell wall (secondary cell wall) and different cell types (vessel and wood fiber) of pretreated biomass were observed by confocal laser scanning microscopy and time of flight-secondary ion mass spectrometry (ToF-SIMS). ToF-SIMS revealed high enzyme adsorption in the middle lamella of wood fibers in the oxalic acid-pretreated biomass. In the sulfuric acid-pretreated biomass, enzyme adsorption was high in the secondary cell wall of wood fibers. G-type lignin showed a high enzyme adsorption capacity. Therefore, irreversible enzyme adsorption, particularly at the middle lamella, was higher in the oxalic acid-pretreated biomass than in the sulfuric acid-pretreated biomass.

Atomistic Details of Chymotrypsin Conformational Changes upon Adsorption on Silica.

Adsorption of enzymes on solid surfaces may lead to conformational changes that reduce their catalytic conversion activity and are thus detrimental to the efficiency of biotechnology or biosensing applications. This work is a joint theoretical and experimental endeavor in which we identify and quantify the conformational changes that chymotrypsin undergoes when in contact with the surface of amorphous silica nanoparticles. For this purpose, we use circular dichroism spectroscopy, standard molecular dynamics, and advanced-sampling methods. Only the combination of these techniques allowed us to pinpoint a destabilization effect of silica on specific structural motifs of chymotrypsin. They are linked by the possibility of theoretically predicting CD spectra, allowing us to elucidate the source of the experimentally observed spectral changes. We find that chymotrypsin loses part of its helical content upon adsorption, with minor perturbation of its overall tertiary structure, associated with changes in the aromatic interactions. We demonstrate that the C-terminal helical fragment is unfolded as an isolated oligopeptide in pure water, folded as an α-helix as terminus of chymotrypsin in solution, and again partly disordered when the protein is adsorbed on silica. We believe that the joint methodology introduced in this manuscript has a direct general applicability to investigate any biomolecule-inorganic surface system. Methods to theoretically predict circular dichroism spectra from atomistic simulations were compared and improved. The drawbacks of the approaches are discussed; in particular, the limited capability of advanced-sampling MD schemes to explore the conformational phase space of large proteins and the dependency of the predicted ellipticity bands on the choice of calculation parameters.

Single-step purification of peroxidases from rice bran: Evaluation of the main expanded-bed chromatography parameters

Peroxidases catalyze the oxidation-reduction reactions of organic substrates and present several applications in the biotechnological, clinical, environmental, and industrial areas. Among the chromatographic strategies applied to peroxidase purification to allow future uses, expanded-bed adsorption (EBA) stands out due to the large volumes of purified enzyme and potential for scale-up. In this paper, the main EBA parameters were evaluated in the purification of rice bran peroxidases. Sodium acetate buffer (pH 4.5) and expansion degree of 2.5 were the most favorable conditions to the enzyme adsorption onto Streamline® SP resin, showing an enzymatic activity at equilibrium on the solid phase (q*) of 2.68 U/mL resin, a partition coefficient (f) of 38.35 and a dynamic binding capacity (Q10%) of 0.19 U/mL resin. Rice bran peroxidase was purified in 14.1-fold (± 1.3) with 49.8% (± 2.5) of recovery by using 25 mmol/L acetate buffer pH 5.5 in the washing step, and elution combining stepwise (0.15 mol/L NaCl) and linear gradient (0.15–1 mol/L NaCl), using acetate buffer pH 5.5 with 1 mmol/L of CaCl2. Based on the results obtained, EBA showed to be a viable single-step strategy for peroxidase purification.

The role of enzyme adsorption in the enzymatic degradation of an aliphatic polyester

The enzyme catalyzed degradation of poly(3-hydroxybutyrate) (PHB) is a two-step process consisting of the adsorption of the enzyme on the surface of a PHB substrate and the cleavage of ester bonds. A deactivated enzyme was prepared by point mutagenesis to separate the two steps from each other. Measurements carried out with active and inactive enzymes on PHB particles proved that mutagenesis was successful and the modified enzyme did not catalyze degradation. Based on the Michaelis-Menten approach a kinetic model was proposed which could describe the processes quantitatively, the agreement between prediction and the measured data was excellent. The separation of the two processes allowed the determination of the adsorption kinetics of the enzyme; the rate constants of the adsorption and desorption process were determined for the first time. Comparison of these constants to reaction rates showed that adsorption is not instantaneous and can be the rate-determining step. The area occupied by an enzyme molecule was also determined (13.1 nm2) and it was found to be smaller than the value published in the literature (17 ± 8 nm2). The separation of the two steps makes possible the prediction and control of the degradation process.

