Enzyme Concentration In Solution Research Articles

Enzymatic Assay of Immobilized β-D-Galactosidase Enzyme on Magnetite Nanoparticle

According to the high operational costs, low stability, and reusability of enzymes, immobilization by nanoparticle gathering has increased in recent years. Iron oxide nanoparticles (magnetite nanoparticles, Fe₃O₄) have been prepared by mixing one volume of iron dioxide ions and two volumes of iron trioxide ions with HCl via the precipitation of iron salts by NH₄OH. The features of magnetic nanoparticles have been studied by Atomic Force Microscopy (AFM), X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy, and Scanning Electron Microscopy (SEM). The prepared Fe3O4 was used in the adsorption method to immobilize the galactosidase enzyme. The immobilized enzyme has been compared with the crude one by optimizing time, PH, temperature, substrate solution concentration, and enzyme solution concentration. As a result, the immobilized enzyme has exhibited higher activity over long storage times, and a wide PH and temperature range than crude enzyme. In addition, whatever the substrate and enzyme concentrations, the activity of the immobilized enzyme increased over the non-immobilized one. As a result, a lower Km value for immobilized D-galactosidase indicates a higher affinity for the substrate than crude D-galactosidase.

Study of the parameters used in the encapsulation of commercial pectinase in calcium alginate and its effect on its catalytic activity

Abstract Despite the high catalytic properties of pectinases, the utilization of free enzymes always presents some hindrances such as low stability, a difficulty for product recovery and the impossibility of continuous use. Enzyme encapsulation is one of the methods used to overcome these limitations; however, some kind of effect is expected to occur on its catalytic activity. The objective of this work was to study the parameters involved in the process of encapsulation of a commercial pectinase product in calcium alginate and the effect of this encapsulation on its catalytic activity. The effect of the parameters of sodium alginate, calcium chloride, enzyme concentration and reaction time on enzymatic activity and encapsulation yield were also evaluated. The effect of pH and temperature on the activity of the free and encapsulated enzyme was studied. The highest yield of immobilization was obtained with a concentration of enzyme solution of 4%. Free and encapsulated enzymes showed similar behavior regarding the catalytic activity. The encapsulated enzyme had a narrower pH range (pH 4.0) than the free enzyme (pH 3.0 to 5.0). Besides, the encapsulated enzyme showed an increase in the stability in the pH range between 7 and 8 and above 10 to 12.

Immobilization of Cellulase on a Functional Inorganic–Organic Hybrid Support: Stability and Kinetic Study

Cellulase from Aspergillus niger was immobilized on a synthesized TiO2–lignin hybrid support. The enzyme was effectively deposited on the inorganic–organic hybrid matrix, mainly via physical interactions. The optimal initial immobilization parameters, selected for the highest relative activity, were pH 5.0, 6 h process duration, and an enzyme solution concentration of 5 mg/mL. Moreover, the effects of pH, temperature, and number of consecutive catalytic cycles and the storage stability of free and immobilized cellulase were evaluated and compared. Thermal and chemical stability were significantly improved, while after 3 h at a temperature of 50 °C and pH 6.0, the immobilized cellulase retained over 80% of its initial activity. In addition, the half-life of the immobilized cellulase (307 min) was five times that of the free enzyme (63 min). After ten repeated catalytic cycles, the immobilized biocatalyst retained over 90% of its initial catalytic properties. This study presents a protocol for the production of highly stable and reusable biocatalytic systems for practical application in the hydrolysis of cellulose.

Titania/lignin hybrid materials as a novel support for α-amylase immobilization: A comprehensive study

α-Amylase from Aspergillus oryzae was immobilized via covalent bonds and by physical interactions on a synthesized titania/lignin novel hybrid support. A temperature of 5°C, a pH of 7.0, an initial enzyme solution concentration of 3.0mg/mL and a 3h process duration were found to be optimal for the highest activity of the immobilized enzyme. Moreover, the effect of temperature, pH, storage time and repeated catalytic cycles on the activity of free and immobilized enzyme was examined. Bound α–amylase showed enhanced thermal and chemical stability, and its reusability was also improved. Immobilized α-amylase retained over 80% of its initial activity when stored for 30days at 4°C. Kinetic parameters of the free and immobilized biocatalyst were calculated and compared. The maximum reaction rate (Vmax) and turnover number (kcat) were slightly lower for the immobilized enzyme than for the free enzyme. It should be clearly stated that this work presents a useful protocol to produce stable and active immobilized α–amylase onto titania/lignin hybrid which may also be applied to immobilization of other enzymes.