An aggregated understanding of cellulase adsorption and hydrolysis for ball-milled cellulose

This study evaluated the effects of physicochemical properties of a series of ball-milled cellulose on cellulase adsorption and glucose yield. The relationship between cellulase adsorption and initial hydrolysis rate was also discussed. We found that hydrophobicity and surface charge are the key factors affecting cellulase adsorption on ball-milled cellulose. The results demonstrated that glucose yield had a positive correlation with specific surface area, while showed a negative correlation with particle size, degree of polymerization and crystallinity. Among these properties, specific surface area and crystallinity are the key factors affecting glucose yield. As ball milling progressed, cellulose showed lower enzyme adsorption capacity/amount of bound enzyme during initial stage of hydrolysis, but had higher initial hydrolysis rate. The enhanced rate is attributed to the fact that the amorphous region produced by ball milling reduces the free energy required for decrystallization thus increases the catalytic efficiency of the bound enzyme.

Enhanced Direct Electron Transfer of Fructose Dehydrogenase Rationally Immobilized on a 2-Aminoanthracene Diazonium Cation Grafted Single-Walled Carbon Nanotube Based Electrode

In this paper, an efficient direct electron transfer (DET) reaction was achieved between fructose dehydrogenase (FDH) and a glassy-carbon electrode (GCE) upon which anthracene-modified single-walled carbon nanotubes were deposited. The SWCNTs were activated in situ with a diazonium salt synthesized through the reaction of 2-aminoanthracene with NaNO2 in acidic media (0.5 M HCl) for 5 min at 0 °C. After the in situ reaction, the 2-aminoanthracene diazonium salt was electrodeposited by running cyclic voltammograms from +1000 to −1000 mV. The anthracene-SWCNT-modified GCE was further incubated in an FDH solution, allowing enzyme adsorption. Cyclic voltammograms of the FDH-modified electrode revealed two couples of redox waves possibly ascribed to the heme c1 and heme c3 of the cytochrome domain. In the presence of 10 mM fructose two catalytic waves could clearly be seen and were correlated with two heme cs (heme c1 and c2), with a maximum current density of 485 ± 21 μA cm–2 at 0.4 V at a sweep rate of 10 mV ...

Development and characterization of bentonite/wGLP systems

Bentonite (Bent) clay is a component of soil with useful properties for enzyme adsorption. In this work, the previously characterized antioxidant superoxide dismutase (SOD) enzyme named wGLP by wheat germin-like protein was immobilized in raw (Bent) and modified bentonites (Bent-DDA). The physicochemical characterizations of both supports were carried out. Successful adsorption of wGLP onto Bent and Bent-DDA occurred on the surface and within the interlayer spacing as was revealed by X-Ray Diffraction (XRD) and Thermogravimetric Analysis (TGA). The release of wGLP at different times and pHs was tested. While wGLP remained almost totally immobilized into Bent-DDA up to 96 h, it was released from Bent to reach nearly 60% after 72 h at pH 7.5 and preserving its SOD activity. Since tomato cell viability under the presence of Bent-wGLP was maintained, Bent-wGLP complexes are potential carriers of antioxidants in tomato cell suspension cultures.

Chemically modified alginate bead matrix for efficient adsorptive recovery of trypsin from fresh bovine pancreas.

The adsorption of commercial trypsin (Try) onto epichlorohydrin cross-linked alginate-guar gum matrix has been studied at equilibrium in batch and in fixed bed column. Experiments were conducted to study the effect of ionic strength, temperature and to obtain a thermodynamic characterization of the adsorption process. The resulting adsorption isotherm fitted the Hill equation. Experimental breakthrough curve profiles were compared with the theoretical breakthrough profiles obtained from the mathematical model, bed depth service time. At pH 5.0, 1.0 g hydrated matrix adsorbed 480.0 milligram of Try per gram of dried bed. The desorption process showed 80% of Try recovery in 50 mM phosphate buffer, pH 7.00-500 mM NaCl-20% propylene glycol. The obtained results were applied to an adsorption/washing/desorption process with fresh pancreas homogenate yielded 20% of recovery and 5.7 purification factor of Try. The matrix remained functional until the fifth cycle of repeated batch enzyme adsorption. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018.

Enhanced enzymatic digestibility of mixed wood sawdust by lignin modification with naphthol derivatives during dilute acid pretreatment

Effects of the addition of 2-naphthol and 2-naphthol-7-sulfonate on the dilute acid pretreatment of mixed wood sawdust were investigated, respectively. Compared to 2-naphthol, 2-naphtnol-7-sulfonate was more effective to enhance delignification and facilitate the enzymatic hydrolysis. The 72 h hydrolysis yield was improved by 47.8% for 2-naphthol-7-sulfone, while only 9.1% was observed for 2-naphthol. The surface charges, enzyme adsorption, and cellulose accessibility of dilute acid pretreated substrates with or without naphthol derivatives were examined. The improved enzymatic hydrolysis by adding 2-naphthol-7-sulfonate was ascribed to the higher negative surface charges, the lower enzyme non-productive binding, and the higher cellulose accessibility of pretreated substrates. Additionally, the HSQC NMR and 31P NMR analysis were carried out on both decomposed lignins and residual bulk lignins. It indicated that the addition of the naphthol derivatives during pretreatment could suppress the lignin repolymerization, which further mitigated the inhibition of residual lignins on enzymatic hydrolysis.

The use of magnetic nanoparticles for immobilization and recycling of enzymes

ABSTRACTThe use of nanostructured materials for enzyme immobilization is an active field of research due to its large surface area and the new emergent properties derived from its size. The present work is focused on the synthesis of magnetic nanoparticles for the adsorption of cysteine-proteolytic enzymes extracted from Bromelia antiacantha Bertol (Bromeliaceae) fruit. The results show that enzyme adsorption is highly dependent on the temperature and pH. The biocatalyst activity increased up to 40 %, once immobilized onto the magnetic nanoparticles. In addition, they can be recovered using a magnet allowing them to be reused up to 5 cycles with a marginal loss (5 %) of the initial activity.

Stepwise pretreatment of aqueous ammonia and ethylenediamine improve enzymatic hydrolysis of corn stover

It is a trade-off between sugar loss in pretreatment and sugar release in hydrolysis for most pretreatment. Here, a stepwise mild pretreatments of aqueous ammonia pretreatment and ethylenediamine was developed to reduce the sugar loss during pretreatment process and improve the sugar release in enzymatic hydrolysis of corn stover. Among four parameters of stepwise pretreatment (NH3 loading, aqueous ammonia pretreatment time, ethylenediamine loading and ethylenediamine pretreatment time), aqueous ammonia pretreatment time and ethylenediamine loading played the key roles in the improving enzymatic hydrolysis. More than 95% glucan and 85% xylan were reserved in the pretreated solid after stepwise pretreatment. The glucan and xylan conversion reached 94.18% and 72.81%, respectively, with a low enzyme loading of 10 mg cellulase/g glucan. The characterization of the pretreated solid suggested that in stepwise pretreatment process, aqueous ammonia pretreatment increased the specific surface area of pretreated solid by 26.4%, while ethylenediamine pretreatment resulted in the collapse of the biomass porous structure and hence reduced the specific surface area. Crystalline index of the biomass increased after aqueous ammonia pretreatment, while the crystalline index was decreased from 53.22% to 32.13% after ethylenediamine pretreatment. Results implied that aqueous ammonia pretreatment enhanced productive enzyme adsorption on biomass mainly by increasing specific surface area, while ethylenediamine pretreatment improved productive enzyme adsorption by the coalescence and re-localization of lignin to expose more accessible surface. As a result, these interactive effects provided by the stepwise pretreatment improved the accessibility of the pretreated corn stover to enzymes and thus increased sugar release significantly.