Luffa cylindrica sponges as a thermally and chemically stable support for Aspergillus niger lipase.

The use of biopolymer compounds as matrices for enzyme immobilization is currently a focus of increasing interest. In the present work we propose the use of Luffa cylindrica vegetable sponges as a support for the lipase extracted from Aspergillus niger. Effectiveness of immobilization was analyzed using Fourier transform infrared spectroscopy, elemental analysis and the Bradford method. An initial enzyme solution concentration of 1.0 mg/mL and an immobilization time of 12 h were selected as the parameters that produce a system retaining the highest hydrolytic activity (84% of free enzyme). The resulting biocatalyst system also exhibited high thermal and chemical stability, reusability and storage stability, which makes it a candidate for use in a wide range of applications. Kinetic parameters for the native and immobilized lipase were also calculated. The value of the Michaelis-Menten constant for the immobilized lipase (0.47 mM) is higher than for the free enzyme (0.21 mM), which indicates that the adsorbed enzyme exhibits a lower affinity to the substrate than native lipase. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:657-665, 2016.

Optimizing the Enzymatic Elimination of Clogging of a Microfiltration Membrane by Parellada Grape Cake

AbstractClogging of the filtration membranes is one of the main problems in the process of obtaining grape must for white wine; therefore, clogging must be reduced to the maximum. The aim of this work was to find the optimal values of the temperature, time and enzyme solution concentration variables to know the best conditions to reduce the clogging of Parellada var. grape pulp in microfilters by enzymatic hydrolysis. Moreover, a study of the effluents produced in this process was also carried out. The unclogging experiments carried out at different temperatures and times led to the activation energy value of 60.3 kJ/mol, and the optimum conditions obtained, those that produced the best filtration, were temperature of 51.8C, time of 35 min and enzyme solution concentration of 379.4 μL/L. Finally, the kinetic equations of the variation in the contents of the effluents generated in this process under the optimal conditions were also determined.Practical ApplicationsThis paper shows an application of a new original funnel reactor designed by the authors to remove the clogging by enzymatic digestion where the experimental parameters of the filtration process can be varied in order to achieve the optimal values of these parameters to improve the efficiency of the process. Using the values obtained for the process variables, a significant reduction of economic costs could be obtained compared with non‐optimized cake degradation, particularly which produced a decrease in the dose of the added enzyme, whose relationship with the amount of pulp in the filter will not change in the scaling up of the process.

Optimization of Enzymatic Treatments for Deskinning of Catfish Nuggets

One collagenase (CLS1®) and two proteases (Proleather FG-F® and Protease N®) were used to deskin the peritoneal membrane of catfish nuggets. The factors were used in a full factorial experiment design: enzyme solution concentration (0.2, 0.3, and 0.4% by weight), temperature of enzyme solution (30, 37, and 44°C), and immersion time (10, 15, and 20 min). Contour plots showed that the enzyme Proleather FG-F® maximized whiteness of catfish nuggets at 40–44°C for 15–19 min. Treatment of the enzyme Proleather FG-F® at 0.2% for 15 min had the highest yield (103.4%) at 44°C. Enzymatic skinning has potential in removing the peritoneal membrane from catfish nuggets, improving color (whiteness), and yield.

Determination of Two Organophosphorus Pesticides Using Electrochemical Sensor in Water Samples

A detection method for organophosphorus pesticides using electrochemical sensor based on enzyme inhibition has been developed. Acetylcholine esterase (AChE) was selected to quantify the pesticides concentration in water samples due to its activity was non-competitively inhibited in the presence of pesticides. To detect parathion and dichlorvos, optimized analytical conditions were that the substrate concentration was 3mmol/L, enzyme solution concentration was 18g/L and reaction time was 20min. Under the optimized detection conditons, a detection limit of 1.2ng/mL and 0.004ng/mL for parathion and dichlorvos was obtained, respectively. The approach was rapid, simple, accurate and of high sensitivity when compared with immunoassays or chromatographic methods.