Comparison of catalytically non-productive adsorption of fungal proteins to lignins and pseudo-lignin using isobaric mass tagging

Catalytically non-productive adsorption of fungal enzymes to pseudo-lignin (PL) was compared to adsorption to lignin preparations derived from different sources (SL, spruce; BL, birch; OL, beech) using different methods [steam pretreatment/enzymatic saccharification (SL, BL) and organosolv processing (OL)]. The protein adsorption to the SL was more extensive than the adsorption to the hardwood lignins, which was relatively similar to the adsorption to the PL. The adsorption patterns of 13 individual proteins were studied using isobaric mass tagging with TMTsixplex reagent and LC–MS/MS analysis. The results suggest that, on an average, adsorption of proteins equipped with carbohydrate-binding modules, such as the cellulases CBHI, EGII, and EGIV, was less dependent on the quality of the lignin/PL than adsorption of other proteins, such as β-Xyl, Xyn-1, and Xyn-2, which are involved in xylan degradation.

Modelling hyaluronan degradation by streptococcus pneumoniae hyaluronate lyase

Hyaluronic acid (Hyaluronan) is a linear, high molecular weight polysaccharide that forms an important component of the extracellular matrix. It is an excellent biomaterial, and it is increasingly being used in biotechnology, biomedical applications, and drug delivery. Polymer chains of hyaluronan occur in many different lengths in nature, and can be as large as multiples of ten thousand. Since the biological function of a hyaluronan chain often depends on its molecular weight, it is of value for applications to develop reliable quantitative descriptions of the degradation processes of hyaluronan. In particular, the development of such models should assist with the rational design of production processes to create polymer chains in a given molecular weight category for a specific application. In this paper, we propose a new mathematical model for the degradation of hyaluronan by the enzyme streptococcus pneumoniae hyaluronate lyase. The model is based on a processive kinetic mechanism and consists of a coupled system of nonlinear ordinary differential equations for the species of interest. The model parameters are estimated using published experimental data, and good agreement between theory and experiment is found. Numerical experimentation and a Sobol global sensitivity analysis reveal that the key model parameters are the initial enzyme concentration and the rate constants for enzyme adsorption and catalysis.

Ethylenediamine pretreatment of corn stover facilitates high gravity fermentation with low enzyme loading

This work investigated the effect of ethylenediamine pretreatment on reducing enzyme loading in high gravity fermentation. At optimal conditions of ethylenediamine pretreatment, 85.5% lignin was removed. Enzyme adsorption analysis using a fluorescent cellulose-binding protein showed 35.2% increase of productive adsorption of enzymes to ethylenediamine pretreated biomass, which was caused by high delignification and dramatically increased surface roughness and porosity. In SScF at 15% glucan loading, up to 82.2 g/L ethanol was achieved with a relatively low enzyme loading of 3.6 FPU/g dry matter. It suggested that the remarkably high digestibility of EDA pretreated corn stover could effectively reduce the enzyme loading in the high gravity fermentation of cellulosic ethanol.

Alternative Low-Cost Additives to Improve the Saccharification of Lignocellulosic Biomass.

A potential strategy to mitigate problems related to unproductive adsorption of enzymes onto lignin during the saccharification of lignocellulosic biomass is the addition of lignin-blocking agents to the hydrolysis reaction medium. However, there is a clear need to find more cost-effective additives for use in large-scale processes. Here, selected alternative low-cost additives were evaluated in the saccharification of steam-exploded sugarcane bagasse using a commercial enzymatic cocktail. The addition of soybean protein, tryptone, peptone, and maize zein had positive effects on glucose release during the hydrolysis, with gains of up to 36% when 8% (w/w) soybean protein was used. These improvements were superior to those obtained using bovine serum albumin (BSA), a much more expensive protein that has been widely reported for such an application. Moreover, addition of soybean protein led to a saving of 48h in the hydrolysis, corresponding to a 66% decrease in the reactor operation time required. In order to achieve the same hydrolysis yield without the soybean additive, the enzyme loading would need to be increased by 50%. FTIR spectroscopy and nitrogen elemental analysis revealed that the additives probably acted to reduce unproductive binding of cellulolytic enzymes onto the lignin portion of the sugarcane bagasse.

Reusable anionic sulfonate functionalized nanofibrous membranes for cellulase enzyme adsorption and separation.

Poly (vinyl alcohol-co-ethylene) nanofibrous membranes (PVA-co-PE NFM) were successfully modified by sodium-3-sulfobenzoate to become negatively charged with sulfonate groups, and the sulfonated (PVA-co-PE) nanofiber membrane SS (PVA-co-PE NFM) was used in non-covalent adsorption of cellulases via electrostatic attraction. The modified NFM showed excellent adsorption to the enzyme molecules due to the incorporated static charge interaction with the fibers, high open-porosity and ultrahigh surface areas of the nanofibers. Such unique morphology and chemical structures lead to the adsorption capacity of 130 mg g-1 and reusability for 5 cycles without significant change in catalytic functions. The morphology changes of the nanofibrous membranes were observed by using a scanning electron microscopy, and chemical structures of the membranes were characterized by using FTIR and water contact angle measurements. SS (PVA-co-PE NFM) is a promising solid support media for enzyme immobilization, and the immobilized enzymes can be applied in industrial applications.

Precipitation-Based Nanoscale Enzyme Reactor with Improved Loading, Stability, and Mass Transfer for Enzymatic CO2 Conversion and Utilization

Enzymatic CO2 conversion has gathered a growing attention due to its fast kinetics in converting CO2 to bicarbonate, but the carbonic anhydrase enzymes easily lose their activities in CO2 conversion processes. Here, we propose a “precipitation-based nanoscale enzyme reactor (p-NER)” approach, which stabilizes the activity of carbonic anhydrase, prepared via the two steps of enzyme adsorption into magnetic mesoporous silica and simultaneous enzyme precipitation/cross-linking. The simple addition of enzyme precipitation during cross-linking step resulted in the formation of cross-linked enzyme aggregates (CLEAs) not only inside the mesopores but also on the surface of mesoporous silica. External CLEAs of p-NER contributed to the improvement of enzyme loading (32.9% (w/w)) and mass transfer (KM = 3.68 mM) compared to those of NER (20.1% (w/w) and 4.29 mM, respectively), prepared without enzyme precipitation step and showing no external CLEAs. p-NER was stable under vigorous shaking (200 rpm) with no activity...

An enzymatic system for decolorization of wastewater dyes using immobilized CueO laccase-like multicopper oxidase on poly-3-hydroxybutyrate.

SummaryThe presence of synthetic dyes in wastewaters generated by the textile industry constitutes a serious environmental and health problem that urges the scientific community on an appropriate action. As a proof‐of‐concept, we have developed a novel approach to design enzymatic bioreactors with the ability to decolorize dye solutions through the immobilization of the bacterial CueO laccase‐like multicopper oxidase from Escherichia coli on polyhydroxybutyrate (PHB) beads by making use of the BioF affinity tag. The decolorization efficiency of the system was characterized by a series of parameters, namely maximum enzyme adsorption capacity, pH profile, kinetic constants, substrate range, temperature and bioreactor recycling. Depending on the tested dye, immobilization increased the catalytic activity of CueO by up to 40‐fold with respect to the soluble enzyme, reaching decolorization efficiencies of 45–90%. Our results indicate that oxidase bioreactors based on polyhydroxyalkanoates are a promising alternative for the treatment of coloured industrial wastewaters.

Adsorptive immobilization of agro-industrially produced crude laccase on various micro-biochars and degradation of diclofenac

Although enzymes are gifted with unique and unprecedented catalytic activity and selectivity over a wide range of pollutants, still their stability related issues often hinder their application in real environmental conditions. In this study, agro-industrially produced crude laccase was concentrated using ultrafiltration. Crude laccase was immobilized on pine wood (BC-PW), pig manure (BC-PM) and almond shell (BC-AS) biochar microparticles. Immobilization of laccase was investigated at various laccase activities on micro-biochars and the release (desorption) of the enzyme has been studied. It was observed that for all the biochars, as the initial concentration of laccase increased in the crude solution, the binding capacity and as result immobilization efficiency also increased. BC-PM was found to be the most effective (31.4 ± 3.1 U g−1) at 10 U mL−1 of enzyme activity followed by BC-AS (24.3 ± 4.8 U g−1) and BC-PW (14.58 ± 3.3 U g−1). In addition, the biochars were functionalized with citric acid for possible surface modifications and the effect of biochars for the adsorption of enzymes has been investigated. Isotherm studies of enzyme loading onto biochar established homogeneous monolayer adsorption as the major mechanism. The desorption of laccase from all biochars followed pseudo-second-order model. Immobilized laccase exhibited superior storage ability and shelf-life which were three times higher than free laccase. Finally, the immobilized laccase was used for the degradation of micropollutant, DCF and near 100% removal was obtained within 5 h at an environmentally relevant concentration (500 μg L−1).