Enzymatic Treatment as a Pre‐Step to Remove Cellulose Films from Sensors

AbstractIn this work an enzymatic treatment is proposed as a preparative, cleaning protocol to remove cellulose films from resonators and sensors. Quartz crystal and surface plasmon gold sensors, coated with ultrathin films of cellulose are used in studies of molecular (for example, polymer and surfactant) adsorption. The sensors are usually recycled after removal of the film, with limited success, after one of two treatments, either hot acid or ammoniac solutions. In the proposed, improved protocol a mixture of cellulases from Aspergillus species, are used as a pre‐treatment to facilitate the release of the cellulose film from the surfaces of the sensors. Two concentrations of NaCl solutions were considered in the enzymatic treatment, 1 and 10 mM, at given enzyme solution concentration, temperature and pH. It was found that after 80 min, the water contact angle after treatment with both salt concentration conditions reached a plateau. The average water contact angle after integration of the enzymatic and ammoniac treatments was found to be low enough, between 6.4 and 7.1 deg to allow reuse the sensors. It is concluded that the use of the ammoniac cleaning solution after the enzymatic treatment is a very convenient, safe and less time consuming way to remove the cellulose films from the sensors to be recycled.

Enzyme:nanoparticle bioconjugates with two sequential enzymes: stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles.

We report the synthesis and characterization of bioconjugates in which the enzymes malate dehydrogenase (MDH) and/or citrate synthase (CS) were adsorbed to 30 nm diameter Au nanoparticles. Enzyme:Au stoichiometry and kinetic parameters (specific activity, kcat, KM, and activity per particle) were determined for MDH:Au, CS:Au, and three types of dual-activity MDH/CS:Au bioconjugates. For single-activity bioconjugates (MDH:Au and CS:Au), the number of enzyme molecules adsorbed per particle was dependent upon the enzyme concentration in solution, with multilayers forming at high enzyme:Au solution ratios. The specific activity of adsorbed enzyme increased with increasing number adsorbed per particle for CS:Au, but was less sensitive to stoichiometry for MDH:Au. Dual activity bioconjugates were prepared in three ways: (1) by adsorption of MDH followed by CS, (2) by adsorption of CS followed by MDH, and (3) by coadsorption of both enzymes from the same solution. The resulting bioconjugates differed substantially in the number of enzyme molecules adsorbed per particle, the specific activity of the adsorbed enzymes, and also the enzymatic activity per particle. Bioconjugates formed by adding CS to the Au nanoparticles before MDH was added exhibited higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also had 3-fold higher per-particle sequential activity for conversion of malate to citrate, despite substantially fewer copies of both enzymes present.

Enzyme immobilization. Part 4. Immobilization of alkaline phosphatase on Na-sepiolite and modified sepiolite

Alkaline phosphatase from calf intestinal mucous membrane was immobilized on the modified and unmodified Na-sepiolite. The effects of various factors such as concentration of enzyme solution, pH and temperature of immobilization medium, magnetic stirring and various thermodynamic parameters on the immobilization rate of alkaline phosphatase (APh) were evaluated. pH = 10 was optimal for enzyme activity for both the free enzyme and the sepiolite. Sepiolite with bilayer surfactant coverage (SBS) had high ability for immobilization of APh. APh immobilized on SBS showed approximately similar V max and K m in comparison with the free enzyme. By immobilization of APh on Na-sepiolite without surfactant (SWS) and sepiolite with monolayer surfactant coverage (SMS), V max decreased and K m increased.

Amylolytic potentiality of fungi isolated from some Nigerian agricultural wastes

Five common Nigerian agricultural wastes: cassava peels, yam peels, banana peels, plantain peels, and Brewery Spent Grains (BSG) were formulated into media on which amylolytic fungal strains were isolated. These isolates were subsequently identified, characterized using standard mycological keys and were thereafter employed in production of
-amylase using submerged and solid-state cultivation regimens. Amount of
-amylase in enzyme unit (E.U.) liberated by each mould was quantified by estimating the amount of reducing sugars produced when specified quantity of starch was hydrolyzed after incubation at 40°C with known concentration of enzyme solution. Results showed the amylolytic isolates to be Helminthosporium oxysporium, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Penicillium frequestans. The mould Helminthosporium oxysporium liberated 10.77 and 10.42 E.U of
-amylase on cassava and yam peels media respectively using submerged cultivation method while A. flavus produced 11.94 E.U. of the enzyme on cassava peels with submerged cultivation method. Production of
-amylase on cassava, yam and plantain peels was facilitated by submerged cultivation method. With exception of banana peels substrate, other investigated Nigerian agricultural wastes favoured
-amylase production at different rates.

Amylolytic potentiality of fungi isolated from some Nigerian agricultural wastes

Five common Nigerian agricultural wastes: cassava peels, yam peels, banana peels, plantain peels, and Brewery Spent Grains (BSG) were formulated into media on which amylolytic fungal strains were isolated. These isolates were subsequently identified, characterized using standard mycological keys and were thereafter employed in production of α-amylase using submerged and solid-state cultivation regimens. Amount of α-amylase in enzyme unit (E.U.) liberated by each mould was quantified by estimating the amount of reducing sugars produced when specified quantity of starch was hydrolyzed after incubation at 40°C with known concentration of enzyme solution. Results showed the amylolytic isolates to be Helminthosporium oxysporium, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, andPenicillium frequestans. The mould Helminthosporium oxysporium liberated 10.77 and 10.42 E.U of α-amylase on cassava and yam peels media respectively using submerged cultivation method while A. flavus produced 11.94 E.U. of the enzyme on cassava peels with submerged cultivation method. Production of α-amylase on cassava, yam and plantain peels was facilitated by submerged cultivation method. With exception of banana peels substrate, other investigated Nigerian agricultural wastes favoured α-amylase production at different rates. Key words: Agricultural wastes, submerged cultivation, solid-state cultivation, α-amylase, fungal isolates.

Enzyme immobilization: Part 2: Immobilization of alkaline phosphatase on Na-bentonite and modified bentonite

Alkaline phosphatase from calf intestinal mucous membrane was immobilized on unmodified and modified bentonites. The activity of the immobilized enzyme was examined under varying experimental conditions. The effects of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were evaluated. The highest enzyme activity, for free and immobilized enzyme, was obtained in 0.1 M phosphate buffer at pH 7. Optimal enzyme activity of both free and adsorbed enzyme was reached at pH = 10. The best time period of magnetic stirring was 2 h at 4 °C. The adsorption isotherm was modeled by Langmuir equation. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited activity comparable to the soluble enzyme after storage at 30 °C. Thermal stability and resistance against proteolytic attack were also evaluated.

Enzyme immobilization: Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase

Immobilization of Candida rugosa lipase onto modified and unmodified bentonites is described. The effect of hydrophilic or hydrophobic nature of the support, the reuse efficiency, and kinetic behavior of immobilized lipase were studied. The modified bentonite with monolayer surfactant (BMS), was the best support, for immobilization. The activity of the immobilized enzyme was examined under varying experimental conditions. The effect of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were also evaluated. The activity of lipase on Na-bentonite, on BMS and on bentonite with bilayer surfactant (BBS) at the optimum pH was 7.2%, 56.6% and 3.6%, respectively. The adsorption isotherm was modelled by the Langmuir equation. The amounts of immobilized lipase on Na-bentonite, BMS and BBS at the highest activity were 42.6%, 61.2% and 28.3%, respectively. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited an activity comparable to the free enzyme after storage at 30 °C. The thermal stability of free and immobilized lipase were also studied.

면사의 효소가공 및 실켓 가공

The cotton yarn was subjected to bio-polishing treatment with three commercial enzymes(Cellusoft L, Denimax-991L and Denimax-acid) to remove the fuzz on the cotton yarn. Also, enzyme treated cotton yarns were compared with singeing cotton yarns. Experimental variables of enzyme treated cotton yarn were as follow: concentration of enzyme solution and NaOH, dipping time, and processing temperature. The enzymatic treatments were evaluated by analyzing the effect on yarn count, twist contraction, evenness and tenacity. As the results, enzymatic treatment on cotton yarn induced same effects as the traditional singeing treatment. Also, silket treatment of cotton yarn after bio-polishing enhanced the tensile properties of the cotton yarn.

Glucose biosensor constructed from capped conducting microtubules of PEDOT

Abstract Electrochemical glucose biosensor based on conducting microcells was constructed by imprisoning the enzymes in the cell arrays on an electrode. This kind of enzyme confinement showed a new way of construction of electrochemical biosensor without allowing modification of enzyme itself. The microtubular array was fabricated through electrochemical polymerization of EDOT into a template membrane attached on an ITO working electrode. The hollow tubules were loaded with glucose oxidase dispersed in aqueous buffer solution. To imprison the glucose oxidase into the cell, the opening of the tubule was sealed with PEDOT/PSS composite cap. The reinforcing electrochemical polymerization of PEDOT or poly(1,3-phenylenediamine) onto the sealing composite reduce the water solubility of the capping. The steady-state amperometric response to glucose was determined by detecting hydrogen peroxide generated by the action of the glucose oxidase. The effects of the applied potential, the gold treatment, the number of capping polymerization cycles, and the concentration of enzyme solution on the sensing properties of the electrode were examined. The calibration curve showed its linearity between 0 and 8 mM and the response time was 20 s. The sensor could retain good selectivity against interfering substances by capping the opening with electrochemically prepared non-conducting poly(1,3-phenylenediamine) layer. In summary, a glucose sensor which shows good selectivity and sensitivity was achieved by this enzyme-imprisonment procedure. This procedure has a wide applicability by just changing the enzyme to be loaded.

Determination of active enzyme concentration using activity-based probes and direct mass spectrometric readout

Activity-based probes (ABPs) are specific covalent inhibitors developed for different classes of enzymes. We have titrated a serine protease and a lipase with their specific ABPs and measured the extent of inhibition using nanoelectrospray mass spectrometry (nanoESI–MS). Because ABPs only interact with the active enzyme form, the approach allows to accurately measure the active enzyme concentration in solution. This is even possible in the presence of contaminants. The concentrations of the two enzymes were also investigated by UV spectroscopy, which appears to give higher concentrations than those measured with the active site titration method.

Studies on α-amylase induced degradation of binary polymeric blends of crosslinked starch and pectin

A blend matrix of crosslinked starch and pectin was prepared and characterized by infra-red (IR) spectroscopy, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). The prepared blends were investigated kinetically for water sorption studies and alpha-amylase induced degradation adopting a gravimetric procedure. Based on the experimental findings, a plausible mechanism including both diffusion and surface enhanced degradation was suggested and degradation profiles were interpreted. The influence of various factors such as chemical architecture of the blend, pH and temperature of alpha-amylase solution were examined for the swelling and degradation kinetics of crosslinked starch-pectin blends. The effect of concentration of enzyme solution was also studied on the degradation profile of the blends. A correlation was established between the extent of degradation and water imbibing capacity of the degrading blends.

Effect of proteolytic enzyme on dyeing of wool with madder

In recent years, the use of low-environmental impact biotechnology giving rises to new types of treatment in the textile industry. The use of protease enzymes to improve some physical and mechanical properties such as smoothness, drapeability, dyeing affinity and water absorbency is particularly interesting. In this research, wool yarns were first treated with different concentrations of protease enzymes in water solution including 1, 2, 4 and 6% o.w.f. for 60 min. The dyeing process was then carried out on the treated yarns with madder (50% o.w.f.). Tensile strength of treated yarns was decreased due to enzyme treatment and it continued to decrease with an increase in enzyme concentration in solution. The L * values decreased for the samples treated with enzyme. The wash and light fastness properties of samples were measured according to ISO 105-CO5 and Daylight ISO 105-BO1. The washing fastness properties of treated samples were not changed. In the case of light fastness properties, it was increased a little for 4% and 6% enzyme treated samples.